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TLR4 gene expression in patients with chronic suppurative otitis media
J. T. Venkataravanappa1, K. C. Prasad2, S. Balakrishna1*
1Department of Cell Biology and Molecular Genetics, Sri Devraj Urs Academy of Higher Education and Research, Karnataka, India;
2Department of Otorhinolaryngology, Sri Devaraj Urs Medical College, Sri Devraj Urs Academy of Higher Education and Research, Kolar-563103, Karnataka, India;
*e-mail: sharath@sduu.ac.in
Received: 23 July 2021; Accepted: 12 November 2021
Chronic suppurative otitis media (CSOM) is an infectious disease of the middle ear that involves inflammation and accumulation of fluid behind the eardrum. The pathogenesis of CSOM involves reduced bacterial clearance due to impairment of Toll-Like Receptor (TLR) 4 pathway. TLR4 receptor serves as the molecular sensor for bacterial endotoxin (lipopolysaccharide) and activates inflammatory cell signaling for clearing the bacteria. Previous studies have shown that the expression of TLR4 gene is reduced in middle ear epithelia of CSOM patients. Whether the expression of TLR4 gene is reduced in leukocytes is not known. In our present study we aim to compare the expression of the TLR4 gene in the blood samples of CSOM patients and healthy controls. A case-control study was carried out by involving 16 participants in each group. The levels of the TLR4 gene expression were measured by using the qRT-PCR method. The median (interquartile range) ΔCt values of the TLR4 gene expression was 4.85 (2.61- 8.55) in the patient group and 2.29 (-1.63-4.85) in healthy controls. Expression of the TLR4 gene in the leukocytes of CSOM patient group was reduced by ~5.9 fold compared to the control group and the difference was found to be significant (P = 0.01).
Properties of alpha-L-fucosidase for serum of patients with hepatocellular cancer and cytotoxicity on some cancer cell lines
Z. M. A. A. Hamodat
University of Mosul, College of Science, Chemistry Department, Mosul – Iraq;
e-mail: zahraahamodat@uomosul.edu.iq
Received: 07 July 2021; Accepted: 12 November 2021
Alpha-L-fucosidase (FUCA) degrades many fucosylated glycans and has long been recognized as a tumor marker associated with the early detection of some cancers. This study aimed to purify and characterize alpha-L-fucosidase from the serum of patients with hepatocellular cancer and estimate its toxic effect against hepatocellular carcinoma HepG2, prostate cancer PC3 cell lines and the standard hepatocyte WRL-68 cell line. SDS-Page Electrophoresis technique was used to determine the purity of the purified alpha-L fucosidase and estimate its molecular weight. Three purification steps were used for FUCA purification: precipitation with 65% ammonium sulfate saturation, DEAE-cellulose ion exchange, Sephadex G-75 gel filtration. The procedure resulted in 54% recovery of the enzyme with 27.5-fold purification and 14 U/mg specific activity. It was demonstrated that FUCA purified from the serum of HCC patients showed a more toxic effect on HepG2 cells (IC50 of 65.74 µg/ml) than on PC3 prostate cancer cells (IC50 of 111.5 g/ml) and less toxic effect against standard hepatocyte WRL-68cells (IC50 of 214.5 µg/ml). We can conclude that the inhibitory effect of the purified FUCA on hepatocellular carcinoma is more than its effect on prostate cancer cells. Also, the purified FUCA may be used in studies on anticancer drug development in liver cancer.
Growth hormone, growth hormone receptor and insulin-like growth factor serum levels in patients with obesity and food addiction
O. Avsar1*, S. Sancak2, I. Koroglu3, E. Avci4
1Hitit University, Department of Molecular Biology and Genetics, Corum, Turkey;
2Fatih Sultan Mehmet Education and Research Hospital, Department of Endocrinology, Istanbul, Turkey;
3Arapgir Ali Özge State Hospital, Department of Internal Medicine, Malatya, Turkey;
4Health Sciences University, Department of Biochemistry, Ankara, Turkey;
*e-mail: orcunavsar@hitit.edu.tr
Received: 27 May 2021; Accepted: 12 November 2021
Obesity is a public health problem that increasingly becomes widespread and causes various complications. Food addiction is a hedonic eating behavior characterized by overconsumption of palatable foods (i.e., foods involve a high amount of salt, sugar and fat). Disturbances in the growth hormone signaling pathway were shown to be associated with increased food intake and adiposity. The study aimed to determine the growth hormone (GH), growth hormone receptor (GHR), insulin, and insulin-like growth factor 1 (IGF-1) serum levels in individuals with obesity and food addiction. The present study involved 30 adults with obesity (23 females and 7 males) and 10 healthy adults (5 females and 5 males). 18 obese adults were diagnosed with food addiction, whereas only 2 individuals with food addiction were in the control group. GH, GHR, IGF-1 and insulin values were analyzed with ELISA kits. It was revealed that the obese subjects had significantly lower serum IGF-1 levels compared to healthy individuals (144.55±22.69 ng/ml vs 338.70±61.90 ng/ml, P < 0.001)). No significant differences in the GH, GHR and insulin levels between obese and control groups were detected (P > 0.05). No significant differences between the group with food addiction and the group without food addiction in terms of gender, age, weight, BMI, GH, GHR, insulin and IGF-1 levels were observed. Our study demonstrates that normal IGF-1 levels may be protective for the development of obesity. The serum levels of GH, GHR, insulin, IGF-1 are not associated with food addiction and, therefore, can not be used as novel markers of food addiction.
