Applications of the Molecular Microbiology of Prokaryotes in Biotechnology and Biopharmacy
Applications of the Molecular Microbiology of Prokaryotes in Biotechnology and Biopharmacy
Prokaryotes represent the largest source of biotechnologically relevant products in nature. New species of prokaryotes are continuously described, and new strains of the “old” species are also continuously isolated. It is known that every new bacterial strain adds dozens of new genes to the genome of its own species, and at least some of these new genes can be exploited for the development of novel, biotechnologically relevant products.
Prokaryotes developed a range of enzymes that degrade polysaccharides, producing oligosaccharides. Different bioactivities useful for human health were reported for oligosaccharides; they are also used as prebiotics in functional food. The enzymatic production of these compounds is the most promising.
Prokaryotes also developed a whole range of structural proteins, and some of them (collagen-like proteins, for example) can be used for the construction of biomaterials with the desirable properties for regenerative medicine.
Most bacteria produce antimicrobial compounds of different nature: volatile compounds, bacteriocins, antibiotics. In practice, they can be used for both the prevention and treatment of infections. Screening for novel antimicrobial compounds is regarded to be the most promising strategy for overcoming the problem of antimicrobial resistance.
- Lukoseviciute, L., Lebedeva, J., Kuisiene, N. Diversity of polyketide synthases and nonribosomal peptide synthetases revealed through metagenomic analysis of a deep oligotrophic cave. Microbial Ecology. 2020, in press.
- Kananavičiūtė, R., Kvederavičiūtė, K., Dabkevičienė, D., Mackevičius, G., Kuisienė, N. Collagen-like sequences encoded by extremophilic and extremotolerant bacteria. Genomics. 2020, 112: 2271–2281.
- Bukelskis, D., Dabkeviciene, D., Lukoseviciute, L., Bucelis, A., Kriaučiūnas, I., Lebedeva, J., Kuisiene, N. Screening and transcriptional analysis of polyketide synthases and non-ribosomal peptide synthetases in bacterial strains from Krubera–Voronja Cave. Frontiers in Microbiology. 2019, 10: 2149.
- Kirtikliene, T., Naugzemys, D., Steponkiene, A., Bogdevic, R., Vizuje, G., Zvingila, D., Kusiene, N. Evaluation of the inter- and intrahospital spread of multidrug resistant Gram-negative bacteria in Lithuanian hospitals. Microbial Drug Resistance. 2019, 25: 326–335.
- Tratulyte, S., Miciuleviciene, J., Kuisiene, N. First genotypic characterization of toxigenic Clostridioides difficile in Lithuanian hospitals reveals the prevalence of the hypervirulent ribotype 027/ST1. European Journal of Clinical Microbiology & Infectious Diseases. 2019, 38: 1953–1959.
Biosynthesis Genes of Bioactive Compounds: Evaluation of Diversity and Expression Analysis in the Unique Environment
The identification of novel compounds with antibacterial, antifungal, anticancer, antiviral, antidiabetic, antiprotozoal and other bioactivities represents an important field of modern biomedical research. Microorganisms are the main targets in this research because of their high potential to produce these bioactive compounds. Bioactive compounds can be difficult to identify phenotypically because of a few reasons: the amount of these compounds can be beyond the detection limits; certain experimental conditions can be inappropriate for the induction of the biosynthesis of these compounds; the coding genes of bioactive compounds can be silent etc. The problem can be solved, and the real potential of bioactivity can be determined through the analysis of biosynthesis genes and not via that of the bioactive compounds themselves. The aim of the current project is to reveal the diversity and prevalence of bioactive compound biosynthesis genes in the bacteria of the deepest cave of the Earth, the Krubera-Voronja Cave. Polyketide synthase, nonribosomal peptide synthetase and bacteriocin biosynthesis genes were under investigation in this project (Lukoseviciute et al. Microbial Ecology. 2020;
Bukelskis et al. Frontiers in Microbiology. 2019).
Fig. 1. Phylogenetic diversity of the adenylation domains of the nonribosomal peptide synthetases in the metagenome of Krubera-Voronja Cave.
Identification, Expression and Characterization of Bacterial Collagen-Like Proteins
During the last decade, a large number of collagen-like proteins have been identified in bacteria mainly through an in silico analysis. Only a few bacterial collagen-like proteins have been expressed in Escherichia coli. It was shown that these recombinant bacterial proteins adopt a classical triple-helix conformation and exhibit high thermal stability. The amino acid composition of bacterial collagen-like proteins varies from species to species, and from protein to protein, conferring the different characteristics to these proteins. Collagen-like proteins can be produced in large quantities by recombinant methods, and the construction of proteins with the desirable characteristics can also be carried out. Therefore, bacterial collagen-like proteins represent an excellent source for the design of new biomaterials with the desirable structural properties and functions. The identification, expression and characterization of bacterial collagen-like proteins represent a highly attractive and important area of research work in the fields of regenerative medicine and biotechnology (Kananavičiūtė et. al. Genomics. 2020).
Fig. 2. Collagen-like sequence proteins having BclA_C (A) and Exospore_TM domains (B).
Molecular Epidemiology of Pathogenic Bacteria in Lithuanian Healthcare Institutions
Bacterial resistance to antimicrobial agents plays an important role in healthcare institutions nowadays. Spread of multidrug resistant bacteria can occur during inter- and intra-hospital transmissions among patients and hospital personnel. One of the highest rates of resistance in healthcare institutions is observed in Acinetobacter spp. isolates, which causes outbreaks around the world and is highly adaptable to changes both in the environment and in the use of antibiotics. These characteristics lead to a high rate of occurrence of multidrug-resistant Acinetobacter spp. cases in the environment.
Methods of molecular epidemiology, such as virulence factors determination, resistance genes distribution and genotyping, are used to better understand the antimicrobial resistance patterns of Acinetobacter spp. and can be used to strengthen the control of multidrug resistant infections in healthcare institutions and to prevent potential outbreaks of this pathogen in the future
(Kirtikliene et al. Microbial Drug Resistance. 2019;
Tratulyte et al. European Journal of Clinical Microbiology & Infectious Diseases. 2019).
Fig. 3. Dendrogram for the BOX-PCR pattern of Acinetobacter spp. isolates resistant to antibiotics.
