Supplementary material for the article: Rašković, B.; Vatić, S.; Andelković, B.; Blagojević, V.; Polović, N. Optimizing Storage Conditions to Prevent Cold Denaturation of Trypsin for Sequencing and to Prolong Its Shelf Life. Biochemical Engineering Journal 2016, 105, 168–176. https://doi.org/10.1016/j.bej.2015.09.018

Supplementary material for: [https://doi.org/10.1016/j.bej.2015.09.018]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2024

Program selektivnog uzgoja šarana (Cyprinus carpio L.) u Srbiji - preliminarni rezultati

The aim of this study was to estimate heritability and genetic correlations between weight, length and height of common carp in Serbia (Cyprinus carpio L.) during 3-year growth period. The 50 families of common carp were produced in 2007 and used for the estimation of genetic parameters. The fish were measured at tagging for weight, length and height (W0, L0, H0), then during the first autumn (W1, L1, H1) and during the second autumn (W2, L2, H2). Based on univariate models heritability estimates were high for all traits (0.39, 0.34 and 0.45 for W1, L1 and H1, respectively) and also for the second production year (0.49, 0.47 and 0.44 for W2, L2 and H2, respectively). The genetic correlations were estimated using multivariate models and they were high between W1 and L1 and H1 (0.81 ± 0.06 and 0.91 ± 0.03 for L1 and H1, respectively), while between H1 and L1 were moderately correlated (0.54 ± 0.12). In the second production year genetic correlations were also high, between W2 and L2 and H2 (0.64 ± 0.09 and 0.74 ± 0.06, respectively), while between length and height they were lower (0.24 ± 0.15). Based on the current results improving growth rate of common carp through genetic selection is expected to be effective.Cilj ove studije je bio da se procene heritabilnost i genetičke korelacije telesne mase, dužine i visine šarana u Srbiji u toku trogodišnjeg ciklusa. U toku 2007. godine proizvedeno je 50 familija šarana i ove jedinke iskorišćene su za procenu heritabilnosti i genetičke korelacije. Merenja riba su obavljena za osobine telesna masa, dužina i visina u momentu označavanja (W0, L0, H0), zatim pri starosti riba prva jesen (W1, L1, H1) i pri starosti druga jesen (W2, L2, H2). Na osnovu jednovarijantnih statističkih modela za svaku osobinu procene heritabilnosti su bile visoke (0.39, 0.34 i 0.45 za W2, L2 i H2). Genetičke korelacije su procenjene na osnovu multivarijacionih statističkih modela i bile su visoke između W1 i, L1 i H1 (0.81 ± 0.06 i 0.91 ± 0.03 za L1 i H1), dok su između H1 i L1 bile umereno korelisane (0.54 ± 0.12). U drugoj produkcionoj godini korelacije su takođe bile visoke, između W2 i, L2 i H2 (0.64 ± 0.09 i 0.74 ± 0.06), dok su između osobina dužina i visina, L2 i H2, bile niže (0.24 ± 0.15). Na osnovu navedenih rezultata očekuje se da bi poboljšanje prirasta u navedenoj populaciji šarana u Srbiji, putem selekcije familija, bilo uspešno

A novel spi1 mutation in a patient with agammaglobulinemia

Agammaglobulinemia is a primary immunodeficiency characterized by a low number or absence of mature B lymphocytes and consequently by immunoglobulin deficiency. In 2021, six patients with pathogenic variants in SPI1 gene associated with agammaglobulinemia type 10 (PU.MA) were described for the first time. This gene encodes the pioneer transcription factor PU.1, which plays an important role in the differentiation of B lymphocytes, monocytes, and conventional dendritic cells. Here we present a female patient with a novel mutation in SPI1 gene which has not been previously found in patients with PU.MA. Case description: A 37-year-old female patient with frequent middle ear infections in early childhood was diagnosed with agammaglobulinemia at the age of 15 when she started immunoglobulin replacement therapy (IgRT). One year later, an allogeneic hematopoietic stem cell transplant from a healthy sibling donor was performed. Unfortunately, chimerism analysis found no DNA material from the donor in the patient's blood, suggesting graft rejection, so she remained dependent on antibody replacement therapy. Years later, she was diagnosed with protein-losing enteropathy, and despite escalating doses of IgRT, IgG levels remained low. Subsequently, the patient developed persistent COVID -19 viremia and bacterial meningoencephalitis. Clinical exome sequencing using the TruSight (Illumina) panel was performed and in comparision with the human reference genome (hg19), has revealed a heterozygous mutation in exon 4 of the SPI1 gene. This mutation is characterized by the insertion of 2 nucleotides (c.441dup), a reading frame shift, and the insertion of a premature stop codon. According to the American College of Medical Genetics and Genomics, this mutation is described as a likely pathogenic-class 2 (PVS1_Very Strong). Conclusion: From analysis of previous literature, we concluded that the mutant sequence in exon 4 encodes the PEST region of the pioneer transcription factor PU.1, which is responsible for interaction with other transcription factors. Immunophenotyping of peripheral blood cells did not reveal CD19+ B cells, suggesting that a differentiation arrest may have developed between the prepro-B and pro-B stages, where there is a high requirement for PU.1 activity. Nextgeneration sequencing can be a very useful tool to uncover the causes of rare primary immunodeficiencies, but further analysis is needed to explain the relationship between patient genotype and clinical presentation

