Tissue Culture Medium Research Articles

Tissue catabolism and donor-specific dexamethasone response in a human osteochondral model of post-traumatic osteoarthritis

BackgroundPost-traumatic osteoarthritis (PTOA) does not currently have clinical prognostic biomarkers or disease-modifying drugs, though promising candidates such as dexamethasone (Dex) exist. Many challenges in studying and treating this disease stem from tissue interactions that complicate understanding of drug effects. We present an ex vivo human osteochondral model of PTOA to investigate disease effects on cartilage and bone homeostasis and discover biomarkers for disease progression and drug efficacy.MethodsHuman osteochondral explants were harvested from normal (Collins grade 0–1) ankle talocrural joints of human donors (2 female, 5 male, ages 23–70). After pre-equilibration, osteochondral explants were treated with a single-impact mechanical injury and TNF-α, IL-6, and sIL-6R ± 100 nM Dex for 21 days and media collected every 2–3 days. Chondrocyte viability, tissue DNA content, and glycosaminoglycan (sGAG) percent loss to the media were assayed and compared to untreated controls using a linear mixed effects model. Mass spectrometry analysis was performed for both cartilage tissue and pooled culture medium, and the statistical significance of protein abundance changes was determined with the R package limma and empirical Bayes statistics. Partial least squares regression analyses of sGAG loss and Dex attenuation of sGAG loss against proteomic data were performed.ResultsInjury and cytokine treatment caused an increase in the release of matrix components, proteases, pro-inflammatory factors, and intracellular proteins, while tissue lost intracellular metabolic proteins, which was mitigated with the addition of Dex. Dex maintained chondrocyte viability and reduced sGAG loss caused by injury and cytokine treatment by 2/3 overall, with donor-specific differences in the sGAG attenuation effect. Biomarkers of bone metabolism had mixed effects, and collagen II synthesis was suppressed with both disease and Dex treatment by 2- to 5-fold. Semitryptic peptides associated with increased sGAG loss were identified. Pro-inflammatory humoral proteins and apolipoproteins were associated with lower Dex responses.ConclusionsCatabolic effects on cartilage tissue caused by injury and cytokine treatment were reduced with the addition of Dex in this osteochondral PTOA model. This study presents potential peptide biomarkers of early PTOA progression and Dex efficacy that can help identify and treat patients at risk of PTOA.

Simulation of thermal field distribution in biological tissue and cell culture media irradiated with infrared wavelengths.

In recent years, there has been a growing interest in the singlet form of oxygen as a regulator of the physiological functions of cells. One of the ways to generate singlet oxygen is direct optical excitation of the triplet oxygen form. Since molecular oxygen weakly absorbs light, high power is required to obtain sufficient concentrations of singlet oxygen. However, the increase in the radiation power of laser can induce a local temperature increase around the laser spot. This may be critical considering the temperature governs every biological reaction within living cells, in particular. Here, the interaction of laser radiation of infrared wavelengths, generating singlet oxygen, with biological tissues and cell culture media was simulated. Using the COMSOL Multiphysics software, the thermal field distribution in the volume of skin, brain tissue and cell culture media was obtained depending on the wavelength, power and exposure time. The results demonstrate the importance of taking temperature into account when conducting experimental studies at the cellular and organismal levels.

New Safety Aspects in Corneal Donation—Studies on SARS-CoV-2-Positive Corneal Donors

In the tissue donation field, to prevent pathogen transmission, all donors are screened by postmortem swabs for SARS-CoV-2 using qRT–PCR. Corneas from donors who tested positive for SARS-CoV-2 were subjected to further investigations. Corneal transplants and culture medium from positive donors were cultured under appropriate safety conditions for further analyses. Cornea tissue samples, including sclera/limbus/cornea, and culture media were taken at different time points for testing for SARS-CoV-2 using qRT–PCR, immunohistochemistry (IHC) and subgenomic RNA (sgRNA) analysis. Between January and May 2021, in four donors with initial negative premortem rapid tests, SARS-CoV-2 was detected post-mortem using qRT–PCR. In these cases, SARS-CoV-2 was observed at the beginning of cultivation in both tissue and culture medium using qRT–PCR and IHC. The virus was mainly localized in the limbus epithelial cells, with a stable detection level. Premortem rapid tests are potentially insufficient to exclude SARS-CoV-2 infection in corneal donors. While, for SARS-CoV-2, the risk of infection via transplants is considered low, a residual risk remains for presymptomatic new infections. However, our investigations provide the first indications that, with organ cultures, the risk of virus transmission is minimized due to the longer minimum culture period.