Applied and Environmental Microbiology
Applied and Environmental Microbiology
Although the potential for microbial degradation is ubiquitous, many organic contaminants are not or often only poorly transformed in natural environmental conditions, thus, organic, and other waste treatment and recycling is an important topic. Therefore, the enhancement of natural microbiological degradative activities at contaminated sites is one of the challenges of the present research group. Through exploitation of advances conventional and molecular biology techniques, search, identification, and characterization of microbial enzymes active towards fatty substances or aromatic compounds are done. Microbial enzymes, especially those exerting activity against ester bonds have a broad range of applications in modern biotechnology. Lipolytic enzymes are among the most industrially relevant and widely used in biocatalysis, both at academic and industrial levels due to their immense versatility regarding catalytic behaviour and great stability in different reaction media. Nevertheless, for the industrial implementations, immobilized enzymes are preferred over their soluble forms. If the enzyme is immobilized properly, it can be considered as a special type of formulation of its properties such as activity, stability, selectivity, purity, and others. Therefore, it is important to examine new types of immobilization sorbents. Ecologically inspired method of immobilization of lipolytic enzymes on industrial waste products as carriers are developed by the group.
Another emerging topic is alternative antibacterial compounds such as bacterial ribosomally synthesized peptides with antibacterial activity (bacteriocins). These natural coumpounds have considerable diversity with respect to their size, structure, mechanism of action, inhibitory spectrum, immunity mechanisms and targeted receptors. In the era of antibiotic resistance, bacteriocins are suggested as a potential alternative to antibiotics in clinics and as food preservatives against spoilage and pathogenic microorganisms.
The research group is also participating in a research regarding safe bacterial biofilm control method development for European Space Agency (ESA). In collaboration with the Institute of Photonics and Nanotechnology, Faculty of Physics (Vilnius University), a novel natural photosensitizers-based antimicrobial photoinactivation (API) technology that is safe for the use in the confined, closed-loop systems such as spacecraft is being developed.
Yeast β-glucans, a diverse group of polysaccharides, exhibiting immunostimulating activity, and algal pigments, which, besides their health benefits, have great commercial value in nutraceutical, cosmetic and pharmaceutical industries, are among the research group’s topics as well.
Enzymes and antimicrobial compounds and systems that are analysed by our research group, are attractive both biotechnologically and in basic research. Some of the competences are achieved not only by introducing publications but also by participating in scientific projects co-financed by ESA, EU funds and collaborating with the regional waste treatment company for the pilot study of biogas production from municipal waste.
Some of our PhD students defended doctoral theses describing the identification of new bacterial lipolytic enzymes and post-translationally modified bacteriocins.
- Buchovec, I., Gricajeva, A., Kalėdienė, L., Vitta, P. Antimicrobial photoinactivation approach based on natural agents for control of bacteria biofilms in spacecraft. International Journal of Molecular Sciences. 2020, 21 (18): 6932.
- Kaunietis, A., Buivydas, A., Citavicius, D., Kuipers, O. Heterologous biosynthesis and characterization of a glycocin from a thermophilic bacterium. Nature Communications. 2019, 10, Article No: 1115.
- Gricajeva, A., Bikute, I., Kalediene, L. Atypical organic-solvent tolerant bacterial hormone sensitive lipase-like homologue EstAG1 from Staphylococcus saprophyticus AG1: Synthesis and characterization. International Journal of Biological Macromolecules. 2019, 130: 253–265.
- Vaičikauskaitė, M., Ger, M., Valius, M., Maneikis, A., Lastauskienė, E., Kalėdienė, L., Kaunietis, A. Geobacillin 26 - high molecular weight bacteriocin from a thermophilic bacterium. International Journal of Biological Macromolecules. 2019, 141: 333–344.
Microbial biofilms are widespread in the environment and form on biotic and abiotic surfaces if constant moisture is present. Biofilms play an important role in human infections and endanger material integrity not only in confined facilities such as hospitals and food settings on Earth but also in spacecraft, which is inhabited by a changing microbial consortium mostly originating from life-supporting devices, equipment collected in pre-flight conditions and crewmembers. Resilient biofilms pose a higher risk to crewmembers’ health and the material integrity of the spacecraft than planktonic cells. Moreover, biofilms in space conditions are characterized by faster formation and acquisition of resistance to chemical and physical control methods than under the same conditions on Earth, making most decontamination methods unsafe. Thus, biofilm control methods that are safe for confined, closed-loop systems such as spacecraft are of high demand. Visible-light irradiation technology - antimicrobial photoinactivation (API) based on natural PSs such as riboflavin (RF) and chlorophyllin - are being developed by the research group, and the technology shows promising results for its use in the mentioned area (confined, closed-loop facilities such as spacecraft and others). API belongs to a multitarget process; therefore, bacteria do not develop resistance, moreover, the process exhibits a very rapid microbial killing.
Fig. 1. Example of natural PSs-based API mechanism; upon activation by light, RF is excited to a singlet state of higher energy (1RF*), followed by intersystem crossing to an excited triplet state (3RF*). 3RF* can be involved in a photosensitized oxidation process of two types. Type I: RF transfers the energy to a substrate (S) and generates RF* free radicals interacting with molecular oxygen in the ground state (GS) to yield oxidation products. Type II: RF transfers the energy to molecular oxygen in the ground state to generate the more reactive 1O2. The process leads to the final products harmful to microbes.
Biocatalysts exerting activity against ester bonds have a broad range of applications in modern biotechnology. Some of the most industrially relevant enzymes of this type are lipolytic. A novel bacterial hormone-sensitive lipase-like (bHSL) family homologue, designated EstAG1, was discovered by mining gDNA of bacteria isolated from fat contaminated soil. EstAG1 was hyperactivated by organic solvents implicating that it could be an industrially applicable enzyme for the organic synthesis of valuable products such as biodiesel, flavour and aroma esters etc. Based on low EstAG1 amino acid sequence identities to closest homologues, by unique catalytic amino acid peculiarities in the sequence and phylogenetic analysis the enzyme belongs to a new family of bacterial lipolytic enzymes.
Fig. 2. Application possibilities of bacterial lipolytic enzymes.
The genome of the thermophilic bacterium, Aeribacillus pallidus 8, encodes the bacteriocin pallidocin belonging to a small class of glycocins and is posttranslationally modified by S-linked glucose on a specific Cys residue. In this study, the pallidocin biosynthetic machinery was expressed in E. coli to achieve its biosynthesis and modification. The characterized biosynthetic machinery was employed to produce two other glycopeptides Hyp1 and Hyp2. Heterologous expression of a glycocin biosynthetic gene cluster with S-glycosyltransferase provides a good tool for production of hypothetical glycocins encoded by various bacterial genomes and allows rapid in vivo screening.
Moreover, geobacillin 26 from Geobacillus stearothermophilus 15 was thoroughly investigated. Our study suggests that this bacteriocin is not a cell wall hydrolyser as most of high molecular weight bacteriocins and has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before.
Fig. 3. Proposed structure of pallidocin. The α-helical structure shown in blue, coil structure in purple.
Fig. 4. Antibacterial activity of Geo26-His. NB-agar medium inoculated with sensitive strain P. genomospecies1 NUB36187.
