Polyacrylamide Gel Research Articles

Study on Internal Standards Applicable to the Detection of Recombinant Erythropoietin.

With the discovery of the variant c.577del in the EPO gene, the procedure for detecting the presence of recombinant erythropoietin (rEPO) has become complicated and time-consuming. To address this situation, an rEPO confirmation method that uses reverse-normal immunopurification coupled with western blotting (WB) and sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE), which can detect the EPO variant (VAR-EPO) and rEPO with anti-VAR-EPO and anti-EPO antibodies, has been developed. Therefore, it is necessary to develop an internal standard (IS) that can be recognized by an anti-VAR-EPO antibody to monitor reverse immunopurification, ensuring the reliability and accuracy of the analysis. In this study, we constructed an IS based on VAR-EPO modified with polyethylene glycol (PEG) and then assessed its reliability and applicability in doping analysis. The data provided here show that the obtained PEGylated VAR-EPO can be used as an IS to monitor the detection of not only rEPO and VAR-EPO but also EPO receptor agonists in urine samples.

Evaluation of a commercial Immunochromatographic Strip Assay (ICT) for rapid detection of bovine, porcine and human Rotavirus A

The most frequent diarrhoeal pathogen “Rotavirus” is commonly detected by Ribo Nucleic Acid- Polyacrylamide Gel Electrophoresis (RNA-PAGE), Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Immunoassay (Enzyme Linked Immunosorbent Assay) in humans. However, use of these tests is limited in animals therefore, penside test like Immunochromatographic test/Lateral Flow Assay (ICT/LFA) is necessary. The present study was carried out to compare the diagnostic efficacy of rapid commercial ICT strip assay, RNA-PAGE and RT-PCR for specific detection of Rotavirus A (RVA) from stool samples of calves, piglets and children. A total of 313 faecal samples were collected between November 2022 to April 2023 from children below 5years of age (n=100), calves (n=100) and piglets (n=113) ≤ 3 months of age and were screened by RNA-PAGE, RT-PCR and commercial ICT kit for rotavirus A.The overall positivity of RVA from human, calves and piglets using RNA-PAGE, RT-PCR and LFA was found to be 35% (35/100), 8% (8/100) and 14.16% (16/113), respectively. Higher positivity of rotavirus was recorded in male children (44.26%, 27/61) than in female children (20.51%, 8/39). The kappa agreement between LFA and RT-PCR was 0.79 (substantial agreement); between LFA and RNA-PAGE was 0.61 (substantially low agreement) and between RNA-PAGE and RT-PCR was found to be 0.75 (substantial agreement). Relative diagnostic sensitivity and specificity of LFA in comparison with RT-PCR was 74.54% and 98.44%, respectively and in comparison with RNA-PAGE was 72.97% and 93.47%, respectively. RNA-PAGE in comparison with RT-PCR is having diagnostic sensitivity and specificity of 65.45% and 99.6%, respectively. In conclusion, the ICT strip assay is a rapid, convenient and effective method with satisfactory efficacy for detection of RVA from children, calves and piglets. ICT fulfilled the WHO ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Delivered) criteria for point-of-care testing. It can be useful in determining rotavirus outbreaks in resource-limited settings.

Determination of diffusion coefficients through gels with non-negligible finite-volume effects in the compartments of the diffusion cell

Diffusion cells are used to measure diffusion coefficients (DM) in gels. These measurements are of interest to understand and predict the availability of nutritive or toxic chemical species in waters, soils and sediments. When the diffusive flux from the donor to the acceptor compartment is constant (steady-state regime), DM is determined from the slope of the linear plot of the acceptor concentration vs time. However, at long enough times, there is a non-negligible concentration depletion in the donor compartment concomitant to a concentration increase in the acceptor compartment. Accordingly, the accumulation plot bends downwards preventing a linear fitting. This is the case of metals whose solubility (especially depending on pH values) limits the concentration in the donor compartment and the time required to reach concentrations above the limit of quantification in the acceptor compartment implies a non-negligible decrease of the concentration in the donor compartment.In this work, a simple linear regression is shown to provide the diffusion coefficient values from experiments exhibiting finite-volume effects. This expression is validated against rigorous numerical simulation as well as reported values in the literature. Diffusion coefficients of Zn, Ni and Pb in agarose cross-linked polyacrylamide (APA) gels (used in Diffusive Gradients in Thin-Film devices, DGT) are determined under finite-volume effects. The resulting values agree with those obtained under the standard linear regime.