The role of auxin and giberalin in the growth of black cumin (Nigella sativa L.) by in vitro

As an herbal plant, black cumin (Nigella sativa L.) has been widely used. The seeds are able to overcome various types of diseases including viruses, able to relieve pain (analgesic), anti-swelling (anti-inflammatory), antiallergic (antihistamines), antioxidant, antidiabetic mellitus, antitumor, and anticancer. Propagation of black cumin by in vitro or known as micropropagation is one of the artificial vegetative propagation that can meet the sustainability production for cumin plants in Indonesia. In vitro propagation of black cumin is able to produce large numbers of seeds in a relatively short time, has the same properties as the parent, with a seedling process that is not seasonally dependent. Currently, the study have started using MS media, and modification of the type of tissue culture media by adding 2,4 D of 10 mg/l combination with GA3 of 1 mg/l can increased the percentage of callus growth. The combination of these two types of growth regulators, also influenced the number of shoots, plant height and number of roots.

Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators.

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

Predictive modeling of Persian walnut (Juglans regia L.) in vitro proliferation media using machine learning approaches: a comparative study of ANN, KNN and GEP models

BackgroundOptimizing plant tissue culture media is a complicated process, which is easily influenced by genotype, mineral nutrients, plant growth regulators (PGRs), vitamins and other factors, leading to undesirable and inefficient medium composition. Facing incidence of different physiological disorders such as callusing, shoot tip necrosis (STN) and vitrification (Vit) in walnut proliferation, it is necessary to develop prediction models for identifying the impact of different factors involving in this process. In the present study, three machine learning (ML) approaches including multi-layer perceptron neural network (MLPNN), k-nearest neighbors (KNN) and gene expression programming (GEP) were implemented and compared to multiple linear regression (MLR) to develop models for prediction of in vitro proliferation of Persian walnut (Juglans regia L.). The accuracy of developed models was evaluated using coefficient of determination (R2), root mean square error (RMSE) and mean absolute error (MAE). With the aim of optimizing the selected prediction models, multi-objective evolutionary optimization algorithm using particle swarm optimization (PSO) technique was applied.ResultsOur results indicated that all three ML techniques had higher accuracy of prediction than MLR, for example, calculated R2 of MLPNN, KNN and GEP vs. MLR was 0.695, 0.672 and 0.802 vs. 0.412 in Chandler and 0.358, 0.377 and 0.428 vs. 0.178 in Rayen, respectively. The GEP models were further selected to be optimized using PSO. The comparison of modeling procedures provides a new insight into in vitro culture medium composition prediction models. Based on the results, hybrid GEP-PSO technique displays good performance for modeling walnut tissue culture media, while MLPNN and KNN have also shown strong estimation capability.ConclusionHere, besides MLPNN and GEP, KNN also is introduced, for the first time, as a simple technique with high accuracy to be used for developing prediction models in optimizing plant tissue culture media composition studies. Therefore, selection of the modeling technique to study depends on the researcher’s desire regarding the simplicity of the procedure, obtaining clear results as entire formula and/or less time to analyze.

High-intensity focused ultrasound (HIFU) therapy for pain palliation in advanced stage pancreatic carcinoma: A Meta-Analysis with recent studies

Background: Day by day, High-intensity focused ultrasound (HIFU) therapy is becoming more familiar in medical field because it is non-invasive technique with fewer side effects and provides promising therapeutic results. Several HIFU therapy applications have approved by many approval authorities of deferent countries since last decade. It is a novel, emerging, therapeutic modality that uses ultrasound waves, propagated through tissue media, as carriers of energy. HIFU has great potential for tumor ablation and the main mechanisms of HIFU ablation involve mechanical and thermal effects. Pancreatic adenocarcinoma is currently the fourth-leading cause of cancer-related death. Up to 60–90% of patients with advanced disease suffer cancer-related pain, severely impacting their quality of life. Current management involves primarily pharmacotherapy with opioid narcotics and celiac plexus neurolysis; unfortunately, both approaches offer transient relief and cause undesired side-effects. High intensity focused ultrasound (HIFU) is a non-invasive thermal ablation technique that has been used to treat pancreatic cancer. This meta-analysis aims to evaluate the role of HIFU in pain palliation of advanced unresectable pancreatic adenocarcinoma. Methods: Paper selection was performed electronically in PubMed up to the end of March 2021, for pain palliation treatment of advanced staged pancreatic cancer with HIFU. Relevant papers were identified through the PubMed search engine using these keywords: HIFU, pancreas, pancreatic cancer, pain and palliation. Additional studies were also done included after manual search of the selected bibliographies. Palliation results reported in studies were analyzed using a logit-transformed random-effects model using the inverse variance method, with the DerSimonian-Laird estimator for t2, and Cochran’s Q test for heterogeneity among studies. The I2 was also calculated to assess the percentage of the total variability in the different effect size estimates that can be attributed to heterogeneity among the true effects and rank correlation test of funnel plot asymmetry was done to assess possible publication bias. Results: In this meta-analysis, we includes only recent 10 year studies i.e. total number of 16 studies with 687total patients with pancreatic cancer. The total patients enrolled ranges from 7 patients in the smallest series, up to 120 in the largest study. The calculated τ2was 0.187, and I2was 41%, the Q test p-value was 0. 026, is indicating significant heterogeneity among studies. The random effects estimate of the proportion of patients with pain reduction was 0.89.08. Conclusions: We concluded that HIFU performs to be an effective tool for pain palliation in advanced staged pancreatic cancer. Prospective randomized and standardized studies are necessary to confirm the effectiveness of HIFU in relieving pain, and to evaluate for any potential impact on tumor control and patient survival.

