Identification of plasma biomarkers for the early diagnosis and therapy of bipolar disorder utilizing mass spectrometry based lipidomics approaches

А.А. Vanyushkina 1, Е.А. Yushina 1,2, А.N. Egorova 1,3, N.А. Anikanov 1, A.I. Tkachev 1, P.E. Khaitovich

1,4 1 Center of Life Sciences, Skolkovo Institute of Science and Technology, 3, ul. Nobelya, Moscow 143026

Russia 2. Pirogov Russian National Research Medical University (RNRMU), 1, ul. Ostrovityanova, Moscow,

117997

3. Moscow Institute of Physics and Technology (State University) (MIPT), 9, ul. Institutskiy per., Dolgoprudny, Moscow Region, 141700

4. ShanghaiTech University, Shanghai, China, 200031

Abstract — Neuropsychiatric diseases, such as bipolar disorder, are highly heterogeneous and

complex disorders, resulting from the interplay between environmental factors and multiple risk

factors. Large-scale -omic approaches such as metabolomics and lipidomics, combined with

genomics in diseased tissues and patient plasma allow to search for biomarker fingerprints that

may help disentangling the biological complexity of these brain diseases and identifying patient

subgroups that are homogeneous in terms of prognosis and drug response. The aim of this study is

to identify plasma biomarkers that uniquely characterize such important and highly represented in

the world neuropsychiatric disease as bipolar disorder (both types). Plasma samples from patient

with this disorder, as well as from control individuals, are collected in collaboration with the

Institute of Psychiatric Phenomics and Genomics (IPPG) at the University of Munich, Germany

(Prof. Thomas G Schulze). Plasma samples are collected at multiple time points (6 months apart

from each other, up to a total number of 4 time-points) from approximately 100 patients from

several clinics in Germany (Münster, Göttingen, Munich). From these samples, approx. 350 age-

and sex-matched plasma samples we analyzed using high-resolution hybrid quadrupole-Orbitrap

mass spectrometer (Q Exactive, Thermo) coupled with UPLC system (Acquity I-Class, Waters).

Identification of Ribosomal Protein L11 as a Novel Biomarker of 5-FU Sensitivity for Gastric Cancer

Takuto Kawahata1,2,*, Kohichi Kawahara1, Michiko Shimokawa1, Akie Sakiyama1, Takehiro Shiraishi1,2,

Kentaro Minami1, Masatatsu Yamamoto1, Yoshinari Shinsato1, Kazunari Arima2, Toshiyuki Hamada2,

Tatsuhiko Furukawa1

1Department of Molecular Oncology, Kagoshima University Graduate School of Medical and Dental

Sciences, Kagoshima 890-8544, Japan; 2Department of Science and Engineering, Kagoshima University

Graduate School of Science and Engineering, Kagoshima 890-8580, Japan

�Abstract — The antimetabolite agent 5-fluorouracil (5-FU) is one of the most frequently used first-line treatments for patients with advanced gastric cancer. However, the 5-year survival rate of patients with gastric cancer remains poor. Therefore, there is an urgent need to identify molecular mechanisms and find novel therapeutic strategies for 5-FU resistance in gastric cancer. 5-FU has been reported to suppress tumor growth through ribosomal protein L11 (RPL11)-mediated activation of P53 in osteosarcoma. However, it is unknown whether 5-FU inhibits tumor growth through RPL11-mediated activation of P53 in gastric cancer. In the present study, we investigated whether RPL11 expression was associated with the drug sensitivity of gastric cancer upon 5-FU treatment. Kaplan-Meier survival analysis indicated that high RPL11 expression in 5-FU treated gastric cancer patients have better prognosis than low RPL11 expression in 5-FU treated gastric cancer patients. We then investigated whether RPL11 expression affects the sensitivity of gastric cancer against 5-FU using four human gastric cancer cell lines, MKN45 (wild-type TP53 gene), NUGC4 (wild-type), MKN7 (mutated), and KE39 cells (mutated). In vitro assays demonstrated that RPL11 knockdown in gastric cancer cell lines carrying the TP53 wild-type gene attenuated 5-FU-induced cell growth suppression and activation of the P53 pathway, but not in cells carrying mutated TP53, suggesting that 5-FU suppresses tumor progression via RPL11-mediated activation of the P53 pathway in gastric cancer. Here, we show that RPL11 expression is a crucial factor affecting the sensitivity of gastric cancer against 5-FU, suggesting that RPL11 may be a potential biomarker for predicting 5-FU sensitivity. Identification of RPL11 as a biomarker helps gastric cancer patients with 5-FU resistance avoid unwanted side effects caused by 5-FU treatment. In addition to the role of RPL11 expression as a biomarker, we found RPL11 is functionally required to modulate sensitivity to 5-FU. Therefore, chemotherapy using a drug to elevate RPL11 expression could improve 5-FU resistance in gastric cancer patients with the TP53 wild-type gene. These findings would greatly contribute to a therapeutic strategy for patients with gastric cancer and other solid tumors treated with 5-FU, including breast cancers, lung cancers, and cancers of the aerodigestive tract.

