Publications by authors named "Algirdas Kaupinis"

33 Publications

Uptake of Upconverting Nanoparticles by Breast Cancer Cells: Surface Coating versus the Protein Corona.

ACS Appl Mater Interfaces 2021 Aug 11;13(33):39076-39087. Epub 2021 Aug 11.

Biomedical Physics Laboratory of National Cancer Institute, Baublio 3B, LT-08406 Vilnius, Lithuania.

Fluorophores with multifunctional properties known as rare-earth-doped nanoparticles (RENPs) are promising candidates for bioimaging, therapy, and drug delivery. When applied , these nanoparticles (NPs) have to retain long blood-circulation time, bypass elimination by phagocytic cells, and successfully arrive at the target area. Usually, NPs in a biological medium are exposed to proteins, which form the so-called "protein corona" (PC) around the NPs and influence their targeted delivery and accumulation in cells and tissues. Different surface coatings change the PC size and composition, subsequently deciding the fate of the NPs. Thus, detailed studies on the PC are of utmost importance to determine the most suitable NP surface modification for biomedical use. When it comes to RENPs, these studies are particularly scarce. Here, we investigate the PC composition and its impact on the cellular uptake of citrate-, SiO-, and phospholipid micelle-coated RENPs (LiYF:Yb,Tm). We observed that the PC of citrate- and phospholipid-coated RENPs is relatively stable and similar in the adsorbed protein composition, while the PC of SiO-coated RENPs is larger and highly dynamic. Moreover, biocompatibility, accumulation, and cytotoxicity of various RENPs in cancer cells have been evaluated. On the basis of the cellular imaging, supported by the inhibition studies, it was revealed that RENPs are internalized by endocytosis and that specific endocytic routes are PC composition dependent. Overall, these results are essential to fill the gaps in the fundamental understanding of the nano-biointeractions of RENPs, pertinent for their envisioned application in biomedicine.
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http://dx.doi.org/10.1021/acsami.1c10618DOI Listing
August 2021

Periosteum-Derived Mesenchymal Stem Cells Secretome - Cell-Free Strategy for Endogenous Bone Regeneration: Proteomic Analysis .

J Oral Maxillofac Res 2021 Apr-Jun;12(2):e2. Epub 2021 Jun 30.

Department of Oral and Maxillofacial Surgery, Faculty of Odontology, Medical Academy, Lithuanian University of Health Sciences, KaunasLithuania.

Objectives: Millions of people worldwide are affected by diseases or injuries which lead to bone/tooth loss and defects. While such clinical situations are daily practice in most of the hospitals, the widely used treatment methods still have disadvantages. Therefore, this field of medicine is actively searching new tissue regeneration techniques, one of which could be stem cell secretome. Thus, the purpose of this research study was to perform the detail proteomic analysis of periosteum-derived mesenchymal stem cells secretome in order to evaluate if it is capable to induce osteo-regenerative process.

Material And Methods: Periosteum-derived mesenchymal stem cells (PMSCs) were extracted from adult male New Zealand White rabbits. Cells were characterised by evaluating their differentiation potential. After characterisation PMSCs secretomes were collected and their proteomic analysis was performed.

Results: PMSCs were extracted from adult male New Zealand White rabbits. In order to characterise the extracted PMSCs, they were differentiated in the directions which mainly describes MSC multipotency - osteogenic, myogenic and adipogenic. A total of 146 proteins were detected. After characterisation PMSCs secretomes were collected and their proteomic analysis was performed. The resulting protein composition indicates the ability to promote bone regeneration to fully mature bone.

Conclusions: Bioactive molecules detected in periosteum-derived mesenchymal stem cells secretome initiates the processes required for the formation of a fully functional bone.
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http://dx.doi.org/10.5037/jomr.2021.12202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8326881PMC
June 2021

Mapping human calreticulin regions important for structural stability.

Biochim Biophys Acta Proteins Proteom 2021 Nov 4;1869(11):140710. Epub 2021 Aug 4.

Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, LT-10257 Vilnius, Lithuania. Electronic address:

Calreticulin (CALR) is a highly conserved multifunctional chaperone protein primarily present in the endoplasmic reticulum, where it regulates Ca homeostasis. Recently, CALR has gained special interest for its diverse functions outside the endoplasmic reticulum, including the cell surface and extracellular space. Although high-resolution structures of CALR exist, it has not yet been established how different regions and individual amino acid residues contribute to structural stability of the protein. In the present study, we have identified key residues determining the structural stability of CALR. We used a Saccharomyces cerevisiae expression system to express and purify 50 human CALR mutants, which were analysed for several parameters including secretion titer, melting temperature (T), stability and oligomeric state. Our results revealed the importance of a previously identified small patch of conserved surface residues, amino acids 166-187 ("cluster 2") for structural stability of the human CALR protein. Two residues, Tyr172 and Asp187, were critical for maintaining the native structure of the protein. Mutant D187A revealed a severe drop in secretion titer, it was thermally unstable, prone to degradation, and oligomer formation. Tyr172 was critical for thermal stability of CALR and interacted with the third free Cys163 residue. This illustrates an unusual thermal stability of CALR dominated by Asp187, Tyr172 and Cys163, which may interact as part of a conserved structural unit. Besides structural clusters, we found a correlation of some measured parameter values in groups of CALR mutants that cause myeloproliferative neoplasms (MPN) and in mutants that may be associated with sudden unexpected death (SUD).
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http://dx.doi.org/10.1016/j.bbapap.2021.140710DOI Listing
November 2021

Bacteriophage vB_PagS_MED16-A Siphovirus Containing a 2'-Deoxy-7-amido-7-deazaguanosine-Modified DNA.