English

Numerous studies have shown that the rhizosphere allelochemicals closely related to the formation of replant problems in Pseudostellaria heterophylla. In this study, ultra-high performance liquid chromatography-quadrupole time-of-flight mass technology was employed to screen the specific compounds in rhizosphere soil induced the formation of replant disease. Simultaneously, the compounds in cultured seedling of P. heterophylla and corresponding medium were identified using the identical methods to obtain compounds that had not been transformed or degraded by soil microbes. As a result, 176 specific molecules were identified in the rhizosphere soil, 1,011 in the rhizosphere tissue culture medium and 1,447 in plant tissue cultures were obtained by matching to the Traditional Chinese Medicine database. Further analysis found that 21 potential allelochemicals might be directly secreted from P. heterophylla plants, which had not been transformed or degraded in theory. Of which, 13 compounds possessed the allelopathic characteristics based on structural analysis that have been identified in other plants. Simultaneously, eight compounds were only present in the rhizosphere tissue culture medium and plant tissue cultures also were found to possess the allelopathic properties. This study provides an important reference for further screening and identification of specific allelochemicals that related closely to the formation of replant problems in P. heterophylla. © 2022 Friends Science Publishers

ANALISA DISTRIBUSI DOSIS PADA FANTOM HOMOGEN DAN INHOMOGEN DENGAN SIMULASI MONTE CARLO

Monte Carlo simulation with electron gamma shower (EGSnrc) code can produce 3-dimensional dose distribution data. The percent depth dose curve (PDD), dose profile, and isodose curve can be extracted through this 3-dimensional data. In this study, a photon source with an energy of 2 MeV is placed at the source to surface distance (SSD) from the phantom surface by adjusting the collimator aperture which is used to control the size of the exposure field. The SSD distance was varied at a distance of 50 cm, 70 cm, 80 cm, 90 cm, and 100 cm. The size of the exposure area is also varied by 2 × 2 cm 2 , 5 × 5 cm 2 , 7 × 7 cm 2 , and 10 × 10 cm 2 . The dose distribution analysis was carried out on homogeneous phantoms containing water and inhomogeneous phantoms containing tissue/bone/lung/bone/tissue material. The PDD curve and dose profile represent dose changes with depth and x or y direction, respectively. The PDD curve on a homogeneous phantom shows an increase in the value on the surface of the phantom to the depth with the maximum dose. This curve then decreases gradually with increasing depth. Inhomogeneous ghosts show spikes in the border region of the two media with different densities. This is due to the contribution of the backscattered electrons generated by the bone media and into the tissue media. This phenomenon is also seen in the isodose curve for inhomogeneous phantoms.

Comparative retrotransposon analysis of mutant and non-mutant rice varieties grown at different salt concentrations

This study investigated the effects of retrotransposon movements on the regeneration of 15- and 30-day-old leaves and roots of four different rice varieties grown in tissue culture medium at different salt concentrations (0, 50, 100, and 200 mmol/L) by Inter Retrotransposon Amplified Polymorphism (IRAP) and Retrotransposon Microsatellite Amplified Polymorphism (REMAP) polymerase chain reaction (PCR)-based marker methods. The study used Hopi, Houba, Osr30, Tos17 primers for IRAP analysis. The (GA)9C ISSR primer was used with four IRAP primers in REMAP analyses. There were different band profiles and polymorphism results between different salts concentrations of the same variety and various rice varieties. We observed that the polymorphism ratios for Houba ranged from 0 to 98% in IRAP and REMAP analyzes in the calculation made with the Jaccard similarity index in mutant samples. We report that mutant varieties give more valuable IRAP and REMAP results than non-mutant varieties. The results reported in this study contribute to understanding the effect of salinity stress on rice in terms of retrotransposon-based molecular markers. These results suggest that all tested retrotransposons are still active, causing genomic polymorphism among rice plantlets.