Functioning and Epigenetic Mechanisms of Human Stem Cells
Functioning and Epigenetic Mechanisms of Human Stem Cells
Epigenetic regulation, when influenced by DNA and histone modifications as well as microRNA expression, causes variances in gene expression and cell phenotype. It has a great influence on the development and functioning of stem cells. These changes could cause cancer and other diseases. An understanding of regulatory and epigenetic molecular mechanisms of stem and cancer cell functioning is the main interest for developing new tools in regenerative medicine as well as novel epigenetic therapeutics. Many factors influence the regulation of stem cell, cancer stem cell and cancer cell proliferation, differentiation and apoptosis, including intracellular signalling molecules, transcription factors and epigenetic events. However, the epigenetic and other regulatory mechanisms, governing stem and cancer cell identity, as well as fate determination are still not well-understood.
Human amniotic fluid-derived stem cells (AFSCs) are a valuable, easily obtainable alternative of stem cells for cell therapy and regenerative medicine. Although this field has gained much research attention, differentiation capacity of AFSCs and epigenetic regulation leading to AFSCs fate determination are still poorly characterized. Therefore, in our study we investigated the differentiation potential and assessed epigenetic factors involved in tissue-specific differentiation.
- Zentelytė, A., Gasiūnienė, M., Treigytė, G., Baronaitė, S., Savickienė, J., Borutinskaitė, V., Navakauskienė, R. Epigenetic regulation of amniotic fluid mesenchymal stem cell differentiation to the mesodermal lineages at normal and fetus-diseased gestation. Journal of Cellular Biochemistry. 2020 Feb, 121(2): 1811–1822. doi: 10.1002/jcb.29416.
- Gasiūnienė, M., Valatkaitė, E., Navakauskienė, R. Long-term cultivation of human amniotic fluid stem cells: The impact on proliferative capacity and differentiation potential. Journal of Cellular Biochemistry. 2020 Jan 3. doi: 10.1002/jcb.29623.
- Gasiūnienė, M., Zentelytė, A., Treigytė, G., Baronaitė, S., Savickienė, J., Utkus, A., Navakauskienė, R. Epigenetic alterations in amniotic fluid mesenchymal stem cells derived from normal and fetus-affected gestations: a focus on myogenic and neural differentiations. Cell Biology International. 2019 Mar, 43(3): 299–312. doi: 10.1002/cbin.11099.
- Gasiūnienė, M., Zentelytė, A., Wojtas, B., Baronaitė, S., Krasovskaja, N., Savickienė, J., Gielniewski, B., Kaminska, B., Utkus, A., Navakauskienė, R. DNMT inhibitors effectively induce gene expression changes suggestive of cardiomyogenic differentiation of human amniotic fluid-derived mesenchymal stem cells via chromatin remodeling. Journal Tissue Engineering and Regenerative Medicine. 2019, 13: 469–481. doi: 10.1002/term.2800.
The Impact of AFSCs on Long-Term Cultivation Capacity and Differentiation Potential
It is crucial to investigate whether the main characteristics of SCs are stably maintained during the long-term cultivation in vitro and whether the passage number of AFSCs culture can have an impact on the future clinical applications. Therefore, we examined the proliferation, differentiation, death and senescence of amniotic-fluid derived stem cells during the long-term culturing in vitro. AFSCs were expanded up to 42 passages and maintained their stemness and mesenchymal characteristics. Alterations in morphology, the expression of pluripotency genes and cell surface markers concomitant with senescence initiation as well as reduced cardiomyogenic differentiation potential were detected at the late passages. These results provide useful insights into the potential use of AFSCs for bio-banking and universal applications requiring large amounts of cells or repeated infusions (Gasiūnienė, Valatkaitė & Navakauskienė, 2020).
AFSCs morphology from different passages of long-term cultivation
Metabolic and Neurogenic Potential of AFSCs from Normal vs Foetus Affected Gestations
Although AFSCs are widely researched, their analysis mainly involves SCs obtained from normal foetus unaffected gestations. However, in clinical setting, the knowledge about AFSCs from normal gestations would be poorly translational, as AFSCs from normal and foetus diseased gestations may differ in their differentiation and metabolic potential. Therefore, in this study the metabolic, neurogenic and neurotrophic potential of AFSCs, obtained from foetus affected gestations with polyhydramnios, in comparison with foetus unaffected gestations, were investigated. Results demonstrated that these cells are similar in gene expression levels of stemness markers. However, they do differ in expression of certain cell surface markers. In addition, AFSCs from “Normal” and “Pathology” groups were found to be different in oxidative phosphorylation rate, as well as in the level of ATP and reactive oxygen species production. AFSCs from normal gestations were found to be more prone to neurogenic differentiation. Overall, these observations provide supplementary insights into the neurogenic potential of AFSCs obtained from foetus unaffected vs foetus affected gestations
(VU funded research No. MSF-LMT-3/2020).
Morphology of AFSCs obtained from healthy and foetus diseased gestations upon neural differentiation induction
Genotoxicity of Anthropogenic and Natural Factors
Genotoxicity of Anthropogenic and Natural Factors
Bioactive molecules from natural sources play an important role in the development of nutraceuticals and pharmaceuticals. Nowadays, bioactive natural products are the sources for >80% of active compounds in foods and >30% of drugs. However, the plants may also produce natural toxic, mutagenic and/or carcinogenic compounds. The increasing demand for plant-derived natural products in cosmetics, medicine and products from the food industry requires a more systematic and comprehensive evaluation of their benefits and possible adverse effects, e.g. such as genotoxicity. However, until now, only a small part of plant species has been screened for their biological activities and genotoxic properties. There is a strong need for a more systematic and comprehensive evaluation of the phytochemical composition and genotoxicity of plant extracts using various genotoxicity assays covering different DNA damage endpoints.
Recent studies have confirmed the usefulness of biomonitoring chromosome damage in groups exposed to genotoxic agents by finding an increased risk of cancer in subjects with high levels of chromosome aberrations and thus proving the chromosome aberration assay as a reliable indicator of cancer risk. UV lasers have provided completely new possibilities for surgery and therapeutic treatments and are increasingly applied in medicine. A number of studies performed in the field of laser treatment and surgery have proved that there are femtosecond laser pulses that have advantages as compared with the longer duration pulses. Although the employment of femtosecond lasers as medical tools opens new possibilities for eye and skin treatment and surgery, the impact of their use on genetic material is not yet fully understood. Such knowledge is especially relevant to ultrashort UV pulses, because radiation in the UV range has the greatest DNA-damaging potential.