APLICATION OF NEW COMBINATION OF PROTEOMICS METHOD FOR FINDING POTENTIAL PANCREATIC CANCER PLASMA BIOMARKERS. TATIANA SMIRNOVA, ŠTĚPÁNKA KUČKOVÁ University of Chemistry and Technology Prague, Department of Biochemistry and Microbiology, Laboratory of Applied Proteomics Technická 5, 166 28 Praha 6 – Dejvice smirnovt@vscht.cz

Pancreatic cancer is one of the most common lethal tumors of the gastrointestinal tract. The treatment of patients with developed pancreatic cancer remains a major challenge because it is often diagnosed at an advanced stage1. Only 5% of patients will survive five years after diagnosis, but most of them will not survive for more than one year2. Over the years, the incidence of this disease in the Czech Republic is growing and mortality of this disease is almost identical to its incidence. In 2016, the Czech Republic was at the second place of the incidence of this disease in the world3. The prognosis is generally negative, because this cancer is often diagnosed at an advanced stage due to long-discrete symptoms4. Unfortunately, there are no specific biomarkers for pancreatic cancer yet.

The focus of this work is to use plasma samples of four groups of patients (with pancreatic cancer, with long-term type II diabetes, with fresh developed type II diabetes and with control group of healthy patients) to try finding new protein biomarkers with increased sensitivity and specificity that would help to detect pancreatic cancer early on. To achieve this goal, the previously untested combination of proteomics techniques will be used. Particularly MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization – Time of Flight Mass Spectrometry) with spectra analysis by Principal Component Analysis5 and two-dimensional electrophoresis followed by identification of proteins by LC-MS/MS (Liquid Chromatography coupled to tandem Mass Spectrometry) would allow us to find the differences between the analysed groups.

Financial support from specific university research (MSMT No 21-SVV/2019). The work was realized within grant no. 16-31028A provided by the Ministry of Health of the Czech Republic REFERENCES [1] Garcea, G., C.P. Neal, C.J. Pattenden, W.P. Steward a D.P. Berry. Molecular prognostic markers in pancreatic cancer: A systematic review. European Journal of Cancer, 2005, 41(15), 2213-2236 [2] Lowenfels, A. B.; Maisonneuve, P., Risk factors for pancreatic cancer. Journal of cellular biochemistry 2005, 95 (4), 649-56. [3] Výskyt nádorů slinivky v ČR, Linkos.cz. Linkos: Česká onkologická společnost České lékařské společnosti J. E. Purkyně, 2019, ČOS ČLS JEP (cit. 15. 1. 2019). Web site: https://www.linkos.cz/pacient-a-rodina/onkologicke-diagnozy/nadory-slinivky-brisni-c25/vyskyt-nadoru-slinivky-v-cr/ (cit. 13. 1. 2019) [4] Li, D. H.; Xie, K. P.; Wolff, R.; Abbruzzese, J. L., Pancreatic cancer. Lancet 2004, 363 (9414), 1049-1057. [5] Cejnar, P., Kuckova, S., Prochazka, A., Karamonova, L., Svobodova, B., Principal component analysis of normalized full spectrum mass spectrometry data in multiMS-toolbox: An effective tool to identify important factors for classification of different metabolic patterns and bacterial strains, Rapid Communications in Mass Spectrometry, 2018, 32(11), 871–881

Positive regulation of human PINK1 and Parkin gene expression by nuclear respiratory factor 1

