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Estimation of salivary proteins in early childhood caries before and after treatment using gel electrophoresis

Posted on February 10, 2022 By Clayton No Comments on Estimation of salivary proteins in early childhood caries before and after treatment using gel electrophoresis
Background: Saliva being an important biological fluid of our body contains both specific and nonspecific protective factors which form an integral part of our immune system. Salivary proteins play a substantial role in protecting humans against infection. Their level in oral cavity is subject to constant variations which is dependent on various factors.
Purpose: The purpose of the study was to compare the levels of salivary proline-rich proteins (PRPs), glycoproteins, amylase bands, and salivary pH in children with early childhood caries before and after treatment using gel electrophoresis.
Materials and methods: The whole salivary pH, mean protein concentrations, and electrophoretic profiles of the salivary proteins were measured using sodium dodecylsulfate-polyacrylamide gel electrophoresis in both pre- and posttreatment groups. Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 15.0 software. Chi-square test and independent t-test were used to further compare the results.
Results: The results were statistically significant in all the groups. There was variation in pre- and posttreatment values seen.
Conclusion: Salivary proteins (glycoproteins, PRPs, and amylase) establish an imperative defense mechanism as antimicrobial agents.
Keywords: Early childhood caries; gel electrophoresis; salivary proteins.

Aerially Applied Zinc Oxide Nanoparticle Affects Reproductive Components and Seed Quality in Fully Grown Bean Plants ( Phaseolus vulgaris L.)

The development of reproductive components in plant species is susceptible to environmental stresses. The extensive application of zinc oxide nanoparticles (nZnO) in various agro-industrial processes has jeopardized the performance and functionality of plants. To understand Gentaur Gel Electrophoresis Systems the response of the developmental (gametogenesis and sporogenesis) processes to nanoparticles (NPs) exposure, the aerial application of nZnO and their ionic counterpart of ZnSO4 at four different levels were examined on bean plants (Phaseolus vulgaris) before the flowering stage.

To evaluate the mentioned processes, briefly, flowers in multiple sizes were fixed in paraffin, followed by sectioning and optical analysis. The possibility of alteration in reproductive cells was thoroughly analyzed using both light and electron microscopes.

Overall, our results revealed the histological defects in male and female reproductive systems of mature plants depend on NPs levels. Furthermore, NPs caused tapetum abnormalities, aberrations in carbohydrate accumulation, and apoptosis. The nZnO induced abnormal alterations right after meiosis and partly hindered the microspore development, leading to infertile pollens. The seed yield and dry weight were reduced to 70 and 82% at 2,000 mg L-1 nZnO foliar exposure, respectively. The sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis pattern showed the increased expression of two proteins at the molecular weight of 28 and 42 kDa at various concentrations of nZnO and ZnSO4. Overall, our results provided novel insights into the negative effect of nano-scaled Zn on the differential mechanism involved in the reproductive stage of the plants compared with salt form.

A Guide to Analysis of Relative Synaptic Protein Abundance by Quantitative Fluorescent Western Blotting

The introduction of fluorescent detection systems has revolutionized the applicability of Western blotting for quantitative protein expression analyses. The fundamental premise behind fluorescent Western blotting is the combination of distinct fluorescent dye-conjugated secondary antibodies and high  performance digital imaging solutions in which the fluorescence signal is directly proportional to the amount of protein enabling quantitative measurements and simultaneous detection of several target proteins.

This aspect of Western blotting is now widely used, especially in preclinical research, to detect quantitative changes in protein levels and phosphorylation status between experimental groups. This chapter provides a detailed step-by-step guide for best practice procedures during the entire process from sample preparation, SDS polyacrylamide gel electrophoresis to electrotransfer of proteins and highlights approaches that can be applied to increase data output.

Overexpression of the dystrophins Dp40 and Dp40 L170P modifies neurite outgrowth and the protein expression profile of PC12 cells

Dp40 is ubiquitously expressed including the central nervous system. In addition to being present in the nucleus, membrane, and cytoplasm, Dp40 is detected in neurites and postsynaptic spines in hippocampal neurons. Although Dp40 is expressed from the same promoter as Dp71, its role in the cognitive impairment present in Duchenne muscular dystrophy patients is still unknown.

Here, we studied the effects of overexpression of Dp40 and Dp40L170P during the neuronal differentiation of PC12 Tet-On cells. We found that Dp40 overexpression increased the percentage of PC12 cells with neurites and neurite length, while Dp40L170P overexpression decreased them compared to Dp40 overexpression.