We use different methods of genotoxicity assessment (cytogenetic tests, the Ames test, the Comet assay) to investigate the genotoxic action of anthropogenic and natural factors. In collaboration with a large international group of researchers, we are studying the effects of ionizing radiation on human chromosomes. Our former and recent study established a link between the incidence of chromosome aberrations and the risk of cancer. In collaboration with industrial partners (Light Conversion Ltd., CC Akių Gydytojų Praktika), we studied the possible harmful impact of the brand-new 206 nm femtosecond laser Pharos on bone marrow, skin and corneal cells. Our investigations demonstrated that the DNA-damaging effect of laser irradiation was mostly dependent on the wavelength, but the influence of such a parameter as beam delivery to the target was also revealed.
- Slapšytė, G., Dedonytė, V., Adomėnienė, A., Lazutka, J. R., Kazlauskaitė, J., Ragažinskienė, O., Venskutonis, P. R. Genotoxic properties of Betonica officinalis, Gratiola officinalis, Vincetoxicum luteum and Vincetoxicum hirundinaria extracts. Food Chem. Toxicol. 2019, 134: 110815.
- Morkunas, V., Urbonaite, G., Gabryte-Butkiene, E., Sobutas, S., Vengris, M., Danielius, R., Ruksenas, O. DNA-damaging effect of different wavelength (206 and 257 nm) femtosecond laser pulses. Photobiomodulation, Photomedicine, and Laser Surgery. 2019, 37: 254–261.
- Pukalskienė, M., Slapšytė, G., Dedonytė, V., Lazutka, J. R., Mierauskienė, J., Venskutonis, P. R. Genotoxicity and antioxidant activity of five Agrimonia and Filipendula species plant extracts evaluated by comet and micronucleus assays in human lymphocytes and Ames Salmonella/microsome test. Food Chem. Toxicol. 2018, 113: 303–313.
Molecular Mechanisms of Cell Death and Survival
Molecular Mechanisms of Cell Death and Survival
Acquired chemoresistance is a major limitation for successful anti-cancer therapy. Some mechanisms of cell chemoresistance are well known: alterations in drug transport and metabolism, modification of drug targets, activation of DNA repair or changes in apoptosis induction. Deeper understanding of chemoresistant cell physiology, in particular cell survival signalling, autophagy and cell death pathways, suggests new possible targets to overcome chemoresistance. We focus our research on molecular mechanisms responsible for cellular chemoresistance of colorectal cancer cells, in particular on the alterations in pathways of cell survival signalling and autophagy. The latter may have both cancer-promoting and cancer-suppressing effects. One focus of our group is to reveal the changes in autophagic machinery in chemoresistant vs chemosensitive cells.
Inflammation and antitumor immunity are important determinants of colorectal cancer progression; it is mediated by cytokine signalling. We have demonstrated that cell stimulation with exogenous interleukin-1 alpha (IL-1α) increased 5-fluorouracil (5-FU) cytotoxicity in both chemosensitive and chemoresistant colorectal cancer cell lines . It was the result of increased cell death, and not of cell cycle arrest. The combined exogenous (IL-1α) and 5-FU treatment changed the expression of cell adhesion molecules that may have an impact on adhesion-dependent chemoresistance and metastatic potential of cells.
We have determined that interleukin-8 (IL-8) and its receptor CXCR2 are upregulated in the chemoresistant colorectal cancer cells. However, chemoresistant cells remained sensitive to blockade of the CXCR2 pathway; it reduced the cell number. IL-1α alpha was found to stimulate production of IL-8 .
Notch and Wnt signalling regulate differentiation of intestinal cells and alterations in these pathways may lead to carcinogenesis. We have determined that Notch and Wnt signalling is upregulated in chemoresistant colorectal cancer cells . The roles of Notch and Wnt pathways for cell survival after 5-FU and oxaliplatin (OxaPt) treatment were different: in the case of 5-FU treatment, Wnt pathway was cytoprotective and supported chemoresistance, while inhibition of either Notch or Wnt pathways increased the cytotoxicity of OxaPt.
In 2020, the researchers of our group together with the scientists from the Institute of Biochemistry participated in two projects: 1) evaluation of cytotoxicity of aromatic nitrocompounds and N-oxides (DOTSUT-34/09.33-LMT-K712-01-0058), 2) elucidation of the mechanisms of bacteriophage-derived nanotube entry to colorectal cancer cells (S-SEN 20-4). We have also implemented a project (MSF-JM-2/2020) with researchers from the Institute of Biomedical Sciences of the Faculty of Medicine, dedicated to molecular mechanisms of pathogenesis of rare genetic diseases. During the year 2020, an invited review article concerning the changes in Notch signalling pathway in endometrial cancer was prepared .
- Grigaitis, P., Jonusiene, V., Zitkute, V., Dapkunas, J., Dabkeviciene, D., Sasnauskiene A. Exogenous interleukin-1α signaling negatively impacts acquired chemoresistance and alters cell adhesion molecule expression pattern in colorectal carcinoma cells HCT116. Cytokine. 2019, 114: 38–46.
- Dabkeviciene, D., Jonusiene, V., Zitkute, V., Zalyte, E., Grigaitis, P., Kirveliene, V., Sasnauskiene, A. The role of interleukin-8 (CXCL8) and CXCR2 in acquired chemoresistance of human colorectal carcinoma cells HCT116. Med Oncol. 2015, 32(12): 258.
- Kukcinaviciute, E., Jonusiene, V., Sasnauskiene, A., Dabkeviciene, D., Eidenaite, E., Laurinavicius, A. Significance of Notch and Wnt signaling for chemoresistance of colorectal cancer cells HCT116. J Cell Biochem. 2018, 119(7): 5913–5920.
- Jonusiene, V., Sasnauskiene, A. Notch and endometrial cancer. Adv Exp Med Biol. 2021, 1287: 47–57.
Plant Polymorphism, Genome Stability and Its Changing Factors
Plant Polymorphism, Genome Stability and Its Changing Factors
Plants as model systems are widely used in molecular-genetic, developmental and environmental studies. The progress of molecular marker techniques and the sequencing of the Arabidopsis genome began the era of plant genomics. However, little is known about the mechanisms that help plants survive and adapt to local and global environmental changes, and how these factors affect the plant’s genome and gene expression. Many adaptation and developmental features have their chemical expressions related to the production of phytohormones, secondary metabolites and signalling molecules. However, chemical changes in the cell and whole organism are controlled by the structure and activity of the genome, its genes and epigenetic changes. Comprehensive studies of plant adaptation strategies should be carried out at the cell, individual and population level. DNA analysis reveals the relationship between the plant genome structure and its functioning as well as the survival and adaptation strategies of the plants. On the other hand, plants have unique developmental and reproductive features; they maintain a close relationship with the soil and its microflora. Therefore, they are often used as a test system to assess the ecological status of the environment, for phytoremediation and as producers of various metabolites.
