Plant Growth Regulation in Cell and Tissue Culture In Vitro.

Precise knowledge of all aspects controlling plant tissue culture and in vitro plant regeneration is crucial for plant biotechnologists and their correlated industry, as there is increasing demand for this scientific knowledge, resulting in more productive and resilient plants in the field. However, the development and application of cell and tissue culture techniques are usually based on empirical studies, although some data-driven models are available. Overall, the success of plant tissue culture is dependent on several factors such as available nutrients, endogenous auxin synthesis, organic compounds, and environment conditions. In this review, the most important aspects are described one by one, with some practical recommendations based on basic research in plant physiology and sharing our practical experience from over 20 years of research in this field. The main aim is to help new plant biotechnologists and increase the impact of the plant tissue culture industry worldwide.

ACTIVITY OF PHOSPHATASES IN THE ORAL LIQUID OF PATIENTS FROM POLLUTED AREAS WHO ARE DIAGNOSED WITH GENERALIZED PERIODONTITIS AND RHEUMATOID ARTHRITIS.

OBJECTIVE: The aim: To study the activity of acid and alkaline phosphatases as markers of bone metabolism in patients from bad environmental areas who are diagnosed with rheumatoid arthritis and are affected by generalized periodontitis. PATIENTS AND METHODS: Materials and methods: A total of 137 residents from Kalush district of the Ivano-Frankivsk region, Ukraine were enrolled in this investigation. There were 105 patients aged between 18 and 60 years with generalized periodontitis of the I-II degree severity selected for further research. Among them were 70 patients diagnosed with rheumatoid arthritis at the stage of remission. The research comprised the study of oral fluid of the examined individuals in order to assess the bone metabolism based on activity of the acid and alkaline phosphatases. RESULTS: Results: The results of the conducted studies testify an increase of acid phosphatase in subgroups of patients diagnosed with rheumatoid arthritis and affected by generalized chronic periodontitis. On the other hand, the obtained results of alkaline phosphatase activity demonstrated a decrease of this indicator in all subgroups. CONCLUSION: Conclusions: The results of biochemical studies suggest that there is a significant violation of bone tissue metabolism observed in patients from environmentally unfavorable areas affected by generalized periodontitis and diagnosed with rheumatoid arthritis.

Therapeutic Potential of an Azithromycin-Colistin Combination against XDR K. pneumoniae in a 3D Collagen-Based In Vitro Wound Model of a Biofilm Infection.

A therapeutic combination of azithromycin (AZM) and colistin methanesulfonate (CMS) was shown to be effective against both non-PDR and PDR Klebsiella pneumoniae biofilms in vitro. These anti-biofilm effects, however, may not correlate with effects observed in standard plate assays, nor will they representative of in vivo therapeutic action. After all, biofilm-associated infection processes are also impacted by the presence of wound bed components, such as host cells or wound fluids, which can all affect the antibiotic effectiveness. Therefore, an in vitro wound model of biofilm infection which partially mimics the complex microenvironment of infected wounds was developed to investigate the therapeutic potential of an AZM-CMS combination against XDR K. pneumoniae isolates. The model consists of a 3D collagen sponge-like scaffold seeded with HEK293 cells submerged in a fluid milieu mimicking the wound bed exudate. Media that were tested were all based on different strengths of Dulbecco's modified Eagles/high glucose medium supplemented with fetal bovine serum, and/or Bacto Proteose peptone. Use of this model confirmed AZM to be a highly effective antibiofilm component, when applied alone or in combination with CMS, whereas CMS alone had little antibacterial effectiveness or even stimulated biofilm development. The wound model proposed here proves therefore, to be an effective aid in the study of drug combinations under realistic conditions.

Azithromycin possesses biofilm-inhibitory activity and potentiates non-bactericidal colistin methanesulfonate (CMS) and polymyxin B against Klebsiella pneumonia.