Two-dimensional gel electrophoresis analysis showed that the protein expression profile was modified in nerve growth factor-differentiated PC12-Dp40L170P cells compared to that of the control cells (PC12 Tet-On). The proteins α-internexin and S100a6, involved in cytoskeletal structure, were upregulated.

The expression of vesicle-associated membrane proteins increased in differentiated PC12-Dp40 cells, in contrast to PC12-Dp40L170P cells, while neurofilament light-chain was decreased in both differentiated cells. These results suggest that Dp40 has an important role in the neuronal differentiation of PC12 cells through the regulation of proteins involved in neurofilaments and exocytosis of synaptic vesicles, functions that might be affected in PC12-Dp40L170P.

Cytotoxic activity of Staphylococcus aureus isolates from a cohort of Mexican children with cystic fibrosis

Background: Cystic fibrosis (CF) is a genetic disease in which thick, sticky mucus is produced in the lungs (and other organs) that impairs ciliary clearance, leading to respiratory problems, increased chronic bacterial infections, and decreased lung function. Staphylococcus aureus is one of the primary bacterial pathogens colonizing the lungs of CF patients. This study aimed to characterize the genetic relatedness of S. aureus, its presence in children with CF, and its cytotoxic activity in THP1 cell-derived macrophages (THP1m).
Methods: Genetic relatedness of S. aureus isolates from a cohort of 50 children with CF was determined by pulsed-field gel electrophoresis (PFGE). The VITEK 2 automated system was used to determine antimicrobial susceptibility, and methicillin-resistance S. aureus (MRSA) was determined by diffusion testing using cefoxitin disk. The presence of mecA and lukPV genes was determined by the polymerase chain reaction and cytotoxic activity of S.aureus on THP1m by CytoTox 96 assay.
Results: From 51 S. aureus isolates from 50 children with CF, we identified 34pulsotypes by PFGE. Of the 50 children, 12 (24%) were colonized by more than one pulsotype, and 5/34 identified pulsotypes(14.7%) were shared between unrelated children. In addition, 3/34 pulsotypes (8.8%) were multidrug-resistant (MDR), and2/34 (5.9%) were MRSA. Notably, 30/34 pulsotypes (88.2%) exhibited cytotoxicity on THP1m cells and 14/34 (41.2%) alteredTHP1m monolayers. No isolate carried the lukPV gene.
Conclusions: Although a low frequency of MRSA and MDR wasfound among clinical isolates, most of the S. aureus pulsotypes identified were cytotoxic on THP1m.
Introducción: La fibrosis quística (FQ) es una enfermedad genética en la que se produce moco espeso y pegajoso en los pulmones (y otros órganos), lo que conduce a problemas respiratorios, incremento de las infecciones bacterianas crónicas y disminución de la función pulmonar. Staphylococcus aureus es uno de los principales patógenos que colonizan los pul-mones de los pacientes con FQ. El objetivo de este trabajo fue caracterizar la relación genética de S. aureus, su presencia en niños con FQ y su actividad citotóxica en macrófagos derivados de células THP1 (THP1m).
Métodos: La relación gené-tica de los aislados de S. aureus provenientes de una cohorte de 50 pacientes con FQ fue determinada por electroforesis en gel de campo pulsado (PFGE). La sensibilidad a los antimicrobianos se determinó mediante el sistema automatizado VITEK 2, y la resistencia a la meticilina (SARM) mediante la prueba de difusión utilizando discos de cefoxitina. La presen-cia de los genes mecA y lukPV se determinó mediante reacción en cadena de la polimerasa, y la actividad citotóxica de S. aureus sobre células THP1m mediante el ensayo CytoTox96®.
Resultados: A partir de 51 aislados de S. aureus provenientes de 50 niños con FQ se identificaron 34 pulsotipos por PFGE. De los 50 niños, 12 (24%) estaban colonizados por más de un pulsotipo y 5 de los 34 pulsotipos (14.7%) los compartían niños que no estaban relacionados. De los 34 pulsotipos, 3 (8.8%) presentaron multirresistencia (MDR) y 2 (5.9%) fueron SARM. Además, 30 pulsotipos (88.2%) fueron citotóxicos sobre células THP1m y 14 (41.2%) alteraron su monocapa. Ninguno de los pulsotipos presentó el gen lukPV.
Conclusiones: Aunque se encontró una baja frecuencia de SARM y MDR en los aislados, la mayoría de los pulsotipos de S. aureus identificados fueron citotóxicos para células THP1m.

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