We studied the natural and induced plant genome variability at the cell, organism and population levels using molecular, biochemical, statistical and bioinformatical methods. One of the traditional trends in our laboratory is studies of barley developmental mutants. Study of lines derived from different cross-combinations confirmed the triggering effect of tweaky mutations on the induction of genetic instability, which may occur due to pleiotropic auxin action in tw mutants on the expression of genes related to developmental processes . Another aspect of our investigation concerns plant evolution and ecology with particular interest on the phenomena of invasiveness and hybridization. Our study confirmed the hybridogenic origin of distinctive Batrachium genotypes  and the role of the multiple introductions on the invasiveness of Bunias orientalis in two climatically different zones . Some of our studies [2–5] were carried out in collaboration with colleagues from other Lithuanian research institutions and abroad. Our team has also a lot of experience in the field of genotoxicity studies on soil contamination by hazardous environmental pollutants using Tradescantia clone #4430 and other test-systems .
- Šiukšta, R., Vaitkūnienė, V., Rančelis, V. Is auxin involved in the induction of genetic instability in barley homeotic double mutants? Planta. 2018, 247(2): 483–498.
- Butkuvienė, J., Sinkevičienė, Z., Naugžemys, D., Žvingila, D., Skridaila, A., Bobrov A. A. Genetic diversity of aquatic Ranunculus (Batrachium, Ranunculaceae) in one river basin caused by hybridization. Plants. 2020, 9: 1455.
- Vaitkūnienė, V., Šiukšta, R., Leistrumaitė, A., Rančelis, V. Prospective use of barley spike/flower homeotic single and double mutants for ornamental purposes. Euphytica. 2019, 215: 127.
- Patamsytė, J., Naugžemys, D., Čėsnienė, T., Kleizaitė, V., Demina, O. N., Mikhailova, S. I., Agafonov, V. A., Žvingila, D. Evaluation and comparison of the genetic structure of Bunias orientalis populations in their native range and two non-native ranges. Plant Ecology. 2018, 219: 101–114.
- Šiukšta, R., Bondzinskaitė, S., Kleizaitė, V., Žvingila, D., Taraškevičius, R., Mockeliūnas, L., Stapulionytė, A., Mak, K., Čėsnienė, T. Response of Tradescantia plants to oxidative stress induced by heavy metal pollution of soils from industrial areas. Environmental Science and Pollution Research. 2019, 26: 44–61.
Genetic Diversity of Aquatic Ranunculus (Batrachium, Ranunculaceae) in One River Basin Caused by Hybridization
Aquatic Ranunculus (sect. Batrachium) include both homophyllous and heterophyllous plants. The development of floating leaves may be induced by genetic mechanisms or/and environmental conditions, and this fact complicates the morphologically based identification of species. DNA-based studies provide the opportunity to expand the knowledge of this complicated group. We studied heterophyllous Ranunculus with well-developed capillary and intermediate leaves and visually homophyllous plants with capillary leaves with the aim to evaluate their genetic polymorphism and taxonomic status: whether the plants with well-developed and weakly expressed intermediate leaves belong to different forms (taxa) or they just express morphological variation of one or two taxa in a specific, highly variable river environment. The molecular analysis did not reveal any inter simple sequence repeat (ISSR) polymorphism associated with the development of intermediate leaves. Analysis of nuclear ribosomal internal transcribed spacers ITS1–2 sequences revealed several ribotypes, which indicated the genetic heterogeneity of the plants studied and indirectly confirmed the hybrid origin of some of them. Hybrid sterile plants between R. circinatus and R. penicillatus were discovered in the Skroblus River; however, identification of the parental species was impeded by the polymorphism detected. For this reason, cytological studies were performed and allowed confirmation of this hybrid (Butkuvienė et al. Plants. 2020, 9: 1455).
Fig. 1. Principal coordinate analysis of supposed parental species (R. circinatus (orange) and R. penicillatus (blue) and Ranunculus hybrid from the Skroblus River (grey).
Genetic Structure of Lithuanian Nuphar lutea River Populations
Currently, in Europe, increasing attention is being paid to the genetic diversity of aquatic macrophytes. Insufficient information exists about the river plants of the Baltic States. Our study aimed to evaluate the genetic diversity of Nuphar lutea individuals growing in the Lithuanian watercourses. The populations were studied in the river catchments of Lithuania: the Nemunas, the Venta and the Lielupė. The genetic structure of the populations was evaluated at microsatellite loci. The population genetic data of N. lutea were analysed by multiple tests, including hierarchical analysis of molecular variance (AMOVA), principal coordinate analysis (PCoA) and the Mantel test. The observed (HO) and expected (HE) heterozygosity values per population were at the corresponding intervals: 0.242–0.655 and 0.503–0.759. Our study revealed significant differentiation among populations (FST=0.162; p<0.001) and a lack of correlation between genetic and geographical distances; this outcome is in agreement with increased inbreeding in populations and implies limited gene flow among subcatchments. Our results also indicate that land-use type in the areas surrounding the river may have an effect on the genetic diversity pattern of N. lutea populations. (Vyšniauskienė et al. Aquat. Bot. 2020, 131: 103173).
Fig. 2. Genetic differentiation between Lithuanian populations of Nuphar lutea based on nuclear microsatellite data and revealed using Arlequin v220.127.116.11.
Single-Cell Transcriptomics and Genomics
Single-Cell Transcriptomics and Genomics
Recent advances in high-throughput single technologies and computational methods have opened new horizons for biological and biomedical sciences. Just over the last few years, we have witnessed significant efforts to develop various analytical techniques to isolate, amplify and sequence the genetic material of individual cells. As the applications of single-cell sequencing continue to expand to all branches of life sciences there is a growing need for technological solutions that can deliver increased molecular sensitivity and reaction throughput at a reduced cost. Droplet microfluidics, a technology that enables pico- and nano-litre volume reactions, plays a major role in this endeavour. Our group are experts in droplet microfluidics technology for single-cell and many biological applications. Our group is pursuing research in cancer and immune system biology, aiming at better understanding of the genetic programs that drive tumour heterogeneity, progression and immune response.
Fig 1. The principle of inDrops technique. Digital micrographs of cell encapsulation together with hydrogel beads and reagents. Scale bars, 100 μm. Cell loading into droplets with hydrogel beads and assay reagents occurs at the flow-focusing junction. Hydrogel bead ferries ssDNA primers attached to hydrogel polymer mesh via UV light-sensitive bond
In collaboration with Harvard University, our group has pioneered the droplet microfluidics technique inDrops (indexing Drops) for barcoding the transcriptome of individual cells (Klein, Cell, 2015). Since then, the technique has triggered immense attention among many scientists across different disciplines. We are applying inDrops and other techniques to better understand the gene expression programs that drive the development of complex diseases (e.g. tumours) and how the immune system responds. In collaboration with the Harvard Medical School (Dr A. Klein), we have studied the pluripotency of mouse embryonic cells , the T-cell activation in tumours  and the osteoblast role in lung adenocarcinoma , all single-cell level. In collaboration with Memorial Sloan Kettering Cancer Center and Columbia University (Dr. D. Pe’er), we have also shown that the T-cell exhibits a continuum of activated states to fight breast cancer  and, in a separate study, we have developed computational tools for recovering gene dropouts that are persistent in scRNA-Seq data .