Dan Wang, Yapeng Lu, Huiwen Kan, Li Zhu*

Institute of Special Environmental Medicine, Nantong University, Nantong 226019,

China

*Corresponding author: Li Zhu

9# Seyuan Road, Nantong 226019, China

Telephone: +86-0513-55003376

Fax: +86-0513-55003376

Email address: zhulizhou@ntu.edu.com

Previous studies have demonstrated that two Parkinson's disease-associated genes

PINK1 and Parkin play a key role in mitochondrial quality control. But until now, the

transcriptional regulation of these two genes under normal physiological conditions is

not well understood. In this study, the transcriptional regulation of PINK1 and Parkin

genes by nuclear respiratory factor 1 (NRF-1) and its effect on PINK1/

Parkin-mediated mitophagy were studied. The NRF-1 binding sites in the promoter

regions of human PINK1 and Parkin genes were analized by JASPER software and

were confirmed by Chromatin Immunoprecipitation (ChIP) assay. Down-regulation of

NRF-1 reduced the transcriptional activities of PINK1 and Parkin genes,

and consequently down-regulated the mRNA and protein levels of PINK1 and Parkin

in HEK293T cells. When NRF-1 was over-expressed in SH-SY5Y cells and

HEK293T cells, the mRNA and protein levels of PINK1 and Parkin were

up-regulated. Furthermore, NRF-1 over-expression up-regulated the protein level of

full-length PINK1 in CCCP-treated HEK293T cells and SH-SY5Y cells�indicating

the enhanced PINK1/Parkin-mediated mitophagy induced by CCCP. When NRF-1

expression was transient or stable knockdown in HEK293T cells, the CCCP-induced

mitophagy was alleviated, characterized by reduced expression of full-length PINK1,

declined ratio of LC3 II to LC3 I, and decreased ratio of

Mt-m-keima fluorescence intensity at 555 nm to 488 nm. In conclusion, NRF-1

positively regulates the transcription of PINK1 and Parkin genes, and consequencely

involves in mitochondrial quality control through regulating PINK1/Parkin-mediated

mitophagy.

Keywords:�Nuclear respiratory factor 1, Transcriptional regulation, PINK1, Parkin,

Mitophagy

� �

A global role for nuclear respiratory factor1 under hypoxia by ChIP-seq

Yapeng Lu, Dan Wang, Xueting Wang, Li Zhu*

Institute of Special Environmental Medicine, Nantong University, Nantong, Jiangsu 226019, China; * Corresponding author: Li Zhu, PhD, 9 Se Yuan Road, Nantong 226019, Jiangsu, China; Tel: +86-13962988532; E-mail: zhulizhou@ntu.edu.cn

Oxygen is essential for almost all animal life that serves as a key substrate in cellular metabolism and bioenergetics. In a variety of physiological and pathological states, organisms encounter insufficient O2 availability, or hypoxia. Consequently, organisms have evolved a number of mechanisms to rapidly adapt to hypoxia. Mitochondria serve as metabolic hubs in the cell and are intimately linked with the adaptive response to hypoxia. Nuclear respiratory factor 1 (NRF1) functions as a key transcription factor linked to machinery in mitochondrial biogenesis, mitochondrial respiration, mitochondrial DNA transcription and replication. Herein, we performed a genome-wide ChIP-seq for NRF1 under hypoxic condition. TM3 cells were treated with hypoxia (1% O2) for 24 h, and then the cells were fixed with formaldehyde to crosslink NRF1-DNA complexes. Sonicated nuclear lysates were processed for immunoprecipitation with a monoclonal anti-NRF1 antibody or normal IgG for input signal. NGS libraries constructed from size-selected and adapter-ligated ChIP DNA fragments were processed for deep sequencing at a 36 bp read length on Genome Ana-lyzer (Illumina). Overall, we identified 3221 highly stringent ChIP-seq peaks on protein-coding genes by hypoxic stimulus. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by NRF1 related to nervous system development, neurogenesis, generation of neurons. Furthermore, some genes located in the core pathways related to long−term potentiation. Finally, we validated several genes belonging to these programs by qPCR. These results suggest a logical hypothesis that aberrant regulation of NRF1 by hypoxia and its targets might contribute to the disorders of the nervous system. Key words: NRF1, hypoxia, ChIP-seq, nervous system �

�

SIRT1 regulates tau exon 10 alternative splicing by modulating

acetylation levels of splicing factors�

Wei Qian1,2*, Xiaomin Yin1,2*, Xiaosu Jiang2, Jia Wang2, Shuo Qian2

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Adult human brain expresses approximately equal levels of 3R-tau (3 repeats-tau) and 4R-tau, which are generated by alternative splicing of tau exon 10. Dysregulation of alternative splicing of tau exon 10 and the imbalance expressions of 3R-tau to 4R-tau have been seen in inherited and sporadic tauopathies, including Alzheimer’s disease (AD). Acetylation is one of the major post-translational protein modifications in the cell by attachment of the acetyl group to either the α-amino group of the N-terminus of proteins or to the ε-amino group of lysine residues. Splicing factor family plays an essential role in promoting the inclusion of tau exon 10. Their function is post-translationally modified by phosphorylation and acetylation modification, though the role of acetylation in each splicing factor-medicated tau exon 10 inclusion remains unclear. Sirtuin type 1 (SIRT1), one member in mammalian Sirtuin family, is an enzyme that deacetylates proteins and associates with age-related disease such as AD. In the present study, we determined the role of SIRT1 in SRFS2 (Serine/arginine-rich splicing factor 2) and SRFS7 acetylation and in the alternative splicing of tau exon 10. We found that SIRT1 interacts and deacetylates SRFS2 or SRFS7, and suppressed their function in regulating tau exon 10 splicing. Substituting K52 residue of SRFS2 by arginine impairs the role of SRFS2 in tau exon 10 inclusion. Mutation of lysine 24 residue of SRFS7 into arginine abolished the inhibition of tau exon 10 inclusion by SIRT1. Therefore, the decrease of Sirt1 level in AD brains may cause dysregulation of tau exon 10 splicing through the acetylation status of SRFS2 or SRFS7, leading to the imbalance of 3R-tau to 4R-tau, which may initiate or accelerate tau pathology and cause neurofibrillary degeneration in AD brain.

INFLUENCE PHYSICAL LOAD TO CHANGE THE PHYSIOLOGICAL

COMPOSITION OF THE BLOOD

Bagirova R.M.

Azerbaijan State Academy of Physical Culture and Sport, Department "Medical and Biological Sciences" Baku, Azerbaijan Republic E-mail: rafiga_bagirova1@mail.ru

The purpose of this work was to study changes in the composition of peripheral blood in athletes

before and after physical load. It has been shown that the number of red blood cells in basketball

athletes after physical load increases from 4.83±0.34 to 6±0.34 h1012 cells/l, which exceeds their

number in comparison with the state of rest by 24%. The growth of red blood cells amounted to

10% of the norm. In the study of hemoglobin indices it was also its increase is revealed from

127.4±5.8 to 158.2 g/l±5.7 g/l, which corresponded to the physical load of its quantity after exercise

compared with the background by 24.2%. Hemoglobin increment was 32% of the norm. We also

identified increase the quantitative value of leukocytes - from 6.19±1.27 to 9.08±0.27x199 cells/l,

which exceeded the background rate of 47%. The increase amounted to 25% of normal. Average

indicators erythrocyte sedimentation rate for basketball players at rest was 5.2±2mm/h, and after

physical load, these parameters reached 16.8±1.3mm/h. As can be seen from the obtained data, an

increase in this indicator after physical load was observed 3 times, which corresponded to 123%

increase from the norm. In our research also noted increase in the number of platelets. In our

research also showed an increase the number of platelets. Thus, in a state of rest in basketball

players of national team quantities platelet count amounted to 226.7±45.6х109 cells/l, and after

performing physical load, their value increased significantly and averaged 300.3±27.7x109 cells/l.

In addition to the above indicators our observations revealed increase of sugar and hematocrit in the

blood of basketball players after the physical load. So, in a calm state the average indicators of the

amount of sugar in the blood on the team was 91.3±14.8 mg%, and after the work performed, this

indicator reached a level of 122±1.85 mg%, which corresponded to a 34% increase in this indicator.

The gain from the norm was 22%. When analyzing the blood picture of basketball players it was

also revealed the increase in hematocrit, the average values of which at rest were 43.5±2.3%, and

after physical load, these values were 49.5±1.36%, which corresponded to an increase of this

indicator by 14%. Gain from the norm was within 10%.

Thus, under the influence of physical activity in the peripheral blood of athletes, erythro-,

thrombotic and leukocytosis, changes in the leukocyte formula, the severity of which depends on

the power and intensity of the load, are observed.

Keywords – red blood cells, hemoglobin, leukocytes, platelets, hematocrit, sugar, physical load.