Effect of pre-acidification induction on the physicochemical features, myofibrillar protein microstructure, and headspace volatiles of ready-to-cook goose meat

This study examined the impact of pre-acidification induction on the quality attributes and flavor retention of ready-to-cook (RTC) goose meat products. The results demonstrated that pre-acidification could influence the eating qualities of RTC goose meat by effectively regulating the physicochemical properties of goose myofibrillar proteins (MP) including solubility and water-holding capacity. Elevated carbonyl content indicated an enhanced gel-forming capacity in RTC goose meat during storage, coupled with reduced total sulfhydryl content from enhanced protonation pretreatment and augmented lipid oxidation. Structural characterization of MP via sodium dodecyl sulfate–polyacrylamide gel electrophoresis, circular dichroism spectroscopy, and intrinsic fluorescence revealed the formation of a dense protein matrix under highly acidic conditions. Furthermore, the headspace concentration of aldehydes increased by 3.23 times upon enhancing the pre-acidification intensity, resulting in the production of esters and acidic flavor compounds with favorable aromas. Correlation analysis demonstrated the dependence of headspace concentrations of volatile constituents on the acidification-enhanced surface hydrophobicity of MP, attributed to the modified binding sites of proteins after pre-acidification. Current results have indicated both the positive and negative influence of pre-acidulation induction on the eating quality of goose meat products, suggesting the necessity of introducing extra processes to modulate the quality of prefabricated products.

Artificial trimerization of β-glucosidase for enhanced thermostability and activity via computational redesign

The improvement of enzyme thermostability is of great significance in the biotechnology industry. Herein, we modified the C-terminus of β-glucosidase via the computer-aided rational design and successfully obtained six variants that exhibit improved optimal temperatures and thermal inactivation half-lives at 65 °C. Among these variants, the thermal inactivation half-life and hydrolytic activity of TrCel1b-H13 at 65 °C were increased by 416 and 3223 folds, respectively, compared to those of the wild type. The optimal catalytic temperature and melting temperature (Tm) of TrCel1b-H13 were elevated by 55 °C and 20 °C respectively, compared to those of the wild type. The Kcat/Km value of TrCel1b-H13 was improved by 13.3-fold, compared to that of the wild type. Furthermore, we found that the modified β-glucosidase, TrCel1b-H13, self-assembles into a trimeric structure, which was confirmed by X-ray crystallography and blue native polyacrylamide gel electrophoresis. The amino acid residues, including E28, H60, E99, K106, and K471, located at the trimeric interface of the adjacent subunits, were found to play an important role in maintaining the crystal structure of the protein trimer. Thus, protein multimerization via the computer-aided rational design method provides a cost-effective tool for simultaneously improving the thermostability and activity of enzymes.

Анализ генетического разнообразия сортов масличного льна селекции ВНИИМК с применением молекулярно-генетических маркеров

