Volume 1 (2017): Issue 1 (January 2017)

Genomics, the study of genes, their functions and related techniques has become a crucial science for developing understanding of life processes and how they evolve. Since the advent of the human genome project, huge strides have been made in developing understanding of DNA and RNA sequence information and how it can be put to good use in the biotechnology sector. Newly derived sequencing and bioinformatics tools have added to the torrent of new insights gained, so that ‘sequence once and query often’ type DNA apps are now becoming reality. Genome editing, using tools such as CRISPR/Cas9 nuclease or Cpf1 nuclease, provide rapid methods for inserting, deleting or modifying DNA sequences in highly precise ways, in virtually any animal, plant or microbial system. Recent international discussions have considered human germline gene editing, amongst other aspects of this technology. Whether or not gene edited plants will be considered as genetically modified remains an important question. This will determine the regulatory processes adopted by different groups of nations and applicability to feeding the world’s ever growing population. Questions surrounding the intellectual property rights associated with gene editing must also be resolved. Mitochondrial replacement therapy leading to ‘3-Parent Babies’ has been successfully carried out in Mexico, by an international team, to correct mother to child mitochondrial disease transmission. The UK has become the first country to legally allow ‘cautious use’ of mitochondrial donation in treatment. Genomics and genome editing will continue to advance what can be achieved technically, whilst society determines whether or not what can be done should be applied.

Extensive use of antibiotics in medicine, veterinary practice and animal husbandry has promoted the development and dissemination of bacterial drug resistance. The number of resistant pathogens causing common infectious diseases increases rapidly and creates worldwide public health problem. Commensal bacteria, including lactic acid bacteria of genera Enterococcus and Lactococcus colonizing gastrointestinal and urogenital tracts of humans and animals may act as vehicles of antibiotic resistance genes similar to those found in pathogens. Lactococci and enterococci are widely used in manufacturing of fermented products and as probiotics, therefore monitoring and control of transmissible antibiotic resistance determinants in industrial strains of these microorganisms is necessary to approve their Qualified Presumption of Safety status. Understanding the nature and molecular mechanisms of antibiotic resistance in enterococci and lactococci is essential, as intrinsic resistant bacteria pose no threat to environment and human health in contrast to bacteria with resistance acquired through horizontal transfer of resistance genes. The review summarizes current knowledge concerning intrinsic and acquired antibiotic resistance in Lactococcus and Enterococcus genera, and discusses role of enterococci and lactococci in distribution of this feature.

For simulations of flow and microbial conversion reactions, related to modeling of simultaneous extraction and fermentation process in a single sugar beet cossette a software package OpenFOAM was used. The mass transfer of the components (sucrose, glucose, fructose and ethanol) in the studied system was controlled by the convection and diffusion processes. Microbial conversion rates and yield coefficients were experimentally determined and/or estimated by mathematical simulation. Dimensions of the model sugar beet cossette (SBC) were: average length of cosettes 40.10 mm, average thickness 3.32 mm and average width 3.5 mm, and represented in the model as a square-shape cross-section mathematical simulation. Dimensions of the model sugar beet cossette (SBC) were: average length of cosettes 40.10 mm, average thickness 3.32 mm and average width 3.5 mm, and represented in the model as a square-shape cross-section used to study the mass transfer and microbial conversion rates on the scale of single sugar beet cossette in the short time scales (up to 25 s). This model can be used for simulation of extractant flow around single sugar beet cossette as well as for description of simultaneous extraction and fermentation process in the studied system.

The aim of this work was the adaptation of a Gas Chromatographic-Flame Ionization Detector (GC-FID) method for detection and quantification of extracellular free amino acids in demineralized water, De Mann Rogosa Sharpe (MRS) medium and corn grits (CG) withdrawn during lactic acid fermentation. In order to analyze free amino acids by the GC-FID method it was necessary to convert free amino acids to volatile compounds. This was accomplished by derivatization of free amino acids with ethylchlor formate in aqueous medium followed by extraction of volatile free amino acid esters with chloroform. It was proven that the combination of derivatization and extraction procedure with developed GC-FID method gave accurate, reproducible and sensitive analytical results. Quantification of 15 (Ala, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Asn, Met, Pro, Lys, His, Asp and Glu) out of 20 ethoxycarbonyl-ethyl esters of free amino acids in demineralized water and MRS medium was achieved by established methods. In corn grits medium all of the above mentioned 15 amino acids, except His, were quantified with this GC-FID method. The established method was efficiently verified in monitoring of extracellular free amino acid concentration during lactic acid production with Lactobacillus rhamnosus DSM 20021^T in MRS medium and Lactobacillus amylovorus DSM 20531^T in corn grits medium.