Serum levels of alpha-melanocyte stimulating hormone, vitamin D, calcium, phosphorus and magnesium in COVID-19 patients
S. Z. Hussein1, M. A. Abdalla2*
1Clinical Biochemistry Unit, Salah Aldeen Health Directorate, Tikrit, Iraq;
2Department of Human Anatomy, Tikrit University College of Medicine, Tikrit, Iraq;
*e-mail: dr.mohammad68@tu.edu.iq
Received: 04 April 2021; Accepted: 12 November 2021
The COVID-19 pandemic occurred and quickly spread throughout the world. To improve the state of COVID-19 patients, it is important to identify the possible clinical differential diagnostic markers and their correlation with the severity of SARS-CoV-2 infection. In this study, the serum level of alpha-melanocyte stimulating hormone (alpha-MSH), vitamin D, calcium, phosphorus and magnesium in the serum of COVID-19 patients were analyzed. Blood samples were collected from 60 patients who attended Isolated Hospital in Tikrit City/Iraq from September to December 2020 and diagnosed by RT-PCR as COVID-19 positive and from 30 healthy individuals. It was shown that COVID-19 patients revealed high serum levels of α-MSH as compared with healthy individuals but low serum levels of vitamin D, calcium, and magnesium which may be recommended as supplements for those patients to increase the innate immune response.
Urine neutrophil gelatinase-associated lipocalin as an early biochemical marker of microalbuminuria in predicting early kidney damage in patients with type 2 diabetes mellitus
P. J. Padmini, V. Ashok*
Shri Sathya Sai Medical College and Research Institute, Sri Balaji Vidyapeeth Deemed to be University, Tamilnadu, India;
*e-mail: dr.ashokmbbs1986@gmail.com
Received: 08 July 2021; Accepted: 12 November 2021
Diabetic nephropathy is one of the microvascular complications of diabetes mellitus. The study was done to evaluate the diagnostic value of neutrophil gelatinase-associated lipocalin (NGAL) urine level as a biomarker for the early detection of nephropathy in type 2 diabetic patients. This study was performed on 150 patients with type 2 diabetes mellitus classified into three equal groups according to their urine albumin/creatinine ratio (ACR), including patients with normoalbuminuria (ACR < 30 mg/g creatinine), microalbuminuria (ACR = 30–300 mg/g creatinine) and macroalbuminuria (ACR > 300 mg/g creatinine). Fifty apparently healthy subjects were selected as a control group. Urine NGAL was estimated by a particle-enhanced turbidimetric immunoassay using Hitachi 917 analyzer. The serum level of glucose and creatinine was also estimated. The results showed that NGAL urine level was significantly elevated in diabetes patient groups with microalbuminuria and macroalbuminuria when compared to the control group and diabetes patient group with normoalbuminuria. The levels of urine NGAL correlated positively with microalbuminuria in patients with diabetes. Receiver operating characteristic curves revealed that urine NGAL had a higher diagnostic value for diabetic nephropathy early detection compared to ACR and microalbuminuria in patients with diabetes mellitus.
Extracellular vesicles produced by mouse breast adenocarcinoma 4T1 cells with up- or down-regulation of adaptor protein Ruk/CIN85 differentially modulate the biological properties of 4T1 WT cells
A. Yu. Zhyvolozhnyi1,2*, I. R. Horak1, D. S. Geraschenko1, M. O. Gomozkova3,
O. O. Hudkova1, S. J. Vainio2, A. A. Samoylenko2, L. B. Drobot1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland;
3Brigham Young University-Idaho, Rexburg, USA;
*e-mail: ppndl2@gmail.com
Received: 02 November 2021; Accepted: 12 November 2021
Extracellular vesicles (EVs) are secreted by most cell types under both physiological and pathological conditions and were proposed to be actively involved in intercellular communication. The mode of EVs action is dependent on their cargos composition. EVs play an important role in tumor initiation, recurrence, metastasis and therapeutic resistance. EVs marker proteins Alix and Tsg101 and cortactin are the binding partners of adaptor protein Ruk/CIN85. The present study aims to analyze the regulatory effects of EVs produced by 4T1 cells with overexpression (RukUp) or down-regulation (RukDown) of adaptor protein Ruk/CIN85 on proliferation rate, migration and invasion activity of parental 4T1 WT cells. EVs from conditioned medium of 4T1 RukUp or RukDown cells were isolated by differential centrifugation followed by further purification using Exo-spin™ kit (Cell Guidance Systems). The number and size of EVs were characterized by NTA (Malvern Panalytical NanoSight NM300) instrument. The content of marker proteins and Ruk/CIN85 in isolated EVs was analyzed by Western-blotting. The viability, migration and invasion activity of 4T1 WT cells were studied using MTT-test, scratch-test and Boyden chamber assay, respectively. It was demonstrated for the first time that adaptor protein Ruk/CIN85 is a constitutive component of EVs produced by 4T1 cells. It was also shown that EVs produced by 4T1 cells with different levels of Ruk/CIN85 expression are characterized by a specific profile of the content of its multiple molecular forms. It turned out that the ability of EVs to modulate the proliferative activity, motility and invasiveness of 4T1 WT cells was tightly correlated with the biological properties of 4T1 cells that produce EVs (highly aggressive 4T1 RukUp cells or weakly invasive 4T1 RukDown cells). Our data suggest that adaptor protein Ruk/CIN85 is not only a constitutive component of cargos composition of EVs produced by tumor cells but, depending on its content in EVs, plays an active role in the control of carcinogenesis.