Oil flax (Linum usitatissimum L.) is one of the most important technical crops. The success of the breeding work is reached mostly by the genetic diversity of used initial germplasm. The molecular-genetic markers determine this diversity. Five cultivars of oil flax: Danik, Snegurok, Nilin, Y 117, and August, bred at the V.S. Pustovoit All-Russian Research Institute of Oil Crops were used as research material. To estimate genetic structure of cultivars we used 40 plants of each cultivar. Ten microsatellite loci markers were used for analysis. The amplification was conducted in a termocycler MiniAmp (Thermo Scientific, USA). The electrophoretic segregation of amplicons was carried out in 8% polyacrilamide gel in the native conditions. The primary indicators of genetic varia-tion (a number of alleles per a locus (Na), an effective number of alleles (Ne), observed (Ho) and wanted (He) heterozigosity, an informative Shennon’s index (I), fixation index (F), percentage of polymorphic loci (%P), genetic distances (D) were determined, analysis of molecular variation and principle coordinate analysis were done. The indicators of intraspecific genetic diversity decreased in the direction of cultivar range Nilin, Danik, August, Y 117, and Snegurok. Snegurok is a cultivar of linear type with some number of non-typical plants. The cultivar Y 117 is divided into two main biotype. Nilin, Danik, and August do not possess clear-marked structure. Most of the total variation is conditioned by differences between cultivars. The genetic distances between cultivars are quite big and equal from 0.752 to 0.986. All cultivars are distant from each other by two main coordinates. The conducted research showed small intravarietal and signif-icant intervarietal genetic diversity of five oil flax cultivars bred at the V.S. Pustovoit All-Russian Re-search Institute of Oil Crops and their potential in as initial material for breeding of new cultivars.

Concise Affinity-Based Purification of Ligated mRNA for Structure-Activity Relationship Studies of Nucleosugar Modification Patterns.

Position-specific nucleoside sugar modifications have been shown to improve the translational activity and stability of chemically synthesized mRNA. For pharmaceutical applications of chemically modified mRNAs, a rapid purification methodology is imperative to identify the optimal modification pattern. However, while the chemical synthesis of mRNAs can be accomplished by splint ligation of oligonucleotide fragments, the current purification method for ligated mRNAs based on denaturing polyacrylamide gel electrophoresis tends to be time consuming. In this study, we developed a two-step affinity purification method for rapid sample preparation. In this method, ligated mRNA is captured by oligo dT magnetic beads and streptavidin magnetic beads with 3'-biotinylated oligo DNA, which are complementary to the 3'-poly(A) and 5' terminal sequences of the target mRNA, respectively. Therefore, the target mRNA can be isolated from a complex mixture of splint ligations. Using this method, six sugar-modified mRNAs were simultaneously purified, and the translational activities of these mRNAs were evaluated immediately after purification. The results demonstrate that this methodology is suitable for the rapid preparation of various chemically synthesized mRNAs to identify their optimal modification patterns.

Testicular enzyme activity alterations in rats with liver cirrhosis induced by alcohol and acetaminophen

Alcohol-induced acetaminophen (APAP) toxicity is one of the numerous factors that might result in liver cirrhosis (LC). The hepatotoxicity of APAP appears to increase in chronic drinkers, according to a number of investigations. These people not only have a greater risk of experiencing acute overdose-related severe and deadly liver damage but are also at risk of experiencing similar substantial liver damage from therapeutic APAP use. The male reproductive system consists of both testes and a few other auxiliary sexual organs. Smaller and lighter testicles are indicative of severe cirrhosis causing testicular atrophy. In the case of severe LC, low testosterone results from hypogonadotropic hypogonadism. Sexual dysfunction is a prevalent condition that is frequently overlooked in individuals suffering from cirrhosis and chronic liver disease. Eighteen rats were randomly divided into three groups. Rats of normal control group received water and normal diet ad libitum; alcohol control and LC group received 4.5% alcohol and a combination of 4.5% alcohol and APAP (300 mg/kg bw) through drinking water, respectively, for 7 days. Several glycolytic and antioxidant enzymes were assessed for their effects through non-denaturing polyacrylamide gel electrophoresis analysis and enzyme activity. The results indicated that long-term alcohol consumption and APAP medication altered the levels of antioxidant and glycolytic enzymes. To the best of our knowledge, this is the first study demonstrating that chronic alcoholism and APAP induce hepatotoxicity, and LC further affects the antioxidant and glycolytic enzyme activities of the testes.

