UNASSIGNED: To evaluate a PCR based method of polyacrylamide gel electrophoresis of short tandem repeats and its quantification for detecting donor chimerism after haematopoietic stem cell transplantation in acute leukaemias.
UNASSIGNED: The descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Feb 2018 - Nov 2020. A total of twenty patients with acute leukaemias having undergone HSCT were selected and assessed for the analysis of chimerism status. DNA extraction from the whole blood was done by chelex method and short tandem repeats were amplified by using conventional STR- PCR assay. Electrophoresis was carried out and 6% polyacrylamide gels were used for the resultant amplified DNA products and then followed by their densitometry. These patients had undergone HSCT from Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre.
UNASSIGNED: The peaks in the PAGE densitometry represented the donor chimerism in all post transplant samples of the patients.
UNASSIGNED: Our study showed that densitometry of STR PCR PAGE is a useful and cheaper method for demonstration of donor chimerism in acute leukaemia patients having undergone HSCT. Hence this method can be a valuable option in the monitoring of chimerism status in these patients and therefore helps in preventing graft failure by fast and early treatment strategies for these patients.

Amyloidosis is an infiltrative disease where amyloid fibrils get deposited in the organs like kidney, liver and spleen. Amyloid deposition in the kidneys classically meant deposition in the glomeruli and mesangium until 2008 when interstitial amyloid deposits were isolated and named as` Leukocyte cell-derived chemotaxin 2-associated amyloidosis. It is a progressive disease which clinically manifests as slowly progressive renal dysfunction and/or proteinuria. Our case 34 year old renal transplant recipient underwent graft biopsy post transplantation which revealed interstitial LECT-2 amyloid deposits. Unfortunately, he developed page kidney post biopsy which was managed conservatively with percutaneous drainage.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s12291-022-01072-6.

Teichoic acids (TA) are anionic polymers comprised of polyol phosphate repeat units that are abundant in the cell wall of Gram-positive bacteria. Both wall teichoic acid (WTA) and lipoteichoic acid (LTA) play important roles in regulating cell wall remodeling as well as conferring antibiotic resistance. To analyze TA, we describe a polyacrylamide gel electrophoresis (PAGE) method for both WTA and LTA. To extract crude WTA, the peptidoglycan sacculus is first isolated and WTA is then liberated by hydrolysis. LTA is extracted by 1-butanol and pre-treated with lipase to prevent aggregation and improve single-band resolution by PAGE. Crude extracts of both TAs are then subjected to PAGE followed by Alcian blue and silver staining. These protocols are easily adoptable by laboratories interested in rapidly analyzing TAs and can be used determine the relative abundance, relative polymer length and whether TAs are glycosylated. More detailed TA structural and compositional information can be obtained using the described purification protocols by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) analysis.

Accumulated lines of evidence have revealed that a large number of circular RNAs are produced in transcriptomes from fruit fly to mouse and human. Unlike linear RNAs shaped with 5\' cap and 3\' tail, circular RNAs are characterized by covalently closed loop structures without open terminals, thus required specific treatments for their identification and validation. Here, we describe a detailed pipeline for the characterization of circular RNAs. It has been successfully applied to the study of circular intronic RNAs (ciRNAs) derived from intron lariats and circular RNAs (circRNAs) produced from back spliced exons in human.

Bacteria in nature and as pathogens commonly face oxidative stress which causes damage to proteins, lipids and DNA. This damage is produced by the action of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen, superoxide anion and hydroxyl radical. ROS are generated by antimicrobials, environmental factors (e.g., ultraviolet radiation, osmotic stress), aerobic respiration, and host phagocytes during infective processes. Pseudomonas aeruginosa, a versatile bacterium, is a prevalent opportunistic human pathogen which possesses several defense strategies against ROS. Among them, two catalases (KatA and KatB) have been well characterized by their role on the defense against multiple types of stress. In this protocol, KatA and KatB activities are detected by polyacrylamide gel electrophoresis (PAGE). It is also suggested that the detection of KatB is elusive.

A white rot fungus Cerrena unicolor has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of C. unicolor culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase\'s transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from C. unicolor, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by C. unicolor are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, β-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5\' or 3\' end or at both ends. The TT-tail at the 5\' end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications, such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion-loaded nickel-nitrilotriacetic acid-based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion-loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion-loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.

The effect of peroxide-induced oxidation of fibrinogen on modification of its primary structure and functional properties was investigated. The oxidation sites were shown to be Met, Trp, and His residues. Using the DLS method, it was found that the oxidative modification of fibrinogen results in the change of microrheological characteristics of fibrin network. The fibrinogen oxidation diminishes its tolerance to plasmin hydrolysis and deteriorates the factor XIIIa ability to stabilize the fibrin gel.

Free-air O3 enrichment was used to investigate the responses of different antioxidant mechanisms in different rice (Oryza sativa L.) cultivars - O3-sensitive hybrid indica (O3-S) cultivars and O3-tolerant conventional japonica (O3-T) cultivars across all growth stages. Elevated [O3] induced increases in reactive oxygen species (ROS) production in O3-S cultivars, which were more pronounced in the later growing stages. In O3-S cultivars, continuous O3 stress decreased catalase (CAT), peroxidase (POD) and glutathione peroxidase (GPX) activities, while in O3-T cultivars, short-term O3 stress decreased superoxide dismutase (SOD), CAT, POD and GPX activities. The same POD isozyme patterns were observed in both O3-S and O3-T cultivars, while SOD and APX isozymes varied by cultivar. The results suggest that O3 tolerance might be improved at different rice development stages through regulating the responses of antioxidant mechanisms to O3 stress.