Int J Mol Sci 2021 Jul 8;22(14). Epub 2021 Jul 8.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Centre, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.

A novel siphovirus, vB_PagS_MED16 (MED16) was isolated in Lithuania using strain BSL for the phage propagation. The double-stranded DNA genome of MED16 (46,103 bp) contains 73 predicted open reading frames (ORFs) encoding proteins, but no tRNA. Our comparative sequence analysis revealed that 26 of these ORFs code for unique proteins that have no reliable identity when compared to database entries. Based on phylogenetic analysis, MED16 represents a new genus with siphovirus morphology. In total, 35 MED16 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. In addition, a gene encoding a preQ DNA deoxyribosyltransferase (DpdA) is present in the genome of MED16 and the LC-MS/MS analysis indicates 2'-deoxy-7-amido-7-deazaguanosine (dADG)-modified phage DNA, which, to our knowledge, has never been experimentally validated in genomes of phages. Thus, the data presented in this study provide new information on -infecting viruses and offer novel insights into the diversity of DNA modifications in bacteriophages.
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http://dx.doi.org/10.3390/ijms22147333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8306585PMC
July 2021

CEACAM6's Role as a Chemoresistance and Prognostic Biomarker for Pancreatic Cancer: A Comparison of CEACAM6's Diagnostic and Prognostic Capabilities with Those of CA19-9 and CEA.

Life (Basel) 2021 Jun 9;11(6). Epub 2021 Jun 9.

Clinic of Gastroenterology, Nephrourology and Surgery, Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, LT-03101 Vilnius, Lithuania.

Survival rates from pancreatic cancer have remained stagnant for decades due to the heterogenic nature of the disease. This study aimed to find a new advanced biomarker and evaluate its clinical capabilities, thus enabling more individualised pancreatic cancer management. Between 2013 and 2020, 267 patients were included in the study. Surgically collected pancreatic tissue samples were analysed via high-definition mass spectrometry. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) was discovered as a possible promising pancreatic cancer biomarker. The predominance of CEACAM6 to pancreatic cancer was validated using antibodies in tissue samples. CEACAM6, carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA) blood serum concentrations were evaluated for clinical evaluation and comparison. Kaplan-Meier survival analyses were used to evaluate disease-free survival (DFS) and overall survival (OS). Poorer overall survival was significantly dependent on increased CEACAM6 blood serum concentrations (17.0 vs. 12.6 months, = 0.017) in pancreatic cancer patients after radical treatment and adjuvant chemotherapy. Increased CEA and CA19-9 concentrations showed no significant dependencies with survival. Thus, CEACAM6 is a promising new biomarker with significant prognostic value and prediction of chemoresistance properties, enabling the improvement of individualised approaches to patients with pancreatic cancer.
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http://dx.doi.org/10.3390/life11060542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226832PMC
June 2021

Assessment of the Bone Healing Process Mediated by Periosteum-Derived Mesenchymal Stem Cells' Secretome and a Xenogenic Bioceramic-An In Vivo Study in the Rabbit Critical Size Calvarial Defect Model.

Materials (Basel) 2021 Jun 24;14(13). Epub 2021 Jun 24.

Department of Oral and Maxillofacial Surgery, Faculty of Odontology, Medical Academy, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania.

The mesenchymal stem cell (MSC) secretome has been considered an innovative therapeutic biological approach, able to modulate cellular crosstalk and functionality for enhanced tissue repair and regeneration. This study aims to evaluate the functionality of the secretome isolated from periosteum-derived MSCs, from either basal or osteogenic-induced conditions, in the healing of a critical size calvarial bone defect in the rabbit model. A bioceramic xenograft was used as the vehicle for secretome delivery, and the biological response to the established biocomposite system was assessed by clinical, histological, histomorphometric, and microtomographic analysis. A comparative analysis revealed that the osteogenic-induced secretome presented an increased diversity of proteins, with emphasis on those related to osteogenesis. Microtomographic and histological morphometric analysis revealed that bioceramic xenografts implanted with secretomes enhanced the new bone formation process, with the osteogenic-induced secretome inducing the highest bone tissue formation. The application of the MSC secretome, particularly from osteogenic-induced populations, may be regarded as an effective therapeutic approach to enhance bone tissue healing and regeneration.
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http://dx.doi.org/10.3390/ma14133512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8269548PMC
June 2021

Menstrual Blood-Derived Endometrial Stem Cells' Impact for the Treatment Perspective of Female Infertility.

Int J Mol Sci 2021 Jun 24;22(13). Epub 2021 Jun 24.

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.