Serum activity of DPPIV and its expression on lymphocytes in patients with melanoma and in people with vitiligo

Background: Dipeptidyl peptidase IV, a multifunctional serine protease, is implicated in regulation of malignant transformation, promotion and further progression of cancer, exerting tumor-suppressing or even completely opposite - tumor-promoting activities. The aim of present research was to determine the serum DPPIV activity, as well as the percentages of CD26+ lymphocytes, CD26+ overall white blood cells and the mean fluorescence intensity of CD26 expression on lymphocytes in patients with melanoma, people with vitiligo and in healthy controls. Methods: The activity of DPPIV in serum was determined by colorimetric test. Expression of DPPIV (as CD26) on immunocompetent peripheral white blood cells was done using flow cytometry analysis. Results: Data from our study show for the first time statistically significant decrease: in the serum DPPIV activity, in the percentage of CD26+ overall white blood cells and in the percentage of lymphocytes in patients with melanoma in comparison to healthy control people. In addition, significantly lower serum DPPIV activity was found in the group of patients with melanoma in relation to people with vitiligo too. Conclusion: This study indicates the need for exploring the cause and the importance of the disturbances in the serum DPPIV activity and in the CD26 expression on immunocompetent cells in complex molecular mechanisms underlying the development and progression of melanomaThe authors are grateful to the Ministry of Education and Science of the Republic of Serbia for the financial support (Project 175011)S

Dynamic stability of a nonlinear multiple-nanobeam system

We use the incremental harmonic balance (IHB) method to analyse the dynamic stability problem of a nonlinear multiple-nanobeam system (MNBS) within the framework of Eringen’s nonlocal elasticity theory. The nonlinear dynamic system under consideration includes MNBS embedded in a viscoelastic medium as clamped chain system, where every nanobeam in the system is subjected to time-dependent axial loads. By assuming the von Karman type of geometric nonlinearity, a system of m nonlinear partial differential equations of motion is derived based on the Euler–Bernoulli beam theory and D’ Alembert’s principle. All nanobeams in MNBS are considered with simply supported boundary conditions. Semi-analytical solutions for time response functions of the nonlinear MNBS are obtained by using the single-mode Galerkin discretization and IHB method, which are then validated by using the numerical integration method. Moreover, Floquet theory is employed to determine the stability of obtained periodic solutions for different configurations of the nonlinear MNBS. Using the IHB method, we obtain an incremental relationship with the frequency and amplitude of time-varying axial load, which defines stability boundaries. Numerical examples show the effects of different physical and material parameters such as the nonlocal parameter, stiffness of viscoelastic medium and number of nanobeams on Floquet multipliers, instability regions and nonlinear amplitude–frequency response curves of MNBS. The presented results can be useful as a first step in the study and design of complex micro/nanoelectromechanical systems

Improving eco-sustainable characteristics and energy efficiency of evaporative fluid cooler via experimental and numerical study

This paper presents an on-going research project that aims to identify possibilities for wider use of evaporative cooling in process industry, especially the use of evaporative fluid cooler units. Experimental study is performed on small scale evaporative fluid cooler, while the correlation based model has been carried out to explore the detailed heat and mass transfer processes inside this unit. Numerical integration of mathematical model is executed by new approach, based on differential, collocation Simpson method. Proposed models have been verified by comparing the computed results with those obtained by the experimental measurements. The results of research will enable the creation of more comprehensive simulation software, with wider range of operating and construction parameters

Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life

Trypsin is a serine protease with widespread applications, including protein sequencing and typsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze-thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in beta-sheet content (band maximum detected at a frequency of 1634 cm in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of ayoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery ( gt 90%) and modest autolysis ( lt 10%). High activity recovery ( gt 90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life. (C) 2015 Elsevier B.V. All rights reserved.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3586

Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage

Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45 degrees C to 60 degrees C with Delta G degrees(321) of 13.9 +/- 0.3 kJ/mol and T-m value of 84 +/- 1 degrees C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular beta-sheet content (band maximum detected at frequency of 1621 cm(-1) in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze-thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry.Peer-reviewed manuscript: [http://cherry.chem.bg.ac.rs/handle/123456789/3367