In order to obtain a natural apiphytotherapeutic formula with anti-parasite effect, as an alternative in the control of Nosema spp. infection, an original formula based on hydro-alcoholic extracts from plants and propolis was tested. Experiments were made on naturally infected adult bees from private apiaries diagnosed with nosemosis (Nosema apis/Nosema ceranae). Initially hydro-alcoholic extracts from plants in Romania’s flora and respectively from propolis (Apis mellifera), with sensorial, physical- chemical and microbiologic characteristics, in conformity with requirements in the European Pharmacopeia VI^th Edition and Romanian Pharmacopeia X^th edition. Parasitosis was demonstrated through laboratory methods, respectively through direct microscopic examination, on bee samples collected from aviaries according to O.I.E. (World Organisation for Animal Health) methodology (2008). „In vitro“ lab testing of plant extracts and of work variants was done firstly on naturally infested polyfloral honey having an average concentration of 7 spores/ microscopic field of Nosema sp., with the purpose of selecting the most effective work variant which was afterwards tested „in vivo“ in private apiaries, on bee colonies. The results of the clinical study showed the absence of disease in 75% of the treated hives (33 hives), bees being declared clinically healthy. In 14 % of the hives (6 hives) infestation was weak, in 6.82% of the hives (3 hives) infestation was medium, and 4.55 % (2 hives) were diagnosed with a massive degree of infestation and the therapeutic protocol continued.

Glycopolymers of two types were isolated from the cell wall of Lactococcus lactis BIM B-1024 by stepwise extraction with cold and hot 10% CCl₃CO₂H and separated by anion-exchange gel chromatography. The following structures of the glycopolymers were established by sugar analysis, dephosphorylation with 48% HF, 1D and 2D NMR spectroscopy, and ESI-MS:

β-D-Galp-(1→4)-β-D-Glcp-(1→6)┐

→4)-β-D-Galp-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→ PSI

β-D-Glcp-(1→3)┐

→60-α-D-GalpNAc-(1→3)-β-D-GalpNAc-(1→5)-Rib-ol-1-P-(O→ PSII

β-D-Glcp-(1→4)┘

Polysaccharides with the same or similar structures to PSI have been found earlier in various Lactobacillus species, whereas, to our knowledge, the structure of PSII is new.

Purpose: To examine brain diffusion characteristics in pediatric patients with attention deficit hyperactivity disorder (ADHD) using diffusion tensor imaging (DTI) and an atlas-based anatomical analysis of the whole brain and to investigate whether these images have unique characteristics that can support functional diagnoses. Materials and Methods: Seventeen children with ADHD and ten control subjects (all age-matched) underwent MRI scans. The Institutional Ethics Board approved this study. Morphometric analysis was performed using MriStudio software. The diffusion images were normalized using a linear transformation, followed by large deformation diffeomorphic metric mapping (LDDMM). For 189 parcellated brain regions, the volume, fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were measured. Results: Children with ADHD were found to have increase in the body of lateral ventricle volumes compared to the control. Increased MD was found in the deep gray matter, amygdala, thalamus, substantia nigra, and also the cerebellum left and right side. Increased RD was found in the deep gray matter, caudate, thalamus, substantia nigra and hippocampus left and right side compared to the control. Significant elevated FA was found in the bilateral splenium of the corpus callosum in ADHD patients. Conclusion: Children with ADHD display abnormal diffusion characteristics and anatomical features compared to healthy controls. DTI can provide sensitive information on integrity of white matter (WM) and intra-WM structures in ADHD.