When looking for the causes and treatments of infertility, much attention is paid to one of the reproductive tissues-the endometrium. Therefore, endometrial stem cells are an attractive target for infertility studies in women of unexplained origin. Menstrual blood stem cells (MenSCs) are morphologically and functionally similar to cells derived directly from the endometrium; with dual expression of mesenchymal and embryonic cell markers, they proliferate and regenerate better than bone marrow mesenchymal stem cells. In addition, menstrual blood stem cells are extracted in a non-invasive and painless manner. In our study, we analyzed the characteristics and the potential for decidualization of menstrual blood stem cells isolated from healthy volunteers and women diagnosed with infertility. We demonstrated that MenSCs express CD44, CD166, CD16, CD15, BMSC, CD56, CD13 and HLA-ABC surface markers, have proliferative properties, and after induction of menstrual stem cell differentiation into epithelial direction, expression of genes related to decidualization (, , and ) and angiogenesis (, and ) increased. Additionally, the p53, p21, H3K27me3 and HyperAcH4 proteins' expression increased during MenSCs decidualization, they secrete proteins that are involved in the regulation of the actin cytoskeleton, estrogen and relaxin signaling pathways and the management of inflammatory processes. Our findings reveal the potential use of MenSCs for the treatment of reproductive disorders.
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http://dx.doi.org/10.3390/ijms22136774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268036PMC
June 2021

Rab40-Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration.

J Cell Biol 2021 Jul 17;220(7). Epub 2021 May 17.

Department of Cell and Developmental Biology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO.

Rab40b is a SOCS box-containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b-Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b-Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b-Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.
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http://dx.doi.org/10.1083/jcb.202008060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129794PMC
July 2021

Bacteriophage vB_PagS_AAS23: A Singleton of the Genus .

Microorganisms 2021 Mar 23;9(3). Epub 2021 Mar 23.

Life Sciences Centre, Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.

A cold-adapted siphovirus, vB_PagS_AAS23 (AAS23) was isolated in Lithuania using the strain AUR for the phage propagation. The double-stranded DNA genome of AAS23 (51,170 bp) contains 92 probable protein encoding genes, and no genes for tRNA. A comparative sequence analysis revealed that 25 of all AAS23 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. Based on the phylogenetic analysis, AAS23 has no close relationship to other viruses publicly available to date and represents a single species of the genus within the family . The phage is able to form plaques in bacterial lawns even at 4 °C and demonstrates a depolymerase activity. Thus, the data presented in this study not only provides the information on -infecting bacteriophages, but also offers novel insights into the diversity of cold-adapted viruses and their potential to be used as biocontrol agents.
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http://dx.doi.org/10.3390/microorganisms9030668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004638PMC
March 2021

Analysis of a Novel Bacteriophage vB_AchrS_AchV4 Highlights the Diversity of Viruses.

Viruses 2021 02 27;13(3). Epub 2021 Feb 27.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.

spp. are ubiquitous in nature and are increasingly being recognized as emerging nosocomial pathogens. Nevertheless, to date, only 30 complete genome sequences of phages are available in GenBank, and nearly all of those phages were isolated on . Here, we report the isolation and characterization of bacteriophage vB_AchrS_AchV4. To the best of our knowledge, vB_AchrS_AchV4 is the first virus isolated from . Both vB_AchrS_AchV4 and its host, RL_4, were isolated in Lithuania. VB_AchrS_AchV4 is a siphovirus, since it has an isometric head (64 ± 3.2 nm in diameter) and a non-contractile flexible tail (232 ± 5.4). The genome of vB_AchrS_AchV4 is a linear dsDNA molecule of 59,489 bp with a G+C content of 62.8%. It contains no tRNA genes, yet it includes 82 protein-coding genes, of which 27 have no homologues in phages. Using bioinformatics approaches, 36 vB_AchrS_AchV4 genes were given a putative function. A further four were annotated based on the results of LC-MS/MS. Comparative analyses revealed that vB_AchrS_AchV4 is a singleton siphovirus with no close relatives among known tailed phages. In summary, this work not only describes a novel and unique phage, but also advances our knowledge of genetic diversity and evolution of bacteriophages.
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http://dx.doi.org/10.3390/v13030374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996906PMC
February 2021

-Infecting Bacteriophage vB_PagS_AAS21: A Cold-Adapted Virus Representing a Novel Genus within the Family .

Viruses 2020 04 23;12(4). Epub 2020 Apr 23.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.

A novel cold-adapted siphovirus, vB_PagS_AAS21 (AAS21), was isolated in Lithuania using as the host for phage propagation. AAS21 has an isometric head (~85 nm in diameter) and a non-contractile flexible tail (~174 × 10 nm). With a genome size of 116,649 bp, bacteriophage AAS21 is the largest -infecting siphovirus sequenced to date. The genome of AAS21 has a G+C content of 39.0% and contains 213 putative protein-encoding genes and 29 genes for tRNAs. A comparative sequence analysis revealed that 89 AAS21 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. In total, 63 AAS21 ORFs were functionally annotated, including those coding for the proteins responsible for virion morphogenesis, phage-host interactions, and DNA metabolism. Proteomic analysis led to the experimental identification of 19 virion proteins, including 11 that were predicted by bioinformatics approaches. Based on comparative phylogenetic analysis, AAS21 cannot be assigned to any genus currently recognized by ICTV and may represents a new branch of viruses within the family .
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http://dx.doi.org/10.3390/v12040479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232348PMC
April 2020

YqfB protein from Escherichia coli: an atypical amidohydrolase active towards N-acylcytosine derivatives.

Sci Rep 2020 01 21;10(1):788. Epub 2020 Jan 21.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio al. 7, Vilnius, LT-10257, Lithuania.

Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid Escherichia coli protein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N-acetylcytidine (ac4C). YqfB has several interesting and unique features: i) it is the smallest monomeric amidohydrolase described to date, ii) it is active towards structurally different N-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (k/K up to 2.8 × 10 M s). Moreover, our results suggest that YqfB contains a unique Thr-Lys-Glu catalytic triad, and Arg acting as an oxyanion hole. The mutant lacking the yqfB gene retains the ability to grow, albeit poorly, on N-acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists in E. coli. Overall, YqfB ability to hydrolyse various N-acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases.
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http://dx.doi.org/10.1038/s41598-020-57664-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972931PMC
January 2020

Molecular Insights into miRNA-Driven Resistance to 5-Fluorouracil and Oxaliplatin Chemotherapy: miR-23b Modulates the Epithelial–Mesenchymal Transition of Colorectal Cancer Cells

J Clin Med 2019 12 2;8(12). Epub 2019 Dec 2.

Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania.

Although treatment of colorectal cancer with 5-florouracil and oxaliplatin is widely used, it is frequently followed by a relapse. Therefore, there is an urgent need for profound understanding of chemotherapy resistance mechanisms as well as the profiling of predictive markers for individualized treatment. In this study, we identified the changes in 14 miRNAs in 5-fluouracil and 40 miRNAs in oxaliplatin-resistant cell lines by miRNA sequencing. The decrease in miR-224-5p expression in the 5-fluorouracil-resistant cells correlated with drug insensitivity due to its overexpression-induced drug-dependent apoptosis. On the other hand, the miR-23b/27b/24-1 cluster was overexpressed in oxaliplatin-resistant cells. The knockout of miR-23b led to the partial restoration of oxaliplatin susceptibility, showing the essential role of miR-23b in the development of drug resistance by this cluster. Proteomic analysis identified target genes of miR-23b and showed that endothelial-mesenchymal transition (EMT) was implicated in oxaliplatin insensibility. Data revealed that EMT markers, such as vimentin and SNAI2, were expressed moderately higher in the oxaliplatin-resistant cells and their expression increased further in the less drug-resistant cells, which had miR-23b knockout. This establishes that the balance of EMT contributes to the drug resistance, showing the importance of the miR-23b-mediated fine-tuning of EMT in oxaliplatin-resistant cancer cells.
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http://dx.doi.org/10.3390/jcm8122115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947029PMC
December 2019

Nitric Oxide Donor NOC-18-Induced Changes of Mitochondrial Phosphoproteome in Rat Cardiac Ischemia Model.

Medicina (Kaunas) 2019 Sep 24;55(10). Epub 2019 Sep 24.

Neuroscience Institute, Lithuanian University of Health Sciences, LT-44307 Kaunas, Lithuania.

: Nitric oxide (NO) is known to exert cardioprotective effects against heart ischemic damage and may be involved in ischemic pre- and postconditioning. NO-triggered cardioprotective mechanisms are not well understood but may involve regulation of mitochondrial permeability transition pore (mPTP). In this study, we aimed to identify differentially phosphorylated mitochondrial proteins possibly involved in the NO/protein kinase G (PKG)/mPTP signaling pathway that can increase the resistance of cardiomyocytes to ischemic damage. : Isolated hearts from Wistar rats were perfused with NO donor NOC-18 prior to induction of stop-flow ischemia. To quantify and characterize the phosphoproteins, mitochondrial proteins were resolved and analyzed by two-dimensional gel electrophoresis followed by Pro-Q Diamond phosphoprotein gel staining, excision, trypsin digestions, and mass spectrometry. Quantitative proteomic analysis coupled with liquid chromatography-tandem mass spectrometry was also performed. : Mitochondrial protein phosphorylation patterns in NOC-18-pretreated ischemic hearts versus ischemic hearts were compared. Pretreatment of hearts with NOC-18 caused changes in mitochondrial phosphoproteome after ischemia which involved modifications of 10 mitochondrial membrane-bound and 10 matrix proteins. Among them, α-subunit of ATP synthase and adenine nucleotide (ADP/ATP) translocase 1, both of which are considered as potential structural components of mPTP, were identified. We also found that treatment of isolated non-ischemic mitochondria with recombinant PKG did not cause the same protein phosphorylation as pretreatment of hearts with NOC-18. : Our study suggests that pretreatment of hearts with NOC-18 causes changes in mitochondrial phosphoproteome after ischemia which involves modifications of certain proteins thought to be involved in the regulation of mPTP opening and intracellular redox state. These proteins may be potential targets for pharmacological preconditioning of the heart.
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http://dx.doi.org/10.3390/medicina55100631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6843668PMC
September 2019

Oxidative phosphorylation inhibition induces anticancerous changes in therapy-resistant-acute myeloid leukemia patient cells.

Mol Carcinog 2019 11 6;58(11):2008-2016. Epub 2019 Aug 6.

Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania.