Novel antibiotic combinations may act synergistically to inhibit the growth of multidrug-resistant bacterial pathogens but predicting which combination will be successful is difficult, and standard antimicrobial susceptibility testing may not identify important physiological differences between planktonic free-swimming and biofilm-protected surface-attached sessile cells. Using a nominally macrolide-resistant model Klebsiella pneumoniae strain (ATCC 10031) we demonstrate the effectiveness of several macrolides in inhibiting biofilm growth in multi-well plates, and the ability of azithromycin (AZM) to improve the effectiveness of the antibacterial last-agent-of-choice for K. pneumoniae infections, colistin methanesulfonate (CMS), against biofilms. This synergistic action was also seen in biofilm tests of several K. pneumoniae hospital isolates and could also be identified in polymyxin B disc-diffusion assays on azithromycin plates. Our work highlights the complexity of antimicrobial-resistance in bacterial pathogens and the need to test antibiotics with biofilm models where potential synergies might provide new therapeutic opportunities not seen in liquid culture or colony-based assays.

eDNA Inactivation and Biofilm Inhibition by the PolymericBiocide Polyhexamethylene Guanidine Hydrochloride (PHMG-Cl).

The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA-PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

Mitochondrial DNA Analysis Clarifies Taxonomic Status of the Northernmost Snow Sheep (Ovis nivicola) Population.

Currently, the intraspecific taxonomy of snow sheep (Ovis nivicola) is controversial and needs to be specified using DNA molecular genetic markers. In our previous work using whole-genome single nucleotide polymorphism (SNP) analysis, we found that the population inhabiting Kharaulakh Ridge was genetically different from the other populations of Yakut subspecies to which it was usually referred. Here, our study was aimed at the clarification of taxonomic status of Kharaulakh snow sheep using mitochondrial cytochrome b gene. A total of 87 specimens from five different geographic locations of Yakut snow sheep as well as 20 specimens of other recognized subspecies were included in this study. We identified 19 haplotypes, two of which belonged to the population from Kharaulakh Ridge. Median-joining network and Bayesian tree analyses revealed that Kharaulakh population clustered separately from all the other Yakut snow sheep. The divergence time between Kharaulakh population and Yakut snow sheep was estimated as 0.48 ± 0.19 MYA. Thus, the study of the mtDNA cytb sequences confirmed the results of genome-wide SNP analysis. Taking into account the high degree of divergence of Kharaulakh snow sheep from other groups, identified by both nuclear and mitochondrial DNA markers, we propose to classify the Kharaulakh population as a separate subspecies.

Models of individual linear dimensions necessary for the construction of the correct form of dental arches in young men with a wide face, depending on the features of odontometric and cephalometric indicators.

OBJECTIVE: The aim is development and analysis of regression models of linear dimensions necessary for the construction of the correct form of dental arches in young men with a wide face, depending on the features of odontometric and cephalometric indicators. PATIENTS AND METHODS: Material and methods: Primary computed tomographic indices of tooth size and cephalometric parameters of 44 young men with normal occlusion were obtained from the data bank of the National Pirogov Memorial Medical University, Vinnytsya. Face type was determined using the Garson morphological index. In licensed statistical package "Statistica 6,0" developed regression models of linear dimensions necessary for the construction of the correct form of dental arches. RESULTS: Results: As a result of researches, it is established that all 18 reliable models of the sizes used for construction of the correct form of dental arches, depending on peculiarities of odontometric and cephalometric indices with a coefficient from 0.645 to 0.944 are constructed. Built models in adolescents with a broad face type more often include odontometric than cephalometric indicators. The most commonly used odontometric indices are: the width of the crowns of the teeth in the mesio-distal and vestibulo-oral directions, as well as the distance from the middle of the cutting edge to the apex of the root of the teeth in the vestibulo-oral direction. CONCLUSION: Conclusions: In adolescents with a broad type of face with normal occlusion, all 18 possible reliable regression models of reproduction of the individual characteristics of the dental arches of the upper and lower jaws were determined and analyzed.

PREVALENCE OF NASAL CARRIAGE OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS AMONG HEALTHCARE WORKERS IN THE DEPARTMENTS OF OTORINOLARYNGOLOGY AND DENTISTRY IN KYIV, UKRAINE.