Gadolinium nanoparticles (GdNPs) are potential agents for MRI of lymph nodes. The aim of this study was to evaluate the in vitro effects of 1 μM, 2.5 μM and 5 μM of GdDOTA⊂CS-TPP/HA and GdDOTP⊂CS-TPP/HA NPs on A20 lymphocyte cells exposed for 6 and 24 hours. The total cellular biomass (SRB), lactate dehydrogenase activity (LDH) and oxidative stress parameters, such as reactive oxygen species generation (ROS), reduced glutathione (GSH), malondialdehyde (MDA) and advanced oxidation protein products (AOPP) were analyzed by spectrophotometric and fluorimetric methods. After cells exposure to 1 μM, 2.5 μM and 5 μM of GdDOTP⊂CS-TPP/HA NPs their viability decreased in a time- and dose-dependent manner, whereas for GdDOTA⊂CS-TPP/HA no significant changes were noticed. Both NPs formulations in doses of 1 μM, 2.5 μM, 5 μM did not affect the plasma membrane at each time point tested. The levels of ROS, MDA and AOPP increased proportionally with the concentration and exposure time. GSH concentration decreased significantly for all doses of both NPs tested. Taken together our data suggest that, GdDOTP⊂CS-TPP/HA and GdDOTA⊂CS-TPP/HA NPs induced oxidative stress in A20 lymphocyte cells which was counteracted by the cells antioxidant defense system to a certain extend.

Background: Nonpathogenic Bacillus strains are used in biotechnology, and pathogenic Bacillus strains are cause of food borne disease. It explains the relevance of the methods of detection and quantification of whole cell and cell components of these bacteria. Aims: Development of methodological approach for investigation of dynamics of specific concentration of cell wall antigens per cell of bacilli without solubilization of cell wall during sample preparation; using of the approach with 6 strains of bacilli as an example. Method: ELISA. Results: Methodological approach for investigation of dynamics of specific concentration of bacilli cell wall antigens has been developed. The distinctive features of the approach are rabbit polyclonal antibodies to genera-specific antigens of bacilli as key reagent and lack of need for solubilization of cell wall during sample preparation. It was shown using 6 strains of Bacilli as an example that specific concentration of cell wall antigens per cell vary according to bacillus strain, stage of culture growth and media composition. The data will find an application in biotechnology of clinical diagnostics and test-systems for food control including detection of whole bacillus cells.

Introduction: A relevant role is now emerging for nutraceuticals and specific functional foods in the treatment of dyslipidemia. The aim of this study was to evaluate the efficacy of a nutraceutical multi-target approach in subjects with moderate cardiovascular risk and to compare it with red yeast rice (RYR) treatment alone. Materials and Methods: Sixty patients with a first diagnosis of moderate dyslipidemia were included in a 6-week open-label, randomized, parallel-group controlled clinical trial and were treated with a nutraceutical supplement of Red Yeast Rice (RYR) extract containing 10 mg of monacolin k or its combination with 48 mg of an improved form of highly bioavailable resveratrol. The dosage of RYR was selected on the basis of its expected efficacy in reducing low-density lipoprotein- cholesterol also approved by the EFSA panel. All differences were assessed by Student’s t test with P values .05 are considered as statistically significant. Statistical analysis was performed by using Excel. Results: Treatment with RYR (10 mg monacolin K) led to a reduction of total cholesterol (20%) and low-density lipoprotein- cholesterol (21%). The combination with resveratrol however, compared to RYR alone significantly reduced triglyceride (-18 %) levels, systolic blood pressure (-2 %) and HOMA index (-17 %). Discussion: These results indicate that the nutraceutical supplementation of RYR associated with resveratrol not only shows lipid-lowering activity but compared to RYR treatment alone significantly also ameliorates other metabolic parameters. Thus, may represent a valid and safe approach, especially in people with moderate cardiovascular risk, in which a pharmacologic intervention may not be appropriate.

Gum exudate was obtained from Prosopis juliflora (Sw.) DC., which is abundantly available in north-west, central, west and south India. It was analysed for its phytochemical composition in aqueous extract and as well as by LCMS, GCMS, TGDTA and SEM to validate it’s potential for use as an excipient (Fig. 1).