Treatment of acute myeloid leukemia (AML) is still a challenge because of common relapses or resistance to treatment. Therefore, the development of new therapeutic approaches is necessary. Various studies have shown that certain cancers, including some chemoresistant AML subsets, have upregulated oxidative phosphorylation. In this study, we aimed to assess treatment-resistant AML patients' cell modulation using oxidative phosphorylation inhibitors metformin and atovaquone alone and in various combinations with cytosine analog cytarabine and apoptosis inducer venetoclax. Metabolic activity analysis using Agilent Seahorse XF Extracellular Flux Analyzer revealed that peripheral blood mononuclear cells' metabolic state was different among treatment-resistant AML patients. We demonstrated that metformin decreased therapy-resistant-AML cell oxidative phosphorylation ex vivo, cotreatment with cytarabine and venetoclax slightly increased the effect. However, treatment with atovaquone did not have a marked effect in our experiment. Cell treatment had a slight effect on cell proliferation inhibition; combination of metformin, cytarabine, and venetoclax had the strongest effect. Moreover, a slightly higher effect on cell proliferation and cell cycle regulation was demonstrated in the cells with higher initial oxidative phosphorylation rate as demonstrated by gene expression analysis using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Proteomic analysis by liquid chromatography-mass spectrometry demonstrated that chemoresistant AML cell treatment with metformin modulated metabolic pathways, while metformin combination with cytarabine and venetoclax boosted the effect. We suggest that oxidative phosphorylation inhibition is effective but not sufficient for chemoresistant AML treatment. Indeed, it causes anticancerous changes that might have an important additive role in combinatory treatment.
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http://dx.doi.org/10.1002/mc.23092DOI Listing
November 2019

Proteomic Analysis of Breast Cancer Resistance to the Anticancer Drug RH1 Reveals the Importance of Cancer Stem Cells.

Cancers (Basel) 2019 Jul 11;11(7). Epub 2019 Jul 11.

Proteomics Center, Institute of Biochemistry, Vilnius University Life Sciences Center, Vilnius University, 10223 Vilnius, Lithuania.

Antitumor drug resistance remains a major challenge in cancer chemotherapy. Here we investigated the mechanism of acquired resistance to a novel anticancer agent RH1 designed to be activated in cancer cells by the NQO1 enzyme. Data show that in some cancer cells RH1 may act in an NQO1-independent way. Differential proteomic analysis of breast cancer cells with acquired resistance to RH1 revealed changes in cell energy, amino acid metabolism and G2/M cell cycle transition regulation. Analysis of phosphoproteomics and protein kinase activity by multiplexed kinase inhibitor beads showed an increase in the activity of protein kinases involved in the cell cycle and stemness regulation and downregulation of proapoptotic kinases such as JNK in RH1-resistant cells. Suppression of JNK leads to the increase of cancer cell resistance to RH1. Moreover, resistant cells have enhanced expression of stem cell factor (SCF) and stem cell markers. Inhibition of SCF receptor c-KIT resulted in the attenuation of cancer stem cell enrichment and decreased amounts of tumor-initiating cells. RH1-resistant cells also acquire resistance to conventional therapeutics while remaining susceptible to c-KIT-targeted therapy. Data show that RH1 can be useful to treat cancers in the NQO1-independent way, and targeting of the cancer stem cells might be an effective approach for combating resistance to RH1 therapy.
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http://dx.doi.org/10.3390/cancers11070972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678540PMC
July 2019

The post-abscission midbody is an intracellular signaling organelle that regulates cell proliferation.

Nat Commun 2019 07 18;10(1):3181. Epub 2019 Jul 18.

Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA.

Once thought to be a remnant of cell division, the midbody (MB) has recently been shown to have roles beyond its primary function of orchestrating abscission. Despite the emerging roles of post-abscission MBs, how MBs accumulate in the cytoplasm and signal to regulate cellular functions remains unknown. Here, we show that extracellular post-abscission MBs can be internalized by interphase cells, where they reside in the cytoplasm as a membrane-bound signaling structure that we have named the MBsome. We demonstrate that MBsomes stimulate cell proliferation and that MBsome formation is a phagocytosis-like process that depends on a phosphatidylserine/integrin complex, driven by actin-rich membrane protrusions. Finally, we show that MBsomes rely on dynamic actin coats to slow lysosomal degradation and propagate their signaling function. In summary, MBsomes may sometimes serve as intracellular organelles that signal via integrin and EGFR-dependent pathways to promote cell proliferation and anchorage-independent growth and survival.
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http://dx.doi.org/10.1038/s41467-019-10871-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639393PMC
July 2019

The Robust Self-Assembling Tubular Nanostructures Formed by gp053 from Phage vB_EcoM_FV3.

Viruses 2019 01 11;11(1). Epub 2019 Jan 11.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Centre, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.

The recombinant phage tail sheath protein, gp053, from infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10⁻1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 °C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences.
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http://dx.doi.org/10.3390/v11010050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357053PMC
January 2019

Bacteriophage vB_PagS_Vid5: A Low-Temperature Siphovirus That Harbors a Cluster of Genes Involved in the Biosynthesis of Archaeosine.

Viruses 2018 10 25;10(11). Epub 2018 Oct 25.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Centre, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.

A novel lowtemperature siphovirus, vB_PagS_Vid5 (Vid5), was isolated in Lithuania using isolate for the phage propagation. The 61,437 bp genome of Vid5 has a G⁻C content of 48.8% and contains 99 probable protein encoding genes and one gene for tRNA. A comparative sequence analysis revealed that 46 out of 99 Vid5 open reading frames (ORFs) code for unique proteins that have no reliable identity to database entries. In total, 33 Vid5 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phagehost interactions, and DNA metabolism. In addition, a cluster of genes possibly involved in the biosynthesis of 7-deazaguanine derivatives was identified. Notably, one of these genes encodes a putative preQ₀/preQ₁ transporter, which has never been detected in bacteriophages to date. A proteomic analysis led to the experimental identification of 11 virion proteins, including nine that were predicted by bioinformatics approaches. Based on the phylogenetic analysis, Vid5 cannot be assigned to any genus currently recognized by ICTV, and may represent a new one within the family of .
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http://dx.doi.org/10.3390/v10110583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266253PMC
October 2018

Proteomic Identification of FLT3 and PCBP3 as Potential Prognostic Biomarkers for Pancreatic Cancer.