OBJECTIVE: The aim: To obtain the first estimates of the current prevalence of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) among healthcare workers (HCWs) in the departments of Otorinolaryngology and Dentistry and to determine of genes virulence factors (Panton Valentine Leukocidine (PVL) genes). PATIENTS AND METHODS: Materials and methods: We performed a multicenter cross-sectional study. The susceptibility to antibiotics was determined by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing. The virulence factor encoding genes, mecA, lukS-lukF, were detected by Polymerase Chain Reaction (PCR). RESULTS: Results: Incidence rate of S. aureus nasal carriage among HCWs was 36.2%, whereas MRSA carriage was 17%. Prevalence of MRSA carriage rate was 34.9% in Otorhinolaryngology departments and 9.7% in Dentistry. PCR testing confirmed that all MRSA strains were mecA gene-positive. The virulence factor encoding genes were detected in 82.3% of the S. aureus isolates from HCWs. Among S.aureus, the lukS-lukF genes were detected in over 59% of the strains. The lukS-lukF genes were detected in 55.5% of MRSA and in 58.9% of MSSA strains. LukS-lukF genes were most commonly co-present in MRSA strains. No significant difference was detected between the occurrences of lukS-lukF genes (P > 0.05). CONCLUSION: Conclusions: Personnell in otorhinolaryngology and dentistry departments have a high rate of nasal colonization of MRSA. This carrier state may be an important risk factor for transmission MRSA from physicians and nurses to patients and vice-versa. Screening for MRSA nasal carriage of HCWs is a key element in enabling infection control measures and early therapeutic decisions.

Enabling magnetic resonance imaging of hollow-core microstructured optical fibers via nanocomposite coating.

Optical fibers are widely used in bioimaging systems as flexible endoscopes that are capable of low-invasive penetration inside hollow tissue cavities. Here, we report on the technique that allows magnetic resonance imaging (MRI) of hollow-core microstructured fibers (HC-MFs), which paves the way for combing MRI and optical bioimaging. Our approach is based on layer-by-layer assembly of oppositely charged polyelectrolytes and magnetite nanoparticles on the inner core surface of HC-MFs. Incorporation of magnetite nanoparticles into polyelectrolyte layers renders HC-MFs visible for MRI and induces the red-shift in their transmission spectra. Specifically, the transmission shifts up to 60 nm have been revealed for the several-layers composite coating, along with the high-quality contrast of HC-MFs in MRI scans. Our results shed light on marrying fiber-based endoscopy with MRI to open novel possibilities for minimally invasive clinical diagnostics and surgical procedures in vivo.

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes.

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.

Different stresses, similar morphogenic responses: integrating a plethora of pathways.

Exposure of plants to mild chronic stress can cause induction of specific, stress-induced morphogenic responses (SIMRs). These responses are characterized by a blockage of cell division in the main meristematic tissues, an inhibition of elongation and a redirected outgrowth of lateral organs. Key elements in the ontogenesis of this phenotype appear to be stress-affected gradients of reactive oxygen species (ROS), antioxidants, auxin and ethylene. These gradients are present at the the organismal level, but are integrated on the cellular level, affecting cell division, cell elongation and/or cell differentiation. Our analysis of the literature indicates that stress-induced modulation of plant growth is mediated by a plethora of molecular interactions, whereby different environmental signals can trigger similar morphogenic responses. At least some of the molecular interactions that underlie morphogenic responses appear to be interchangeable. We speculate that this complexity can be viewed in terms of a thermodynamic model, in which not the specific pathway, but the achieved metabolic state is biologically conserved.

The involvement of reactive oxygen species (ROS) in the cell cycle activation (G(0)-to-G(1) transition) of plant cells.

Reactive oxygen species (ROS) are involved in various cellular processes in plants. Among those, resistance to abiotic stress, defence mechanisms and cell expansion have been intensively studied during the last years. We recently demonstrated that ROS, in concert with auxin, have a role in cell cycle activation of differentiated leaf cells.1 In this addendum we provide further evidence to show that oxidative stress/ROS accelerate auxin-mediated cell cycle entry (G(0)-to-G(1)) and may have a positive effect on the plant cell cycle machinery. A generalized model for concentration-dependent synergistic effect of auxin and ROS on differentiated plant cells is also shown.

Stress-induced morphogenic responses: growing out of trouble?