Application of polysaccharides isolated from non-medical parts of Bletilla formosana

Mei-kuang Lu1, Chi-Hsein Chao1 and Jing-Jy Cheng1,*

1. National Research Institute of Chinese Medicine, Ministry of Health and Welfare,

Taipei, Taiwan Bletilla formosana, one kind of the Taiwan peculiar plants, is also used as common

Bletilla tuber like B. striata. Bringing B. formosana into cultivation through controlled growth systems is a strategy to mass produce this species for its commercial value. More, the usage of common Bletilla tuber requires at least 3-year growth, the development of the non-medical aerial part provide another chance to find therapeutic applications. Wound repair is highly complex, comprising a series of coordinated and overlapping processes. The management of a chronic wound has become a major therapeutic challenge, and it is a problem that will only escalate with the increasing incidence of conditions that impede wound healing, such as diabetes, obesity and vascular disorders. In this study, we propagate the culture of B. formosana up to 540 days, isolation of its polysaccharide (BFP) and to identify the sugar compositions and MW distribution. The molecular weight distribution profile of the lyophilized polysaccharides was determined as characterized for five-groups distribution. Sorbitol, mannitol, galactose, glucose, and mannose were the major sugars composed in the polysaccharides. Moderate IL-1β and TNF-α secreation was found after treatment with BFP for 24 hrs in macrophages. BFP also showed significant potency on EC migration and also increased angiogenesis in vitro. VE-cadherin, ß-catenin and Wnt1 expression were increased while E-cadherin expression decreased after BFP incubation on ECs. The moderate inflammatory effect, enhancement of EC migration and alternation of Wnt/ß-catenin pathway might correlate with the wound-healing activity of BFP. Working on the study and development of Taiwan peculiar plants is valuable especially using tissue culture is a convenient way to propagate plants fast and efficient. As chronic wound is a problem that will escalate with the increasing incidence of conditions that impede wound healing, such as diabetes, obesity and vascular disorders, we anticipate our results will further provide evidence of BFP as a new therapeutic reference for wound repair.

Key words: wound healing, Bletilla formosana, polysaccharide, Taiwan peculiar plants

Hexavalent Chromium Bioreduction may Enhanced Microbial

Bioremediation of Organic Pollutants.

$EVWUDFW�²�Bioremediation of organic pollutants by different microorganisms has been reported

with tremendous improvement. Nevertheless, long term microbial activity is still at halt due to

interfering heavy metals with duration of organism’s survival. Most of these heavy metal can be

converted to their less toxic form through reduction reaction. Reduction of hexavalent chromium

to less toxic trivalent form was carried out simply using plant extract. Effects of process

parameters, reaction kinetics and thermodynamics were investigated. Tamarindus indica aqueous

pulp extract demonstrate a very powerful reduction capacity under friendly conditions. The

reaction follows second order kinetics and it’s thermodynamically feasible. Thus, conversion of

toxic to less toxic chromium can favors the growth of microorganism used for bioremediation of

organic pollutant, hence better performance.

Keywords: Bioreduction; Chromium; Kinetics; Thermodynamics; Process parameters

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TYROSINASE AND α-AMYLASE INHIBITION EFFECIENCIES OF SOME NEWLY SYNTHESIZED HYBRIDE TYPE ORGANIC COMPOUNDS

Ummuhan Cakmak1, Ahmet Colak*,1, Serap Basoglu Ozdemir1, Ilke Demir2,

Neslihan Demirbas1, Safiye Sag Erdem2

1 Karadeniz Technical University, Faculty of Science, Department of Chemistry, 61080 Trabzon

2 Marmara University, Faculty of Arts and Sciences, Chemistry Department, Goztepe, İstanbul

Tyrosinase (EC 1.14.18.1) is a crucial enzyme in melanin biosynthesis in mammals,

bacteria, plants, and fungi [1]. It is well known that melanin protects the skin from UV damage but its excessive production causes skin cancer, freckles, melasma and age spots [2,3,4]. The enzyme is involved in development of Parkinson’s disease by causing neurodegeneration through decreasing dopamine level. Besides, tyrosinase participates in cuticle formation in insects. Therefore, tyrosinase inhibitors have potential applications in medicine and cosmetics as whitening and depigmentation agents, and in agriculture as bioinsecticides [5].

α-Amylase plays a major role in the digestion of starch yielding glucose, leads to increased postprandial glucose levels. Hence, reducing the starch digestion rate by inhibition of enzymes as α-amylase the best way for the management of diabetes [6].