Anticancer Res 2018 Oct;38(10):5759-5765

Center of Abdominal Surgery, Vilnius University Hospital Santaros Klinikos, Vilnius, Lithuania.

Background/aim: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer, particularly due to its aggressive course and challenging diagnostics in early-stage disease. The aim of this study was to discover new potential prognostic and diagnostic pancreatic cancer biomarkers.

Materials And Methods: The proteomes of 37 samples from pancreatic cancer, inflammatory or healthy pancreatic tissue derived through in-depth differential proteomic analysis were compared.

Results: A set of candidate proteins as pancreatic cancer-specific diagnostic or prognostic biomarkers were identified. Survival data of patients after two-year follow-up indicated FLT3 and PCBP3 proteins as potential biomarkers for favourable pancreatic cancer prognosis. The levels of PCBP3 correlated with tumour stage and FLT3 levels, were evaluated as independent prognostic marker.

Conclusion: FLT3 and PCBP3 represent potential biomarkers for improved individualized pancreatic cancer prognosis. Moreover, FLT3 may play a role in future treatment selection.
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http://dx.doi.org/10.21873/anticanres.12914DOI Listing
October 2018

Mycobacteria produce proteins involved in biofilm formation and growth-affecting processes.

Acta Microbiol Immunol Hung 2018 Aug 19;65(3):405-418. Epub 2018 Jul 19.

1 Department of Immunology, State Research Institute, Centre for Innovative Medicine , Vilnius, Lithuania.

The aim of this study was to determine the effect of mycobacterial proteins on mycobacterial biofilm formation and growth processes. We separated growth-affecting proteins (GEPs) from wild type of Mycobacterium bovis and ATCC strain of Mycobacterium avium subsp. avium. Our results showed that these mycobacteria-secreted GEPs are involved in biofilm formation, growth stimulatory, and inhibitory processes. Our findings suggest that GEP stimulated M. avium subsp. avium growth in vitro. Stimulation process was observed in mycobacteria affected with GEP extracted from M. avium subsp. avium. We found that both GEPs inhibited the growth of the M. bovis. Optical density measurement and visual analysis confirm that GEP plays an important role in biofilm formation process. Most of M. bovis GEP are associated with the type VII secretion and general secretion pathways. Our results contribute to a better understanding of the mechanisms underlying mycobacterial biofilm formation and growth-affecting processes and better characterization of mycobacterial proteins and their functions. It is noteworthy that this finding represents the first demonstration of GEP-mediated growth effects on a solid and liquid medium.
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http://dx.doi.org/10.1556/030.65.2018.033DOI Listing
August 2018

Molecular analysis of the low-temperature Escherichia coli phage vB_EcoS_NBD2.

Arch Virol 2018 Jan 10;163(1):105-114. Epub 2017 Oct 10.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio al. 7, 10257, Vilnius, Lithuania.

A novel low-temperature Escherichia coli phage vB_EcoS_NBD2 was isolated in Lithuania from agricultural soil. With an optimum temperature for plating around 20 °C, vB_EcoS_NBD2 efficiently produced plaques on Escherichia coli NovaBlue (DE3) at a temperature range of 10-30 °C, yet failed to plate at temperatures above 35 °C. Phage vB_EcoS_NBD2 virions have a siphoviral morphology with an isometric head (65 nm in diameter), and a non-contractile flexible tail (170 nm). The 51,802-bp genome of vB_EcoS_NBD2 has a G + C content of 49.8%, and contains 87 probable protein-encoding genes as well as 1 gene for tRNA. Comparative sequence analysis revealed that 22 vB_EcoS_NBD2 ORFs encode unique proteins that have no reliable identity to database entries. Based on homology to biologically defined proteins and/or proteomics analysis, 36 vB_EcoS_NBD2 ORFs were given a putative functional annotation, including 20 genes coding for morphogenesis-related proteins and 13 associated with DNA metabolism. Phylogenetic analysis revealed that vB_EcoS_NBD2 belongs to the subfamily Tunavirinae, but cannot be assigned to any genus currently recognized by ICTV.
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http://dx.doi.org/10.1007/s00705-017-3589-5DOI Listing
January 2018

Construction, Purification, and Characterization of a Homodimeric Granulocyte Colony-Stimulating Factor.

Mol Biotechnol 2017 Oct;59(9-10):374-384

Institute of Biotechnology, Vilnius University, Sauletekio al. 7, 10257, Vilnius, Lithuania.