In Vitro and In Vivo Digestibility of Putative Nutraceutical Common-Bean-Derived Alpha-Amylase Inhibitors

The inhibition of the alpha-amylase digestive enzyme impedes starch digestion by blocking access to the active site of the enzyme, thereby playing a role in the prevention of obesity and type 2 diabetes. Plant-derived alpha-amylase inhibitors (αAIs) are promising nonpharmacological alternatives for the prevention of these diseases. Alpha-amylase inhibitor-1 (αAI-1) present in common bean (Phaseolus vulgaris) is derived from a precursor protein. In this study, the effect of digestion on the digestibility, immune reactivity, and bioactivity of αAI-1 was assessed from four varieties of Hungarian common bean (Phaseolus vulgaris), with special regard to the precursor protein. For this purpose, αAI-1 was tested in both matrix (native flour and cooked flour) and purified forms under in vitro and acute rat in vivo digestion experiments. The effect of digestion on αAI-1s was monitored by lab-on-a-chip (LOC) electrophoresis, SDS-PAGE/immunoblot, and inhibitory activity analyses by native PAGE. After both in vitro and in vivo digestion, we established that αAI-1 was not degraded even after 60 min gastric digestion and showed immune-reactive properties as well. Although the activity of the purified αAI-1 was lost, that of αAI-1 in the flour matrix (noncooked and cooked) was retained in the stomach. Presumably, in the beans, αAI-1 polypeptides became active due to the pepsin digestion of the precursor protein. The latter samples were also tested in vivo in the small intestine and their resistance and immune reactivity were observed, but αAI-1 did not show activity, as αAI-1 polypeptides were probably complexed by pancreatic amylases. From these results, we can assume that the αAI-1-rich bean protein preparation can affect the carbohydrate metabolism; thus, it could be a promising ingredient for weight loss purposes.

Moringa oleifera Leaf Extract Functions as a Potent Inhibitor of Snake Venom

<i>Moringa oleifera</i> Leaf Extract Functions as a Potent Inhibitor of Snake Venom

A CD Study of a Structure-Based Selection of N-Heterocyclic Bis-Carbene Gold(I) Complexes as Potential Ligands of the G-Quadruplex-Forming Human Telomeric hTel23 Sequence.

Herein, we report the structure-based selection via molecular docking of four N-heterocyclic bis-carbene gold(I) complexes, whose potential as ligands for the hTel23 G-quadruplex structure has been investigated using circular dichroism (CD) spectroscopy, CD melting, and polyacrylamide gel electrophoresis (PAGE). The complex containing a bis(1,2,3,4,6,7,8,9-octahydro-11H-11λ3-pyridazino[1,2-a]indazol-11-yl) scaffold induces a transition from the hybrid (3 + 1) topology to a prevalent parallel G-quadruplex conformation, whereas the complex featuring a bis(2-(2-acetamidoethyl)-3λ3-imidazo[1,5-a]pyridin-3(2H)-yl) moiety disrupted the original G-quadruplex structure. These results deserve particular attention in light of the recent findings on the pathological involvements of G-quadruplexes in neurodegenerative diseases.

Self-induced anaerobiosis fermentation in coffees inoculated with yeast: Effect on key enzymes of the germination process and its relationship with the decrease in seed germination

Our objective was to monitor the main enzymes of coffee germinal metabolism and chemical composition during Self-Induced Anaerobiosis Fermentation (SIAF) with yeasts (Saccharomyces cerevisiae (CCMA0543), Candida parapsilosis (CCMA0544) and Torulospora delbrueckii (CCMA0684)) evaluating their relationship with seed germination. The starter cultures were assessed by qPCR. The organic acids were analyzed by liquid chromatography. Catalase (CAT), Esterase (EST), Alcohol dehydrogenase (ADH), and Isocitrate Lyase (ICL) enzyme activity was confirmed by the presence of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-Page) gel bands. The formation of a white halo confirmed the activity of the enzyme endo-β-mannanase, and its quantification was performed using the diameter of the halo of both the samples and the standard curve. At the end of the fermentation process, S. cerevisiae and T. delbrueckii presented the highest populations (>7 log10 cells/g). Succinic acids (average −1.11 g/kg) were consumed during SIAF. Lactic acid increased after 180 h in coffees fermented by the SIAF method (average 3.57 g/kg). CAT and EST showed high activity in the conventional process. ADH activity was detected in both processes after 180 h of the SIAF method. Yeast inoculation during the SIAF method increased the activity of ICL andshowed more intense activity in the first 96 h of fermentation, especially the pulped coffee. Endo-β-mannanase activity was intense during conventional coffee processing (9.89–10.99 pmol/min/g). Natural processing tends to preserve a higher percentage of viable seeds. Therefore, the processing and fermentation methods impact seed quality differently.

