In vitro assessment of genotoxic and cytotoxic effects of Artemisia annua L. tincture

The genus Artemisia (fam. Asteraceae) is one of the largest and widely distributed with around 500 species, majority used as aromatic and medicinal plants. Artemisia annua L. is widely used as a dietary spice, herbal tea, as a supplement, and in a non-pharmaceutical form for treatment of malaria and fever. It is orally consumed as capsules, extracts and tinctures and topically applied as an essential oil diluted in lotions and ointments. Artemisinin is the main constituent of Artemisia annua L. extracts. Since the discovery that the artemisinin is efficient in malaria treatment, there is also a growth in consumption of A. annua extracts for antitumour and even recently for antiviral treatments against SARS-CoV-2 infections. This study aimed to investigate genotoxic effect in peripheral blood culture and cytotoxic effects in cancer and normal cell lines, of commercially available A. annua L. tincture in series of dilutions. Both comet and neutral red uptake assays revealed dose-dependent genotoxicity and cytotoxicity of A. annua tincture dilutions. Comet assay revealed significantly increased DNA damage in peripheral blood cells while neutral-red assays showed increase in cytotoxicity (p<0.001) in both normal and cancer cell cultures treated with the lowest extract dilution compared to the highest one applied. Obtained results indicate caution needed in A. annua L. tincture use, especially when poorly diluted.

Assessment of Ion S5 NGS protocol for SARS-CoV-2 genome sequencing

Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.

Effects of Silymarin on Blood Glucose Concentration, Hepatic Histopathological Changes and FOXA2 and FOXA3 Gene Expression in Streptozotocin-Induced Diabetic Male Wistar Rats

Since the liver is among the primary organs susceptible to the effects of hyperglycaemia, diabetes mellitus (DM) could be a risk factor for the development and progression of liver damage. In present study, since no side-effects from the herbal medicine have been reported, the effect of silymarin on blood glucose concentration, hepatic histopathological changes and FOXA2 and FOXA3 gene expression, which are key genes in liver regeneration, was investigated. In this fundamental with experimental approach study, 40 male Wistar rats weighing 180-220 g were used. Rats were kept under the standard conditions of temperature of 20-22°C and humidity of 50% and consecutive 12-hour periods of light and darkness. Rats were randomly divided into five different groups (n=8 each), including healthy control rats, diabetic control rats, diabetic rats receiving silymarin (50, 100 and 150 mg/kg). Diabetes was induced by injecting streptozotocin (50 mg/kg B.W., i.p.). For 4 weeks silymarin groups received the drug once every three days through gavage and fasting blood glucose concentration measured once every 10 days. At the end of a month experiment, livers were harvested for hepatic histopathological and FOXA2 and FOXA3 gene expression changes analysis. In the diabetic rats treated with silymarin (50, 100 and 150 mg/kg), by comparison with the diabetic control group (p<0.05), glucose levels decreased significantly. Moreover, FOXA2 and FOXA3 expression in diabetic groups treated with silymarin significantly increased compared to diabetic control group (p<0.05). Hepatic histopathological changes were improved in the treated groups.The present study indicates that silymarin significantly decreased blood glucose concentration and increased the FOXA2 and FOXA3 gene products level. Hence, silymarin is able to improve some of the symptoms associated with diabetes and possesses hepatoprotective effects in streptozotocin-induced diabetic rats.

Production of Human Pancreatitis-Associated Protein (hPAP) in Komagataella phaffii (Pichia pastoris)

Pancreatitis-associated protein (PAP) is a pancreatic stress protein that is not produced in a healthy pancreas but is highly synthesized in pancreatic acinar cells in response to acute and chronic pancreatitis, hypoxia, toxins, diabetes, lipopolysaccharides hypotransferrinemia and organ transplantation. Changes in the PAP levels in serum are an important biological marker in the early stage of pancreatic diseases. In this study, the recombinant human PAP protein, which has the potential to be used as a diagnostic marker and as research material in proliferation, apoptosis, cell migration, cell invasion, and immunoassay studies, was expressed efficiently under the control of the AOX1 gene promoter in the Komagataella phaffii (Pichia pastoris) (K. phaffii) X33 strain. We describe the conditions required for the efficient production of PAP protein by methanol induction and its use without purification. The produced unpurified protein was tested in sandwich ELISA and showed consistent results with the commercial product. These results are encouraging that the protein produced can be used as a biomarker standard in ELISA tests without the cost and labor of purification.