Between 1996-2015, at V.R.D.S Buzau were conducted researches regarding the acclimatization, breeding and elaboration of crop technology for a new vegetable species, Momordica charantia (bitter cucumber). There were purchased and studied a large number of genotypes which were assessed for a long period; 4 of those have demonstrated increased adaptability, for which they were retained and multiplied in lineage. In time, by specific cucurbits improvement techniques were obtained 4 genetically stabilized accessions noted as G1, early accessions with large fruits,G2, medium fruit size, G3, medium fruit size and L4 with small crunchy fruits, semi tardy. Of these, L3 was homologated and patented under the name of Rodeo, and the other 3 will be proposed for homologation in the future. The studies demonstrated that this species has well adapted to our pedoclimatic conditions; it can be cultivated both in greenhouses and in open fields, in warmer areas, mentioning that for both crop variants it is recommended a drilling system. The new genotypes support various technological variants and can be ecologically cultivated with remarkable results.

Control of plant bacterial diseases remains difficult due to the limited availability of efficient plant protection products with reduced negative effects either in the environment or with human and animal health. In order to reduce the usage of chemical pesticides alternative strategies for controlling plant pathogens and improve plant disease resistance are promoted. The aim of the study was to investigate the antibacterial activity of some natural compounds (plant extracts of Tamarix ramosissima, Rosmarinus officinalis, Chelidonium majus, Silybum marianum, Satureja hortensis essential oil and propolis) against bacterial ring rot pathogen, Clavibacter michiganensis ssp. sepedonicus (Cms). An agar diffusion method was used for the screening of the inhibitory effect of natural compounds on bacterial strains’ growth. Minimum inhibition concentrations (MICs) were determined by a twofold serial dilution method. The anti-pathogenic activity was investigated by the study of anti-biofilm activity of natural substances. The analyzed natural substances showed a good microbicidal activity and anti-biofilm activity. The results obtained from this study may contribute to the development of new bio-control agents as alternative strategies for prevention and control of ring rot pathogen.

From fresh and fermented vegetables (cucumber, sauercraut, eggplant), cereals (wheat, rice), legumes (soy) 37 cultures of lactic acid bacteria were isolated. Based on biochemical characteristics, MALDI-TOF MS protein profiling and 16S rRNA gene sequencing they were identified as representatives of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Enterococcus genera. Six members of Lactobacillus genera and two members of Leuconostoc genera actively producing EPS were selected for further investigation as components of starters for production of special dietetic foodstuffs.

In the series of experiments Wistar rats(n=14) after intranasal implantation 35 thousand mesenchymal stem cells in 50 μl of buffer and modeling damage in sensorimotor there are no differences between ipsilateral and contralateral administration. In the series of experiments Wistar rats(n=18) after implantation in Meckel cavity of 35 thousand mesenchymal stem cells in 50 μl of buffer and modeling damage in cerebellar cortex it was shown that ipsilateral administration more efficient than contralateral administration.

The main objective of the work was to clarify the question - how will cell cultures functional state change after microgravity simulation when the shift in full strength direction takes place? Proliferation processes and apoptosis intensity in cell lines of rat glioma and human fibroblasts were compared in changing the position of flasks with cell culture in relation to the horizon. The detection of apoptosis and necrosis processes was carried out using flow cytofluorimetry. It was found that the change in full strength direction provides an inhibitory effect on tumor glial cells and fibroblasts’ proliferative activity enhances along with inhibition of apoptotic processes. Intensification of apoptotic processes in glioma cells and attenuation of cell death processes in normal cells - fibroblasts - are the result of cell cooperation disturbance.

Here we provide our pipeline for antibody discovery process. Starting from de novo isolation from custom made phage display libraries to expression and downstream applications; it allows us to obtain recombinant antibodies with desired properties to vast set of antigens. We also developed the CYB5-fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. The combination of redox dependent absorbance spectrum of red-colored cytochrome b5 with its high value molar extinction coefficient allows us to monitor antibody fusion proteins localization, redox state and quantify them in reliable way in turbid solutions. Moreover, it was revealed that due to outstanding stability and solubility, cytochrome b5 significantly enhances expression level of antibody fragments in Escherichia coli periplasm.

Process schemes yielding β-galactosidase of different purity grade were designed, the enzyme properties were determined. It was the first isolation and investigation of the gene encoding Arthrobacter sulfonivorans extracellular β-galactosidase.

The technology for in vitro anther culturing was optimized in hexaploid triticale using combination of external factors that allowed to obtain more than 100 doubled haploid lines. Investigation of genetic variation among anther culture derived doubled haploids of triticale showed the occurrence of heterozygous plants.