Granulocyte colony-stimulating factor (G-CSF) has found widespread clinical application, and modified forms with improved biopharmaceutical properties have been marketed as well. PEGylation, the covalent modification of G-CSF with polyethylene glycol (PEG), has a beneficial effect on drug properties, but there are concerns connected to the immunogenicity of PEGylated compounds and bioaccumulation of the synthetic polymer. To overcome challenges connected with chemical modifications, we developed fusion proteins composed of two G-CSF molecules connected via different peptide linkers. Three different homodimeric G-CSF proteins were purified, and their in vitro and in vivo activities were determined. A G-CSF dimer, GCSF-Lα, was constructed using an alpha-helix-forming peptide linker, and it demonstrated an extended half-life in serum with a stronger neutrophil response as compared to the monomeric G-CSF protein. The GCSF-Lα protein, therefore, might be selected for further studies as a potential drug candidate.
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http://dx.doi.org/10.1007/s12033-017-0026-7DOI Listing
October 2017

Molecular Analysis of Arthrobacter Myovirus vB_ArtM-ArV1: We Blame It on the Tail.

J Virol 2017 04 29;91(8). Epub 2017 Mar 29.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Centre, Vilnius University, Vilnius, Lithuania.

This is the first report on a myophage that infects A novel virus, vB_ArtM-ArV1 (ArV1), was isolated from soil using sp. strain 68b for phage propagation. Transmission electron microscopy showed its resemblance to members of the family : ArV1 has an isometric head (∼74 nm in diameter) and a contractile, nonflexible tail (∼192 nm). Phylogenetic and comparative sequence analyses, however, revealed that ArV1 has more genes in common with phages from the family than it does with any myovirus characterized to date. The genome of ArV1 is a linear, circularly permuted, double-stranded DNA molecule (71,200 bp) with a GC content of 61.6%. The genome includes 101 open reading frames (ORFs) yet contains no tRNA genes. More than 50% of ArV1 genes encode unique proteins that either have no reliable identity to database entries or have homologues only in phages, both sipho- and myoviruses. Using bioinformatics approaches, 13 ArV1 structural genes were identified, including those coding for head, tail, tail fiber, and baseplate proteins. A further 6 ArV1 ORFs were annotated as encoding putative structural proteins based on the results of proteomic analysis. Phylogenetic analysis based on the alignment of four conserved virion proteins revealed that myophages form a discrete clade that seems to occupy a position somewhat intermediate between myo- and siphoviruses. Thus, the data presented here will help to advance our understanding of genetic diversity and evolution of phages that constitute the order Bacteriophages, which likely originated in the early Precambrian Era, represent the most numerous population on the planet. Approximately 95% of known phages are tailed viruses that comprise three families: (with short tails), (with long noncontractile tails), and (with contractile tails). Based on the current hypothesis, myophages, which may have evolved from siphophages, are thought to have first emerged among Gram-negative bacteria, whereas they emerged only later among Gram-positive bacteria. The results of the molecular characterization of myophage vB_ArtM-ArV1 presented here conform to the aforementioned hypothesis, since, at a glance, bacteriophage vB_ArtM-ArV1 appears to be a siphovirus that possesses a seemingly functional contractile tail. Our work demonstrates that such "chimeric" myophages are of cosmopolitan nature and are likely characteristic of the ecologically important soil bacterial genus .
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http://dx.doi.org/10.1128/JVI.00023-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5375659PMC
April 2017

Tailored Dual PEGylation of Inorganic Porous Nanocarriers for Extremely Long Blood Circulation in Vivo.

ACS Appl Mater Interfaces 2016 Dec 23;8(48):32723-32731. Epub 2016 Nov 23.

Institute of Biochemistry, Vilnius University , Saulėtekio al. 7, LT-10257 Vilnius, Lithuania.

Drug carrier systems based on mesoporous inorganic nanoparticles generally face the problem of fast clearance from bloodstream thus failing in passive and active targeting to cancer tissue. To address this problem, a specific dual PEGylation (DPEG) method for mesoporous silicon (PSi) was developed and studied in vitro and in vivo. The DPEG coating changed significantly the behavior of the nanoparticles in vivo, increasing the circulation half-life from 1 to 241 min. Furthermore, accumulation of the coated particles was mainly taking place in the spleen whereas uncoated nanoparticles were rapidly deposited in the liver. The protein coronas of the particles differed considerably from each other. The uncoated particles had substantially more proteins adsorbed including liver and immune active proteins, whereas the coated particles had proteins capable of suppressing cellular uptake. These reasons along with agglomeration observed in blood circulation were concluded to cause the differences in the behavior in vivo. The biofate of the particles was monitored with magnetic resonance imaging by incorporating superparamagnetic iron oxide nanocrystals inside the pores of the particles making dynamic imaging of the particles feasible. The results of the present study pave the way for further development of the porous inorganic delivery system in the sense of active targeting as the carriers can be easily chemically modified allowing also magnetically targeted delivery and diagnostics.
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http://dx.doi.org/10.1021/acsami.6b12481DOI Listing
December 2016

Incomplete LPS Core-Specific Felix01-Like Virus vB_EcoM_VpaE1.

Viruses 2015 Nov 27;7(12):6163-81. Epub 2015 Nov 27.

Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University, Vilnius LT-08662, Lithuania.