Allelic variation at the PPD-A1 locus and its associations with heading time and winter wheat (Triticum aestivum L.) agronomic traits in the northern Black Sea region

Aim. The identification of Ppd-1-alleles in winter bread wheat varieties of various origin, including the ones of Ukrainian plant breeding and recombinant-inbred lines Orenburgskaya 48//Cappelle Desprez/2В Chinese Spring, and the evaluation of the effects of allele Ppd-А1_del303, including the interaction with different alleles of gene Ppd-B1, by the duration of the period before heading and the related agronomically valuable traits. Methods. DNA extraction, allele-specific PCR, agarose and polyacrylamide gel electrophoresis, phenological observations, evaluation of frost resistance in seedlings, and analysis of morphobiological traits and elements of yield structure. Statistical analysis of the obtained data was carried out in Microsoft Excel. The significance of the difference between samples was assessed by Fisher's F test. A difference of p &lt; 0.05 was considered statistically significant for all indicators. Results. The marking of 30 varieties of different origin and 64 recombinant-inbred lines of Orenburgskaya 48//Cappelle Desprez/2B Chinese Spring winter bread wheat was carried out to identify the alleles of the Ppd-A1 gene. The polymorphism of varieties and populations of recombinant-inbred lines in the northern Black Sea region (Odesa) was evaluated for ten traits: frost resistance of plants in the seedling phase, winter hardiness, duration of the period before heading, plant height, grain number per spike, grain weight per spike, thousand grain weight, number of productive tillers per unit area, harvest index and grain yield. The comparison of the lines evaluation data in terms of agronomic traits and the results of the genotypes identification allowed us to identify the influence of Ppd-A1 gene alleles and various combinations of the alleles of Ppd-A1 and Ppd-B1 genes on these traits. Conclusions. A higher prevalence of the Ppd-A1_del303 allele was found both among varieties and recombinant-inbred lines. The genetic differences by the Ppd-A1 gene (Ppd-A1_del303 or Ppd-A1b) are significantly related only to frost resistance of seedlings in the absence of significant differences in other traits. The interaction between Ppd-A1b and Ppd-B1c alleles contributed to the acceleration of early maturity and the formation of the highest indicators of grain weight per spike, thousand grain weight, harvest index, and grain yield. The replacement of the Ppd-A1b allele with Ppd-A1_del303 led to a decrease in the effect of the dominant Ppd-B1c allele on accelerating heading and negatively affected the grain weight per spike, thousand grain weight, harvest index and grain yield compared to the Ppd-A1b Ppd-B1c genotype.

Chondroitin Sulfate-Coated Heteroduplex-Molecular Spherical Nucleic Acids.

Molecular Spherical Nucleic Acids (MSNAs) are atomically uniform dendritic nanostructures and potential delivery vehicles for oligonucleotides. The radial formulation combined with covalent conjugation may hide the oligonucleotide content and simultaneously enhance the role of appropriate conjugate groups on the outer sphere. The conjugate halo may be modulated to affect the delivery properties of the MSNAs. In the present study, [60]fullerene-based molecular spherical nucleic acids, consisting of a 2'-deoxyribonucleotide and a ribonucleotide sequence, were used as hybridization-mediated carriers (''DNA and RNA-carriers'') for an antisense oligonucleotide, suppressing Tau protein, (i.e. Tau-ASO) and its conjugates with chondroitin sulfate tetrasaccharides (CS) with different sulfation patterns. The impact of the MSNA carriers, CS-moieties on the conjugates and the CS-decorations on the MSNAs on cellular uptake and - activity (Tau-suppression) of the Tau-ASO was studied with hippocampal neurons in vitro. The formation and stability of these heteroduplex ASO-MSNAs were evaluated by UV melting profile analysis, polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and size exclusion chromatography equipped with a multi angle light scattering detector (SEC-MALS). The cellular uptake and - activity were studied by confocal microscopy and Western blot analysis, respectively.