Plants exposed to sub-lethal abiotic stress conditions exhibit a broad range of morphogenic responses. Despite the diversity of phenotypes, a generic 'stress-induced morphogenic response' can be recognized that appears to be carefully orchestrated and comprises three components: (a) inhibition of cell elongation, (b) localized stimulation of cell division and (c) alterations in cell differentiation status. It is hypothesized that the similarities in the morphogenic responses induced by distinct stresses, reflect common molecular processes such as increased ROS-production and altered phytohormone transport and/or metabolism. The stress-induced morphogenic response (SIMR) is postulated to be part of a general acclimation strategy, whereby plant growth is redirected to diminish stress exposure.

Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures.

It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper experimental data are presented which indicate that NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than 3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells, however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent signaling pathways in plant cells.

Electromechanical model of cardiac resynchronization in the dilated failing heart with left bundle branch block.

Experimental studies have shown that biventricular pacing can improve systolic function in the failing heart with bundle branch block. The goal of this study was to develop and validate a three-dimensional computational model of the dilated failing heart with left bundle branch block to investigate how biventricular pacing can improve systolic mechanical performance and synchrony. In an anatomically detailed model of canine ventricular geometry, fiber architecture and Purkinje fiber network structure, a monodomain solution for anisotropic impulse conduction gave rise to electrical activation sequences that were consistent with experimentally observed patterns. Coupling this with regional myocardial mechanics computed for left branch bundle block and biventricular pacing showed good agreement with published regional fiber strains measured in dogs by using magnetic resonance imaging tagging. Biventricular pacing improved mechanical synchrony and systolic function in the computational model. The model may be a useful tool for investigating the pacing conditions required to achieve optimal mechanical improvement in the failing heart, especially because electrical synchrony does not correlate directly with mechanical synchrony and performance.

Relationship between regional shortening and asynchronous electrical activation in a three-dimensional model of ventricular electromechanics.

INTRODUCTION: Asynchronous electrical activation can cause abnormalities in perfusion and pump function. An electromechanical model was used to investigate the mechanical effects of altered cardiac activation sequence. METHODS AND RESULTS: We used an anatomically detailed three-dimensional computational model of the canine ventricular walls to investigate the relationship between regional electrical activation and the timing of fiber shortening during normal and ventricular paced beats. By including a simplified Purkinje fiber network and anisotropic impulse conduction in the model, computed electrical activation sequences were consistent with experimentally observed patterns. Asynchronous time courses of regional strains during beats stimulated from the left or right ventricular epicardium showed good agreement with published experimental measurements in dogs using magnetic resonance imaging tagging methods. When electrical depolarization in the model was coupled to the onset of local contractile tension development by a constant time delay of 8 msec, the mean delay from depolarization to the onset of systolic fiber shortening was 14 msec. However, the delay between the onset of fiber tension and initial shortening varied significantly; it was as late as 60 msec in some regions but was also as early as -50 msec (i.e., 42 msec before depolarization) in other regions, particularly the interventricular septum during free-wall pacing. CONCLUSION: The large variation in delay times was attributable to several factors including local anatomic variations, the location of the site relative to the activation wavefront, and regional end-diastolic strain. Therefore, we conclude that these factors, which are intrinsic to three-dimensional ventricular function, make the regional sequence of fiber shortening an unreliable surrogate for regional depolarization or electromechanical activation in the intact ventricles.

The Role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa.

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.

Muscle LIM protein deficiency leads to alterations in passive ventricular mechanics.

Accumulating evidence indicates that cytoskeletal defects may be an important pathway for dilated cardiomyopathy and eventual heart failure. Targeted disruption of muscle LIM protein (MLP) has previously been shown to result in dilated cardiomyopathy with many of the clinical signs of heart failure, although the effects of MLP disruption on passive ventricular mechanics and myocyte architecture are not known. We used the MLP knockout model to examine changes in passive ventricular mechanics and laminar myofiber sheet architecture. Pressure-volume and pressure-strain relations were altered in MLP knockout mice, in general suggesting a less compliant tissue in the dilated hearts. Transmural laminar myocyte structure was also altered in this mouse model, especially near the epicardium. A mathematical model of the heart showed a likely increase in passive tissue stiffness in the MLP-deficient (-/-) heart. These results suggest that the disruption of the cytoskeletal protein MLP results in less compliant passive tissue and concomitant structural alterations in the three-dimensional myocyte architecture that may in part explain the ventricular dysfunction in the dilated heart.