- Klein, M., Mazutis, L., Akartuna, I., Tallapragada, N., Veres, A., Li, V., Peshkin, L., Weitz, D. and Kirschner, M. Droplet barcoding for single cell transcriptomics applied to embryonic stem cells. Cell. 2015, 161(5): 1187–1201.
- Zemmour, D., Zilionis, R., Kiner, E., Klein, A. M., Mathis, D., Benoist, C. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nat Immunol. 2018, Mar; 19(3): 291–301.
- Engblom, C., Pfirschke, C., Zilionis, R., Da Silva Martins, J., Bos, S. A., Courties, G., Rickelt, S., Severe, N., Baryawno, N., Faget, J., Savova, V., Zemmour, D., Kline, J., Siwicki, M., Garris, C., Pucci, F., Liao, H. W., Lin, Y. J., Newton, A., Yaghi, O. K., Iwamoto, Y., Tricot, B., Wojtkiewicz, G. R., Nahrendorf, M., Cortez-Retamozo, V., Meylan, E., Hynes, R. O., Demay, M., Klein, A., Bredella, M. A., Scadden, D. T., Weissleder, R., Pittet, M. J. Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils. Science. 2017, 12 01: 358(6367).
- Azizi, E., Carr, A., Plitas, G., Cornish, E. A., Konopacki, C., Prabhakaran, S., Nainys, J., Wu, K., Kiseliovas, V., Setty, M., Choi, K., Dao, P., Mazutis, L., Rudensky, Y. A. & Peer, D. Single-cell map of diverse immune phenotypes in the breast tumor microenvironment. Cell. August 23, 2018, 174: 1–16.
- van Dijk, D., Sharma, R., Nainys, J., Yim, K., Kathail, P., Carr, A., Burdziak, C., Moon, R. K., Chaffer, C., Pattabiraman, D., Bierie, B., Mazutis, L., Wolf, G., Krishnaswamy, S., Peer, D. Recovering gene interactions from single-cell data using data diffusion. Cell. July 29, 2018, 174: 716–729.
Screening and Isolation of B-cells Producing Therapeutic Antibodies
Monoclonal antibodies constitute important tools for scientific research and are the basis of numerous successful therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and, due to fusion inefficiency and display bias, respectively, are unable to sample the entire antibody repertoire. We are working on a new platform to rapidly generate recombinant monoclonal antibodies. We use droplet microfluidics and a bead-based binding assay to directly identify and isolate individual rare cells that secrete target-binding antibody from a primary cell population with high throughput, screening more than one million cells per day. We then perform RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences. We verify target-specific binding using ELISA. Our platform can facilitate rapid screening of an animal’s IgG-secreting cell repertoire to generate antigen specific recombinant antibodies and can also be adapted to isolate cells based on virtually any secreted product.
Transcriptional Profiling of Tumour
Tissue homeostasis is maintained by stem cells, whereas damaged tissues are repaired by facultative progenitors that are activated upon injury. The role of developmental plasticity in tumour progression and metastasis remains poorly understood and the extent to which tumour cells subvert regenerative processes during metastatic progression is unknown. In collaboration with biomedical and computational scientists, we applied inDrops technology to explore tumour cell heterogeneity through in lung cancer metastasis and to assess tumour cell plasticity. Using patient tumours as well as a mouse model of lung cancer metastasis, we identified the regenerative cell types and lineage promiscuity in untreated primary tumours and revealed a range of embryonic lung morphogenic states in metastases. We demonstrate an unexpected, developmental stage-specific differential sensitivity to natural killer cells that shapes the phenotypic landscape of latent metastasis-initiating cells.
Hydrogel Capsules for Single-Cell Multi-Step Processing
Droplet microfluidics technology provides a powerful approach to isolate and process millions of single cells simultaneously. However, multi-step reaction, including molecular biology and cell-based phenotypic screening assays, cannot be easily adapted to droplet format. To circumvent these limitations, we combined advantages offered by droplet-based and hydrogel-based systems to create capsules containing a thin, semi-permeable shell. The shell acts as a passive sieve retaining encapsulated, large molecular weight compounds while allowing smaller molecules (such as proteins) to diffuse through. We used an aqueous two-phase system (ATPS) composed of dextran and acrylate-modified polyethylene glycol to generate the biocompatible hydrogel particles and showcased a few examples of sequential reactions on encapsulated species. Specifically, we compared genome amplification reaction efficiency on Gram-negative and Gram-positive bacterial cells and found that the DNA amplification yields tend to be higher in the capsule-based system. Capsules readily sustained multiple pipetting steps when performing complex biochemical reactions, and, in contrast to solid-hydrogel beads, retained a significantly larger fraction of encapsulated bacteria.
Stem Cell Technologies for Bone Tissue Engineering
Stem Cell Technologies for Bone Tissue Engineering
Tissue engineering or the fabrication of artificial tissue is a promising field of regenerative medicine, which meets a lot of scientific and technological challenges. Artificial tissues could be produced by using different biofabrication techniques, which depend on the specifics of the tissue being created.
Fabrication of an artificial biocompatible, osteoconductive and osteoinductive bone graft using special scaffold still remains a major issue in bone tissue engineering approaches. It is obvious that the newly produced bone graft should not only stimulate cells to regenerate damaged bone tissue, it also should mimic patient-specific bone defect morphology and should be low in price and high in production speed. One of the most important factors for constructing an artificial bone tissue is the morphology of the bone scaffold. It is known that the material and topography of the scaffolds have an impact on cell focal adhesions (FA) formation, which in turn modifies cell shape and morphology, leading to various signalling pathways activation and eventually influencing cell adhesion, proliferation, and differentiation.
We have previously demonstrated that low-cost 3D printed polylactic acid (PLA) macro structures (larger than cell diameter) without additional surface modifications could promote spontaneous stem cell osteogenic differentiation. Currently, we aimed to improve the osteoinductivity of these scaffolds and attenuate the negative effects of PLA degradation by creating PLA composites with 10% of hydroxyapatite (HA) or 10% of bioglass (BG) filaments, which were further used for scaffold production.