RNA Blotting by Electrotransfer and RNA Detection.

Northern blot, or RNA blot, is a widely used technique in molecular biology to detect and semi-quantify specific RNAs. The advantage of this method is its ability to simultaneously estimate the sizes and quantities of degraded or processed RNA products. Northern blotting involves the use of electrophoresis to separate RNA samples by size. These RNAs are then transferred and immobilized on a membrane, and the RNAs of interest are detected by hybridization using a sequence-specific labeled DNA probe. Agarose gels are typically used in routine procedures to allow for the separation and specific detection of high molecular weights (ranging from tRNA size to several kb RNAs). However, acrylamide gels are preferred when the separation of lower weight RNAs (ranging from 20 to 200 nucleotides) is required. Both approaches for RNA separation are presented here. The term "northern blot" originally referred to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However, RNA transfer to the membrane by electrotransfer has also been proven to be a suitable and fast method for transferring RNAs from acrylamide and agarose gels to the membrane.

Assessing Deadenylation Activity by Polyacrylamide Gel Electrophoresis Using a Fluorescent RNA Substrate.

The poly(A) tail is a dynamic structure at the 3'- end of the majority of RNA polymerase II transcripts. It is a critical feature, particularly for mRNAs, as the length of the poly(A) tail regulates their translational efficiency and lifespan. The shortening of the tail is catalyzed by deadenylases that trim and finally remove it, triggering mRNA degradation. Conventional assays to evaluate and measure deadenylase activity rely on radiolabeling of the substrate while colorimetric alternatives lack sensitivity. In this chapter, we describe a poly(A)-shortening assay using a 5'-Cy3-labeled RNAsubstrate bearing a 3'-oligo(A) sequence. The reaction products representing substrates with shortened tails are analyzed by denaturing acrylamide electrophoresis and are visualized using a gel documentation system. This assay is efficient, it requires standard lab equipment, including a fluorescence-detecting gel documentation system, and presents a quantifiable and safer alternative to radioactivity-based methods for evaluating deadenylase activity.

Levilactobacillus brevis 47f: Bioadaptation to Low Doses of Xenobiotics in Aquaculture.

Agricultural and industrial activities are increasing pollution of water bodies with low doses of xenobiotics that have detrimental effects on aquaculture. The aim of this work was to determine the possibility of using Levilactobacillus brevis 47f culture in fish aquaculture under the influence of low doses of xenobiotics as an adaptogen. An increase in the survival of Danio rerio individuals exposed to the xenobiotic bisphenol A solution and fed with the L. brevis 47f was shown compared to control groups and, at the same time, the cytokine profile in the intestinal tissues of Danio rerio was also investigated. Analysis of differential gene expression of the L. brevis 47f grown under the action of high concentrations of bisphenol A showed changes in mRNA levels of a number of genes, including genes of various transport proteins, genes involved in fatty acid synthesis, genes of transcriptional regulators, genes of the arabinose operon, and the oppA gene. The identification of L. brevis 47f proteins from polyacrylamide gel by mass spectrometry revealed L-arabinose isomerase, Clp chaperone subunit, ATP synthase subunits, pentose phosphate pathway and glycolysis enzyme proteins, which are likely part of the L. brevis 47f strain's anti-stress response, but probably do not affect its adaptogenic activity toward Danio rerio.

Dynamic Evolution of Poly-A Tail Lengths Visualized by RNAse H Assay and Northern Blot Using Nonradioactive Probes in Yeast.