Production of recombinant SARS-COV-2 proteins and diphtheria toxoid CRM197-based fusion
O. I. Krynina1, S. I. Romaniuk1, O. B. Gorbatiuk1,2,
O. H. Korchynskyi1,3,4, А. V. Rebriiev1, Ya. S. Kulyk1,
Ye. O. Kozadaieva1, A. A. Siromolot1,5, M. M. Guzyk1,
D. V. Kolybo1*, S. V. Komisarenko1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2State Institute of Genetic and Regenerative Medicine, National Academy of Medical Sciences of Ukraine, Kyiv;
3Centre for Innovative Research in Medical and Natural Sciences, Faculty of Medicine, University of Rzeszow, Rzeszow, Poland;
4S. Gzhytskyi National University of Veterinary Medicine and Biotechnologies, Lviv, Ukraine;
5ESC “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Ukraine;
*e-mail: kolibo@biochem.kiev.ua
Received: 10 October 2021; Accepted: 12 November 2021
The quickly emerged global COVID-19 pandemic raised a desperate need in the development of protecting vaccines targeting this disease. Therefore, a generation of effective producers of recombinant SARS-CoV-2 proteins became an urgent task. Its resolving contributes to the study of functional SARS-CoV-2 properties, as well as will allow developing the domestic COVID-19 vaccine in Ukraine, thus playing an important strategic role in tackling the pandemics. The aim of the study was to generate prokaryotic and eukaryotic producers of recombinant SARS-CoV-2 proteins and to isolate nucleocapsid (N) protein, receptor-binding domain (RBD) of spike (S) protein, as well as RBD fused to the carrier – diphtheria toxoid CRM197. For this purpose, appropriate genetic constructs, in particular, replication deficient recombinant AdvC5-based adenoviral vectors expressing the SARS-CoV-2 proteins and CRM197-fused conjugate were created through methods of molecular biology and genetic engineering. Restriction analysis and/or DNA sequencing confirmed that we created the correct constructs. Immobilized metal affinity chromatography was used to purify the recombinant proteins. Compliance of their properties was confirmed by the results from polyacrylamide gel electrophoresis, Western blotting, immunoenzymatic assay and MALDI-TOF mass spectrometry. As a result, we generated E. coli Rosetta (DE3) bacterial strain and HEK293 cell line producing recombinant SARS-CoV-2 proteins and CRM197-based fusion. In addition, pure N protein, RBD of S protein and RBD-CRM197 fusion protein were isolated. The obtained recombinant SARS-CoV-2 proteins can be used to study immunogenic and antigenic properties of the SARS-CoV-2 proteins. Cells producing recombinant SARS-CoV-2 proteins and RBD-CRM197 fusion protein are able to provide cheap and safe synthesis of the antigenic substances for domestic development and production of immunodiagnostics for COVID-19 and COVID-19 vaccines in Ukraine.
Biochemical and molecular-physiological aspects of the nitric oxide action in the utera
H. V. Danylovych, Yu. V. Danylovych
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
Received: 18 May 2021; Accepted: 12 November 2021
The sources of the nitric oxide (NO) formation in the uterus and the dynamics of changes in its content in different periods of organ functioning in human and animals are analyzed. The biochemical mechanisms of NO action on the myometrium contractile activity, the significance of NO in the physiological processes during pregnancy and labor, the importance of mitochondria as a reliable NO source in the smooth muscle and the possible ways of NO influence on Ca2+ transport and bioenergetic processes in mitochondria are considered. The authors’ data concerning ionic and membrane mechanisms of NO action on Ca2+-homeostasis of uterine myocytes, identification of nitric oxide in uterine smooth muscle mitochondria, biochemical characteristics of the NO-synthase reaction and the possible role of NO in the regulation of Ca2+ transport in these subcellular structures and in the electron transport chain functioning are presented and discussed.