The tasks of our group are (1) to develop the best way of selected composite material microstructurization, (2) to compare the physical and osteoinductive properties of 3D printed PLA+ hydroxyapatite (HA), PLA+bioglass (BG), (3) to elucidate the fate of cells grown on these scaffolds and (4) to evaluate the influence of biodecoration effect on cell differentiation.
We have analysed various fused filament fabrication (FFF) 3D-printed PLA scaffold modifications’ impact on the fate of rat dental pulp stem cells (DPSC). Primary rat DPSCs were selected as a model, which helped to understand the cellular response to different substrate modifications. PLA scaffolds were modified by altering their chemical composition (PLA composites with 10% of HA or PLA with 10% of BG), and surface coating with proteins to determine the impact of each substrate modification on cell function and properties, focusing on cell osteogenesis processes.
- Alksne, M., Kalvaityte, M., Simoliunas, E., Rinkunaite, I., Gendviliene, I., Locs, J., Rutkūnas, V., Bukelskienė, V. In vitro comparison of 3D printed polylactic acid/hydroxyapatite and polylactic acid/bioglass composite scaffolds: Insights into materials for bone regeneration. J Mech Behav Biomed Mater. 2020, 104: 103641.
- Gendviliene, I., Simoliunas, E., Rekstyte, S., Malinauskas, M., Zaleckas, L., Jegelevicius, D., Bukelskiene, V., Rutkūnas, V. Assessment of the morphology and dimensional accuracy of 3D printed PLA and PLA/HAp scaffolds. J Mech Behav Biomed Mater. 2020, 104:103616.
- Grigaleviciute, G., Baltriukiene, D., Bukelskiene, V., Malinauskas, M. Biocompatibility evaluation and enhancement of elastomeric coatings made using table-top optical 3D printer. Coatings. 2020, 10: 254.
- Han, Y., Baltriukiene, D., Kozlova, E. Effect of scaffold properties on adhesion and maintenance of boundary cap neural crest stem cells in vitro. J Biomed Mater Res A. 2020, 108: 1274–1280.
- Meškyte, E., Keskas, S., Ciribilli, Y. MYC as a multifaceted regulator of tumor microenvironment leading to metastasis. Int J Mol Sci. 2020, 21: 3234.
Impact of Extracellular Environment on Cell Fate
Analysis of substrate macrophotography (>100 µm) impact on the fate of DPSCs revealed that for spontaneous cell osteogenesis induction, the precise structuring of the scaffold (nano- and micro-surface patterns) is not necessarily required. Nevertheless, we have found that PLA+HA filaments printed with FFF 3D printer produced equal or even better accuracy than scaffolds printed with pure PLA filaments (Gendviliene et al. 2020). After evaluating the impact of surface chemical composition on cell behaviour it was observed that PLA+HA composite was more suitable for DPSC attachment and proliferation, meanwhile the minimum number of focal adhesions were formed in the cells grown on PLA+BG scaffolds, which means that DPSCs adhesion strength on PLA+BG scaffolds was the weakest compared with PLA+HA (p<0.05) and pure PLA (p<0.05) (Fig.1). Despite this, PLA+BG composites promoted the earliest and strongest DPSC osteogenesis.
Fig. 1. DPSCs adhesion on 3D printed composite scaffolds. A – immunofluorescence staining of nucleus (DAPI, blue) and FA spots (vinculin, green) in DPSCs 24 h post-seeding; B – cell surface area after culturing for 0.5, 2 and 24 h on the scaffolds; C – quantitative FA evaluation within the cells after culturing for 24 h.
Further investigation has shown that PLA surface coated with DPSC-derived extracellular matrix (ECM) network significantly improved the osteoinductive properties of the surface. The obtained results of DPSC migration and proliferation showed that ECM proteins had a positive impact on both cell migration and proliferation (Fig. 2).
Fig. 2. DPSC migration and proliferation on ECM coated scaffolds. A – evaluation of vertical cell migration onto the scaffolds using crystal violet assay; B – relative DPSC proliferation rate.
The in vitro assay of scaffolds (PLA, PLA+HA, PLA+BG, and PLA+ECM) and DPSCs ability to initiate angiogenesis revealed that DPSC-PLA+BG and DPSC-PLA+ECM constructs would be the most suitable candidates for bone engineering. According to our data, the combination of DPSCs, PLA+BG composite microstructured scaffold and DPSC-derived ECM network can be expected to reach successful bone tissue regeneration in vivo (Alksne et al. 2020).
Molecular Mechanisms of Intracellular Trafficking
Molecular Mechanisms of Intracellular Trafficking
Intracellular trafficking distributes newly synthesized and endocytosed material to diverse cellular destinations and, by doing so, ensures cellular homeostasis. The functionality of the secretory trafficking and endocytosis are regulated in a highly complex manner with hundreds of molecular machineries and multiple pathways acting simultaneously. A precise coordination of transport carriers’ formation, directionality of their movement, fusion to the acceptor membranes and the morphology of intracellular organelles is tightly regulated in a temporal and spatial manner. Trafficking is also closely linked to multitude of other cellular processes: autophagy, cell death, regulation of transcription and translation. Deregulation of cargo trafficking leads to ever-increasing list of such diseases as cancer, cardio-vascular or neurodegenerative ones. Regulation of trafficking as a cellular response function to changes in the surrounding is little understood, and I am especially interested to understand this relation in detail.
To dissect the complexity of trafficking and signalling pathways, we use fluorescent microscopy-based assays, cell biology and biochemistry techniques to identify novel intracellular and extracellular regulators, inter-connections among them. We modify gene, transcript and protein expression function by geneediting, RNA interference and antibody-mediated approaches, respectively. We develop techniques to perform these experiments in highresolution and on a largescale, thereby, extracting high-content information from varying biological scales.
- Grigaitis, P., Starkuviene, V., Rost, U., Serva, A., Pucholt, P., Kummer, U. miRNA target identification and prediction as a function of time in gene expression data. RNA Biology. 2020, Apr 22: 1–11.
- Liu., S. J., Majeed, W., Grigaitis, P., Betts, M. J., Climer, L. K., Starkuviene, V., Storrie., B. Epistatic analysis of the contribution of rabs and kifs to CATCHR family dependent Golgi organization. * Front Cell Dev Bio. 2019, Aug 2,7: 126.
- Starkuviene, V., Kallenberger, S. M., Beil, N., Lisauskas, T., Schumacher, B. S., Bulkescher, R., Wajda, P., Gunkel, M., Beneke, J., Erfle, H. High-density cell arrays for genome-scale phenotypic screening. SLAS Discov. 2019 Mar, 24(3): 274–283.
- Bulkescher, R., Starkuviene, V., & Erfle, H. Solid-phase reverse transfection for intracellular delivery of functionally active proteins. Genome Res. 2017 Oct, 27(10): 1752–1758.