Comparative analysis of two L-carnitine preparations and their concentration effects on CAT expression in healthy human peripheral blood lymphocyte cultures

CAT gene encodes catalase, a key antioxidant enzyme in the body against oxidative stress. This enzyme plays an important role in the molecular mechanisms of inflammation, apoptosis, mutagenesis and tumorigenesis. Anti-oxidant L-carnitine is used in food supplementation, medical co-treatment and bodyweight regulation. We aimed to investigate molecular basis of L-carnitine commercial preparations supplementation in reducing oxidative stress with customized CAT gene assay in vitro. Human lymphocytes cell culture was established using standard procedure and treated with range of concentrations of L-carnitine in two preparations. We tested two preparations: 500 mg tablets of L-carnitine and liquid L-carnitine with vitamin B6. L-carnitine significantly reduced the expression of CAT gene in cultured lymphocytes at concentrations of 50 μmol/l and 250 μmol/l compared to negative control, (p = 0,001; p = 0,001; respectively). The L-carnitine liquid supplement with vitamin B6 also reduced the transcription of CAT gene at concentrations of 50 μmol/l and 250 μmol/l as compared to the negative control (p = 0,018; p = 0,006; respectively). Selected L-carnitine preparations modulated the transcriptional activity of the antioxidant enzyme gene in human lymphocyte culture, indicating its possible effects in inhibition of pro-inflammatory processes that involve catalase activity.

Third International Green Biotechnology Congress

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The 2nd Congress of Geneticists in Bosnia and Herzegovina with International Participation

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New insertions-induced Hb H disease

In spite of high incidence of α-thalassemia as the most common inherited disorder of hemoglobin (Hb) production in Southeast Asia, the role of point mutations in this aspect not still well known. This fact can lead to missing rare variants of α-thalassemia mutations by the routine screening, which they may need to be screened for possibility of causing Hb H disease. In this study we found two insertions in alpha1 gene which cause to Hb H disease. One of the insertions, 108/109, is a new findings and another one, codon 44, is the mutation which has been followed for the first time. These new molecular findings about changes in α-globin production which results in decreased of hemoglobin (Hb) value, have high-impact clinical importance.

Expression of the apelinergic system and its influence on functional properties of tumor cells

Small peptide Apelin and its cognate receptor APJ, are known to play a role in tumor angiogenesis and overall cancer progession. Certain authors suggest that the Apelin receptor is also a factor in cancer immunotherapy. In this article, our goal was to study the effects of in vitro targeting of the Apelin/APJ system on the tumor cells functional properties. Protein surface and mRNA expression of Apelin and APJ had been largely examined in various tumor-derived cell lines. In contrast to the tumor tissue, the results of this study demonstrated that most tumor cell lines exhibited somewhat moderate expression of Apelin/APJ. Similar effects of APJ stimulation and inhibition had been observed in in vitro functional assays, which was due to their unusually low expression levels. Low APJ expression in cell lines has been overcome by stable APJ overexpression. In such conditions, stimulation of apelinergic system APJ-overexpressed cells affected cell functional properties in comparison to the wildtype cell lines, where overexpression of APJ receptor resulted in increased migration. On the other hand, no effect on cell proliferation was observed. Consequently, Apelin/APJ signaling in tumor-derived cell lines is not expected to play a direct and crucial role in in vitro cancer survival. Further investigation should focus on in vivo role of the apelinergic system, as demonstrated in the recently published studies, where apelinergic system is claimed to be a promising target for anti-cancer therapy.