Poly-A tail length dynamics have been extensively studied from yeast to human, mostly using reporter transcripts. Recent studies have been carried out genome-wide to determine the status of poly-A tails at steady state. However, poly-A tail measurement at equilibrium gives an overall length that reflects a mixture of the different poly-A tail sizes for a single transcript. New genome-scale techniques are emerging to estimate dynamic of poly-A tails lengths, but they are not yet routine and individual validation experiments are useful. In this chapter we describe a protocol for visualizing poly-A tail lengths following transcription inhibition for a reporter mRNA using denaturing poly-acrylamide gel electrophoresis and northern blot assay. This protocol is quick to set up, requires the purchase of only a few specific reagents, does not rely on radioactivity for RNA monitoring, and can be easily implemented in any molecular biology laboratory.

Immuno-PET/CT Imaging of Trop2 with [18F]AlF-RESCA-T4 Differentiates Lung Cancer from Inflammation.

Immuno-PET/CT imaging, a branch of molecular imaging, can noninvasively and specifically visualize biomarker expression across the body. Trophoblast cell surface antigen 2 (Trop2) is a pan-cancer biomarker and plays a crucial role in tumorigenesis through multiple signaling pathways. The study aims to develop and translate novel Trop2 single-domain antibody (sdAb) tracers for clinical use. Methods: Two sdAbs (i.e., His-tagged T4 and His-tag-free RT4) are recombinantly expressed in Chinese hamster ovary cells. The purities and binding kinetics are determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, high-performance liquid chromatography, and surface plasmon resonance assays. The AlF restrained complexing agent (RESCA) method is applied to develop 18F-labeled sdAb tracers ([18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4), followed by thorough preclinical imaging and blocking studies on tumor-bearing mice and a pilot clinical trial evaluating the clinical imaging safety and feasibility of [18F]AlF-RESCA-T4 immuno-PET/CT. Results: [18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4 possess high radiochemical purities. Preclinical imaging in the T3M-4 tumor model revealed prominent uptake (percentage injected dose/g) of [18F]AlF-RESCA-T4 (11.13 ± 1.53, n = 4) and [18F]AlF-RESCA-RT4 (8.83 ± 1.22, n = 4), which were significantly reduced by coinjection of unlabeled T4 and RT4 in blocking studies. The His-tag removal strategy further optimized the probe's invivo pharmacokinetics and reduced renal radioactivity accumulation without significantly decreasing tumor uptake. In a pilot clinical trial, [18F]AlF-RESCA-T4 immuno-PET/CT showed promising potency in annotating Trop2 expression and differentiating tumors from inflammatory diseases such as tuberculosis. Conclusion: [18F]AlF-RESCA-T4 and [18F]AlF-RESCA-RT4 can specifically annotate Trop2 expression. Clinical [18F]AlF-RESCA-T4 immuno-PET/CT imaging can screen patients for Trop2-targeted therapies and differentiate lung inflammation from cancer.

Key Proteins in Rat Cerebral Cortex: Application of Cornu aspersum Extract as a Neuroprotective Agent in Alzheimer's Type Dementia.

Alzheimer's disease (AD) is the most widespread neurodegenerative disorder. Recently, it was found that mucus extract from Cornu aspersum has beneficial effects on memory and cognitive processes in a rat scopolamine model of AD. The present study elucidated the mechanisms of action of standardized mucus snail extract (SE) enriched with a fraction above 20 kDa on Alzheimer-type dementia in rats. Using proteomic analysis on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) on rat cortex extracts, we compared protein expression in both groups: the first group was treated intraperitoneally with scopolamine (Sco, 2 mg/kg, 11 days) and the second (Sco + SE) group was treated intraperitoneally with Sco (Sco, 2 mg/kg) and protected by SE (0.5 mL/100 g bw) applied daily orally for 11 days. Brain cortex was separated and the expressions of various proteins related to memory and cognitive functions were identified. We found that the expression of Ubiquitin carboxyl-terminal hydrolase isozyme L1, Calbindin, Vacuolar ATP synthase catalytic subunit A, Tropomyosin beta chain, 14-3-3 zeta/delta, Kinesin-1 heavy chain, and Stathmin-4 significantly differs in SE-protected rats as compared to dement animals treated only by Sco, and these brain proteins might be potential therapeutic targets for Alzheimer's-type dementia treatment.