- Gunkel, M., Erfle, H., & Starkuviene, V. High-content analysis of the Golgi complex by correlative screening microscopy. Methods Mol Biol. 2016, 111–121.
Strategies in Antimicrobial Therapy and Protein Engineering
Strategies in Antimicrobial Therapy and Protein Engineering
In biocontrol of the skin pathogens and analysis of their physiology we are focusing on the application of pulsed electric field (PEF) in combination with the various chemical compounds to achieve the successful elimination of the skin pathogens, both bacteria and yeasts. The skin pathogens, Candida genera yeast, are capable of undergoing morphology switches and form pseudohyphae structures with highly increased resistance to the antifungal compounds. We discovered that, after growth in a rotary cell cultivation system (RCCS), a new, super-resistant and morphology-switching-unrelated phenotype of Candida is formed . RCCS is changing the pattern of the antibiotic resistance of Pseudomonas aeruginosa and Staphylococcus aureus as well. A combination of the novel chemical compounds with the pulsed electric field (PEF) and pulsed electromagnetic fields (PEMF) technologies allows us to perform a wide scale biocontrol of the drug resistant skin pathogens .
In 2020, we started a new project “The influence of intensive fish farming on aquatic microbiome and resistome”, analysing and comparing the microbial communities in the fresh aquatic systems and fish farms. These studies provide information not only concerning the microbial communities present in the environmental samples, but also regarding the pathogens and antibiotic resistant strains that can be spread in the population. In this research, we are also focusing on the bacteria genotypes that could be related to the microplastic degradation, synthesis of the antimicrobial compounds, capability to grow on different carbon sources.
Protein engineering (directed evolution, rational design, enzyme fusion) is a powerful tool for developing new biocatalysts for different industrial fields. Lipolytic enzymes are extensively used in chemistry, food, pharmaceutical, detergent, cosmetics industry and biodiesel production. Our research team apply different protein engineering methods (random and site-specific mutagenesis, DNA shuffling, SHIPREC, epPCR, the design of new fused biocatalysts) to investigate structure-function relationships of lipolytic enzymes produced by Geobacillus bacteria. In our research, we predicted a few amino acids, which strongly affected the activity of these enzymes, constructed several fused lipolytic enzymes and developed new Geobacillus lipase variant with improved kinetics and physicochemical characteristics (4–6).
1. Lastauskiene, E., Novickij, V., Zinkevičiene, A., Girkontaite, I., Paškevičius, A., Svediene, J., Markovskaja, S., Novickij, J. Application of pulsed electric fields for the elimination of highly drug-resistant Candida grown under modelled microgravity conditions. International Journal of Astrobiology. 2019, 18: 405–411.
2. Novickij, V., Staigvila, G., Gudiukaitė, R., Zinkevičienė, A., Girkontaitė, I., Paškevičius, A., Švedienė, J., Markovskaja, S., Novickij, J., Lastauskienė, E. Nanosecond duration pulsed electric field together with formic acid triggers caspase-dependent apoptosis in pathogenic yeasts. Bioelectrochemistry. 2019, 128: 148–154.
3. Novickij, V., Lastauskienė, E., Staigvila, G., Girkontaitė, I., Zinkevičienė, A., Švedienė, J., Paškevičius, A., Markovskaja, S., Novickij, J. Low concentrations of acetic and formic acids enhance the inactivation of Staphylococcus aureus and Pseudomonas aeruginosa with pulsed electric fields. BMC Microbiology. 2019, 19: 73.
4. Druteika, G., Sadauskas, M., Malunavicius, V., Lastauskiene, E., Statkeviciute, R., Savickaite, A., Gudiukaite, R. New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations. World Journal of Microbiology and Biotechnology. 2020, 36: 41. doi: 10.1007/s11274-020-02816-3.
5. Kumar, A., Gudiukaite, R., Gricajeva, A., Sadauskas, M., Malunavicius, V., Kamyab, H., Sharma, S., Sharma, T., Pant, D. Microbial lipolytic enzymes – promising energy-efficient biocatalysts in bioremediation. Energy. 2020, 192: 116674. https://doi.org/10.1016/j.energy.2019.116674.
6. Druteika, G., Sadauskas, M., Malunavicius, V., Lastauskiene, E., Taujenis, L., Gegeckas, A., Gudiukaitė, R. Development of a new Geobacillus lipase variant GDlip43 via directed evolution leading to identification of new activity-regulating amino acids. Int J Biol Macromol. 2020, 151: 1194–1204.
The Candida genera yeast-caused infections are frequent and difficult to treat, as the physiology and metabolisms of yeast are similar to the host . Induction of the programmed cell death is of great medical relevance, since during apoptosis peptides, nucleotides, amino acids and other compounds are released to the surrounding media and can contribute to the regeneration of the human tissues. Discovery of the apoptogenic substances for the biocontrol of pathogenic yeasts as well as optimization of the apoptogenic conditions is the main task of our research group. PEF can be also successfully applied to inactivate bacterial pathogen colonizing human skin. Best results are achieved by combining PEF with the week organic acids [2, 3].
PEF treated yeast cells. Scanning electron microscopy.
The biodiversity of fishery ponds is changed towards fulfilling the industrial needs, therefore reducing the microbial biodiversity and precautions should be taken to keep the system sustainable and protect the adjacent environment from possible damage. The metagenome analysis allows us to assess prokaryotic diversity in aquatic environment and evaluate the influence of intensive fish farming on the environment. We aim to evaluate the current situation in the fishery ponds, fresh water environments and adjacent aqua systems in order to understand the microbial population dynamics.
Bacterial genus diversity in the fish farm ponds.
Microbial lipolytic enzymes have gained attention for the ability to catalyse biotransformation reactions of different esters-bond containing compounds. Conversion of the latter into high-energy products like biofuel and other value-added products (fatty acid esters, mono- and diacylglycerols, etc.) in an energy-efficient and ecologically-friendly way makes these biocatalysts an important tool for sustainable biotechnology. Protein engineering, immobilization and application of Geobacillus lipases and carboxylesterases is one of the major research fields of our group [4–6].
The main advantages and disadvantages of lipolytic microbial enzymes usage in immobilized, free and chimeric forms .
Microbially induced calcite precipitation (MICP) is an effective and eco-friendly technology that can be applied to solve soil problems, including soil erosion, pollution with heavy metals and radionuclides or for CO2 sequestration. In geotechnical engineering, bioconsolidation is an effective technique to increase slope stability. The success of this process depends on ureases producing microorganisms. We have shown that using both 0.5–1 M concentration of urea with CaCl2 and urease positive Staphylococcus sp. H6 cells successful MICP process can be carried out.
Bioconsolidated sand particles obtained during MICP using Staphylococcus sp. H6