Bacteriophages represent a valuable source for studying the mechanisms underlying virus-host interactions. A better understanding of the host-specificity of viruses at the molecular level can promote various phage applications, including bacterial diagnostics, antimicrobial therapeutics, and improve methods in molecular biology. In this study, we describe the isolation and characterization of a novel coliphage, vB_EcoM_VpaE1, which has different host specificity than its relatives. Morphology studies, coupled with the results of genomic and proteomic analyses, indicate that vB_EcoM_VpaE1 belongs to the newly proposed genus Felix01likevirus in the family Myoviridae. The genus Felix01likevirus comprises a group of highly similar phages that infect O-antigen-expressing Salmonella and Escherichia coli (E. coli) strains. Phage vB_EcoM_VpaE1 differs from the rest of Felix01-like viruses, since it infects O-antigen-deficient E. coli strains with an incomplete core lipopolysaccharide (LPS). We show that vB_EcoM_VpaE1 can infect mutants of E. coli that contain various truncations in their LPS, and can even recognize LPS that is truncated down to the inner-core oligosaccharide, showing potential for the control of rough E. coli strains, which usually emerge as resistant mutants upon infection by O-Ag-specific phages. Furthermore, VpaE1 can replicate in a wide temperature range from 9 to 49 °C, suggesting that this virus is well adapted to harsh environmental conditions. Since the structural proteins of such phages tend to be rather robust, the receptor-recognizing proteins of VpaE1 are an attractive tool for application in glycan analysis, bacterial diagnostics and antimicrobial therapeutics.
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http://dx.doi.org/10.3390/v7122932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690856PMC
November 2015

Quantitative proteomic analysis of anticancer drug RH1 resistance in liver carcinoma.

Biochim Biophys Acta 2016 Feb 17;1864(2):219-32. Epub 2015 Nov 17.

Vilnius University Institute of Biochemistry, Vilnius, Lithuania. Electronic address:

Unlabelled: Acquired resistance of tumor cells to the therapeutic treatment is a major challenge in virtually any chemotherapy. A novel anticancer agent 2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is designed to be activated by

Nad(p)h: quinone oxidoreductase, an enzyme expressed at high levels in many types of tumors. Here we investigated the potential mechanisms of acquired RH1 drug resistance in cancer cells by applying high-throughput differential quantitative proteomic analysis of the newly established RH1-resistant hepatoma cell lines. Over 400 proteins display significantly altered levels between drug-sensitive and drug-resistant cell lines. Differentially expressed proteins were clustered into more than 14 groups according to their functional annotation and protein-protein interactions. Bioinformatic analysis highlights the biological processes that might be responsible for acquired resistance to RH1. The level of several xenobiotic metabolism enzymes (total n=17) involved in RH1 activation and detoxification is decreased (Nqo1, catalase, Gst, Gsr), corresponding with the decrease in their catalytic activity. The altered biological processes also include the decrease of cell cycle positive regulators (n=15) and the increase of DNA repair proteins (n=5) as well as annexin family members (n=5) in the RH1-resistant cells. Drug-resistant hepatoma cell proteomes are also distinguished by the altered level of proteins involved in energy production and metabolism (n=55). Our data provide the basis for in-depth study of molecular mechanisms of tumor cell resistance to the promising anticancer drug RH1 enabling the further validation of protein biomarkers for the drug insusceptibility and of potential secondary pharmacological targets of RH1 resistant cells.
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http://dx.doi.org/10.1016/j.bbapap.2015.11.005DOI Listing
February 2016

Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis.

Stem Cells Int 2015 17;2015:319238. Epub 2015 Aug 17.

Department of Molecular Cell Biology, Vilnius University, LT-08662 Vilnius, Lithuania.

Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.
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http://dx.doi.org/10.1155/2015/319238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553339PMC
September 2015

Roscovitine in cancer and other diseases.

Ann Transl Med 2015 Jun;3(10):135

1 CALIPHO Group, Swiss Institute of Bioinformatics, Geneva, Switzerland ; 2 MAP Kinase Resource, Bern, Switzerland ; 3 Proteomics Centre, Vilnius University Institute of Biochemistry, Vilnius, Lithuania ; 4 Systems Biomedicine Division and Department of Virology and Immunology, Haffkine Institute for Training Research and Testing, Mumbai, India ; 5 Department of Biochemistry, Vilnius University, Vilnius, Lithuania ; 6 RTI International, Research Triangle Park, NC, USA ; 7 Lithuanian Centre of Non-Formal Youth Education Vilnius, Lithuania ; 8 National Cancer Institute, Vilnius, Lithuania ; 9 Vilnius University, Vilnius, Lithuania.

Roscovitine [CY-202, (R)-Roscovitine, Seliciclib] is a small molecule that inhibits cyclin-dependent kinases (CDKs) through direct competition at the ATP-binding site. It is a broad-range purine inhibitor, which inhibits CDK1, CDK2, CDK5 and CDK7, but is a poor inhibitor for CDK4 and CDK6. Roscovitine is widely used as a biological tool in cell cycle, cancer, apoptosis and neurobiology studies. Moreover, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, inflammation, viral infections, polycystic kidney disease and glomerulonephritis. This review focuses on the use of roscovitine in the disease model as well as clinical model research.
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http://dx.doi.org/10.3978/j.issn.2305-5839.2015.03.61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4486920PMC
June 2015

Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling.

J Cell Mol Med 2015 Jul 11;19(7):1742-55. Epub 2015 Apr 11.

Department of Molecular Cell Biology, Institute of Biochemistry, Vilnius University, Vilnius, Lithuania.

Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases (HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia (APL) treatment. Belinostat (Bel, PXD101), a hydroxamate-type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid (RA) on human promyelocytic leukaemia NB4 and HL-60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers (HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up-regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro-apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells.
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http://dx.doi.org/10.1111/jcmm.12550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511371PMC
July 2015
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