Because of these reasons, new inhibitor molecules should be designed and synthesized to investigate tyrosinase and α-amylase inhibition in order to treat certain diseases. In this study, eight novel molecules containing benzimidazole, triazole and quinolone skeletons were designed and synthesized. Mushroom tyrosinase and porcine pancreatic α-amylase inhibitory potentials of these molecules have been studied. Kojic acid and acarbose were used as reference standard inhibitors, respectively. Among the 8 studied molecules, the 16th molecule efficiently inhibited the tyrosinase and the IC50 value of this molecule was calculated as 523.4±3.6 µM. It was also observed that 14th and 15th molecules were able to inhibit α-amylase activity. The IC50 values of these molecules were determined as 28.7±1.1 and 359.3±0.9, respectively.

The results of docking studies of both two enzymes were in good agreement with that of in vitro studies. 14th and 15th compounds bind to the active site of α-amylase with high affinities (-10.1 and -8.9 kcal/mol). Asp300, Glu233, Asp197, Val163 and Trp59 are common residues in the active site which play role in the binding of both ligands via H-bonding and hydrophobic interactions. Compound 16 binds to the active site of tyrosinase with highest affinity (-9.4 kcal/mol). Val283, Pro284, His285, Leu275 and Arg268 are common residues in the active site which play role in the binding of the ligand via H-bonding, π-alkyl and π-σ interactions. ADME properties of investigated molecules as drug candidate molecules were found to be quite well.

The authors are grateful to TUBITAK for their financial support (Project No:117Z199). References

1.� Decker, H., Tuczek, F. 2000. Trends in Biochemical Sciences, 25, 392-397. 2.� Nguyen, H., Nguyen, N., Nguyen, M., Lee, T., Do, T., Hung, T., Nguyen, M. 2016.

Chemistry Central Journal, 10, 1-6. 3.� Zhang, L., Zhao, X., Tao, G., Chen, J., Zheng, Z. 2017. Food Chemistry, 223, 40-48. 4.� Ortiz-Ruiz, C.V. , Berna, J. , Tudela, J. , Varon, R., Garcia-Canovas, F. 2016. Journal

of Dermatological Science, 82 (2), 115-122. 5.� Goua, L., Lee, J., Yang J. M., Parke, Y. D., Zhoue, H. M., Zhanf, Y., Lüe, Z. R. 2017.

International Journal of Biological Macromolecules, 105, 1663-1669. 6.� Sudha, P,, Zinjarde, S. S., Bhargava, S. Y., Kumar, A. R. 2011. BMC Complement

Altern Medicine, 11:5.

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Involvement of Sperm Nuclear Basic Proteins in copper/H2O2-induced DNA damage

Marina Piscopo1*, Gennaro Lettieri1, Elena Mele1, Ferdinando Febbraio2

1Department of Biology, University of Naples, Federico II, Naples, Italy

2Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy

Successful reproduction is a determining factor for the survival of species. Thus, sperm DNA integrity is essential for the accurate transmission of genetic information, because, sperm chromatin abnormalities or DNA damage may result in male infertility. Many studies have pointed out a central role of oxidative stress, leading to the formation of ROS, in the etiology of sperm DNA damage. Is well known that DNA is protected by the sperm nuclear basic proteins (SNBPs), that are the chromosomal proteins associated with DNA in sperm nuclei at the end of spermiogenesis. These highly specialized proteins are lysine and arginine rich and can be classified into three major types: histone type, protamine-like type, and protamine type. However, in literature there are no studies that have investigated their possible participation in DNA oxidative damage in particular conditions, such as in the presence of some heavy metal. �In the past few decades, several ecosystems are polluted by increased levels of heavy metals resulting from anthropogenic activities, industrial production and domestic/agricultural use of metals and metal-containing compounds, negatively affecting reproductive health of several species including humans. Copper is one of the most interesting heavy metals, because in small quantities, it is essential - being involved in several physiological functions such as redox reactions, oxygen transport, cellular respiration, free radical defense - but can be toxic above certain threshold concentrations. Here, we reported evidences that, both in marine organisms and in humans, in presence of copper, arginine-rich SNBPs can contribute to oxidative DNA damage. The experiments were performed by using the three different types of SNBPs: sperm H1 histones extracted from the sperm chromatin of the annelid worm Chaetopterus variopedatus; protamine-like from sperm chromatin of the mussel Mytilus galloprovincialis unexposed and exposed to a subtoxic copper concentration and protamines from sperm chromatin of man living in area with low and high environmental impact, such as the well known "Land of Fires".�� In particular, in spermatozoa of men living in high impact area, we found a copper level of about 95% respect to that found in spermatozoa of individuals living in other areas. Exploiting the conversion from supercoiled to relaxed form of a plasmid in presence of SNBPs and H2O2, we developed an in vitro assay for the determination of DNA oxidative damage. We found that DNA damage was observed only in the presence of SNBPs deriving from organisms (human and mussels) exposed to copper. In order to understand the mechanisms, SNBPs mediated, of copper/H2O2-induced DNA damage, we performed experiments of in vitro oxidative DNA damage using sperm and somatic H1 histones in presence of increasing copper concentrations. Sperm H1 histones, like protamine and protamine-like, are arginine-richer in comparison to somatic ones. The results of our studies support the existence of Cu(II) effects arginine-dependent, that provides new insight in copper toxicity mechanisms.��

Cytotoxic and anti-mitotic effects of novel combretastatin A-4 benzothiazolone analogs

G. Atanasov*1, R. Rusev2, B Shivachev2, O. Petrov3, M. D. Apostolova1

1Medical and Biological Research Lab., Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria 2Institute of mineralogy and crystallography, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl.17, 1113 Sofia, Bulgaria

3Department of Applied Organic Chemistry, Faculty of Chemistry, University of Sofia “St. Kliment Ohridski”, 1, James Bourchier Blvd., 1164 Sofia, Bulgaria

Tubulin-binding agents represent an important group of microtubule-targeting chemotherapeutics. Combretastatin A-4 (CA-4) is well appreciated for it’s potent cytotoxic activity directed towards tumor vasculature and has shown promising results in pre-clinical studies against different cancers. However, its cardiotoxicity and poor bioavailability are the main reasons for clinical studies withdrawal.

We have selected the benzothiazolone moiety as a modification fragment of pharmacological relevance to synthesize new series of CA-4 analogs. The aim of the study was to characterize the biological effects of 26 newly synthesized structural analogs. Thirteen different cell lines with cancer (K-562, THP-1, U-937, LNCap, PC-3, HT-29, Colon-26, HepG2, MCF-7, MDA-MB-231,) and control (EA.hy926, HaCaT, MCF-10A) origin were studied for anti-proliferative activity of the most active compounds by MTT-assay. The formation of microtubules was analyzed by ex-vivo tubulin polymerization assay. The effects of CA-4 analogues on cell proliferation and death were analyzed by flow cytometry, nucleosomal DNA-fragmentation assays and Western blot analyses.

Six cis-analogues of CA-4 showed an anti-proliferative /cytotoxic activity in a concentration range of 10 nM to 25 µM in the studied cell lines. For most cell lines, the analog S19 showed very good anti-proliferative activity with IC50 values between 15-40 nM, and similar or better activity in comparison to CA-4 for HT-29, THP-1 and LNCap. These are very encouraging results considering the weaker cytotoxicity shown in control cells. Ex-vivo microtubule polymerization was blocked or increased following application of most active CA-4 analogues. The disturbances in microtubules formation were connected with the inhibition of cell cycle in G2/M phase, cell death and formation of multinucleated cells detected with flow cytometry. Apoptotic cell death was confirmed by activation of caspase 3 and PARP-1 fragmentation.

The results showed that CA-4 modification with pharmacologically beneficial benzothiazolone heterocycle resulted in analogs with strong anti-proliferative/cytotoxic activity. Further elucidation of benzothyazolone CA-4 analogs mode of action would be valuable knowledge for better anti-cancer therapeutics design.

Acknowledgments: The research was supported under contracts DFNP-17-124 (BAS) and DFN-19-13 (NSF, Bulgaria).

Hepatoprotective effect of Watercress extract and quercetin on bile duct ligated-induced cholestasis in rats

Hossein Sadeghi1, Nahid Azarmehr2, Fatemeh Razmkhah2, Nazanin Danaei1, Navid Omidifar3, Hossein Vakilpoor2, Amir Hossein Doustimotlagh1,5*

1Medicinal Plants Research Center, Yasuj University of Medical Sciences, Yasuj, Iran 1Student Research Committee, Yasuj University of Medical Sciences, Yasuj, Iran 3Senior Resident of Pathology, Department of Pathology, Transplant Research Center, Nemazee Hospital, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 5Department of Clinical Biochemistry, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran *The main (presenting) author: Amir Hossein Doustimotlagh #Corresponding author: Amir Hossein Doustimotlagh Email: amirhosseindoustimotlagh@gmail.com Abstract

Cholestatic liver disease is recognized by extreme collagen formation and deposition, which is mediated by free radicals. The aim of the current study was to investigate the probable hepatoprotective effects of watercress (WC) extract and quercetin against oxidative stress and liver injury in bile duct ligation (BDL)- induced cholestatic rats. A total of 34 male Wistar rats were divided into four groups; sham control (SC), BDL, BDL+ WC and BDL+ quercetin. WC extract and quercetin- treated BDL rats received daily WC 500 mg/kg/day and quercetin 50 mg/kg/day for 10 days after BDL. Biochemical tests, hepatic oxidative stress markers and antioxidant enzymes activity were estimated. Also, Liver hydroxyproline content and hematoxylin and eosin staining were determined. The BDL model obviously elevated the protein carbonyl (PCO) and hydroxyproline contents, as well as decreased the glutathione peroxidase (GPx) activity. WC extract and quercetin administration drastically decreased the increased liver PCO and hydroxyproline levels and increased the reduced GPx enzyme activity contents in the hepatic tissue. As determined by hematoxylin and eosin staining, BDL considerably induced the liver necrosis. Also, these changes were markedly alleviated by WC extract and quercetin treatment. The data indicate that the WC extract and quercetin administration attenuated liver damage in BDL rats by decreasing hydroxyproline content and histopathological indexes. Also, both of them reduce oxidative stress by preventing the hepatic protein oxidation and renewing the activity of the GPx enzyme via antioxidative effect and free radical scavenging. Our finding recommend that WC extract and quercetin could be a beneficial new curative agent for cholestatic liver damage.

Keywords: Cholestatic; Watercress; Quercetin; Oxidative stress; BDL rats

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PCB 126 exposure disrupts hepatic iron homeostasis and induces NAFLD

through downregulation of hepatic STAMP2

Hye Young Kim*, Woo Young Kwon, Joon Beom Park, Hwa Jin Kim and Young Hyun Yoo

Department of Anatomy and Cell Biology, Dong-A University College of Medicine, Busan, Republic of Korea

�

Polychlorinated biphenyls (PCBs), with 209 congeners, are a large family of persistent organic pollutants (POPs)

and have been associated with neurotoxicity, hepatoxicity, oncogenicity, and endocrine-disrupting effects.

Although epidemiological and experimental studies demonstrated that PCBs lead to non-alcoholic fatty liver

disease (NAFLD), the underlying mechanism has remained unsolved. In this study, we examined in vivo and in

vitro effects of dioxin-like PCB 126 on liver. For in vivo studies, 8-weeks-old C57BL/6 mice were fed either a

standard diet (SD) or a high fat diet (HFD) for 4 weeks and then were administered vehicle (corn oil), PCB 126

(1 or 5 mg/kg) by intraperitoneal injection for a total of four injections (2, 3, 4 and 5 weeks) during the 6-week

study duration. The detailed molecular mechanism was investigated by using HepG2 hepatocytes. PCB 126

significantly promoted hepatic fat accumulation in HepG2 cells treated with oleic acid. In mice, PCB 126 induced

hepatic steatosis, inflammation and fibrosis. Because our previous study suggested that STAMP2 may be a

suitable target for treating NAFLD, we examined whether hepatic STAMP2 involves in PCB 126-induced

NAFLD. Expression of hepatic STAMP2 was decreased in PCB 126 treated HepG2 cells and C57BL/6 mice.

Overexpression of hepatic STAMP2 using adenoviral delivery resulted in attenuation of hepatic fat accumulation

in HepG2 cells treated with oleic acid. Recent studies demonstrated that exposure to environmental pollutants

could lead to disruption of the hepcidin–ferroportin axis along with disordered systemic iron homeostasis and

diseases. Also, STAMP2 protein has been identified as ferrireductases responsible for the reduction of Fe3+. Thus,

we next investigated the effects of PCBs exposure on hepatic iron homeostasis. We observed that exposure to

PCB 126 significantly induced hepatic iron overload in vivo and in vitro. Noticeably, overexpression of hepatic

STAMP2 attenuated effects of PCB 126-induced hepatic iron overload in HepG2 cells. This study suggests that

PCB 126 disrupts hepatic iron homeostasis by decreasing hepatic STAMP2 expression, resulting in induces

NAFLD. Our ndings indicate that enhancing STAMP2 expression represents a potential therapeutic avenue for

treatment of PCB 126-induced NAFLD.

Keywords:

PCBs, STAMP2, iron homeostasis, hepatic iron overload, NAFLD