Effect of LRRK2 protein and activity on stimulated cytokines in human monocytes and macrophages

Leucine-rich-repeat kinase 2 (LRRK2), a potential therapeutic target for the treatment of Parkinson’s disease (PD), is highly expressed in monocytes and macrophages and may play a role in the regulation of inflammatory pathways. To determine how LRRK2 protein levels and/or its activity modulate inflammatory cytokine/chemokine levels in human immune cells, isogenic human induced pluripotent stem cells (iPSC) with the LRRK2-activating G2019S mutation, wild-type LRRK2, and iPSC deficient in LRRK2 were differentiated to monocytes and macrophages and stimulated with inflammatory toll-like receptor (TLR) agonists in the presence and absence of LRRK2 kinase inhibitors. The effect of LRRK2 inhibitors and the effect of increasing LRRK2 levels with interferon gamma on TLR-stimulated cytokines were also assessed in primary peripheral blood-derived monocytes. Monocytes and macrophages with the LRRK2 G2019S mutation had significantly higher levels of cytokines and chemokines in tissue culture media following stimulation with TLR agonists compared to isogenic controls. Knockout of LRRK2 impaired phagocytosis but did not significantly affect TLR-mediated cytokine levels. Interferon gamma significantly increased the levels of LRRK2 and phosphorylation of its downstream Rab10 substrate, and potentiated TLR-mediated cytokine levels. LRRK2 kinase inhibitors did not have a major effect on TLR-stimulated cytokine levels. Results suggest that the LRRK2 G2019S mutation may potentiate inflammation following activation of TLRs. However, this was not dependent on LRRK2 kinase activity. Indeed, LRRK2 kinase inhibitors had little effect on TLR-mediated inflammation under the conditions employed in this study.

DAT and PRX1 gene expression modulates vincristine production in Catharanthus roseus L. propagates using Cu, Fe and Zn nano structures

Underlying mechanism of nanostructures upon monoterpene induction in Catharanthus roseus has not been explored yet. In the current study, Copper, Iron and Zinc nanoparticles were biosynthesized by Eriobotrya japonica seed extract and capped with reduced glutathione. Biosynthesized nanoparticles and their capped analogues were characterized by UV–visible spectrophotometer, FTIR, XRD and SEM. Selected concentration of nanostructures were used in plant tissue culture media which instigated the production of alkaloids, tannins and flavonoids without significantly affecting the growth index of propagated calli and shoots cultures of C. roseus. Accelerated vincristine production was noticed in propagated calli and shoots under copper and zinc nanostress (1645–1865 μg/ml respectively) with the least effect by iron nanostructure. Highest concentration of calcium was recorded in in vitro shoots under capped (3.42 mg/ml ± 7.16) and uncapped (4.41 mg/ml ± 20.44) Zn nanoparticles compared to control (2.82 mg/ml ± 13.41). Real time PCR depicts nano-zinc mediated increased expression of DAT and PRX1 genes of TIA pathway. Significant correlation among PRX1/DAT gene expression with vincristine production and calcium accumulation in the presence of nanostress validate by PCA. This study paved way the opportunities of metal biogenic nanomaterials as an ideal drug modulator in plant tissue culture studies.

Viable human brain microvessels for the study of aging and neurodegenerative diseases.

The brain microvasculature is altered in normal aging and in the presence of disease processes, such as neurodegeneration or ischemia, but there are few methods for studying living tissues. We now report that viable microvessels (MV) are readily isolated from brain tissue of subjects enrolled in studies of neurodegenerative diseases who undergo rapid autopsy (performed with <12h postmortem interval - PMI). We find that these MV retain their morphology and cellular components and are fairly uniform in size. Sufficient MV (~3-5000) are obtained from 3 to 4g of tissue to allow for studies of cellular composition as well as extracellular matrix (ECM). Using live/dead assays, these MV are viable for up to 5days in tissue culture media (2D) designed to support endothelial cells and up to 11days post-isolation in a 3-dimensional (3D) matrix (Low Growth Factor Matrigel™). Assays that measure the reducing potential of live cells \demonstrated that the majority of the MV maintain high levels of metabolic activity for a similar number of days as the live/dead assays. Functional cellular components (such as tight junctions and transporter proteins) and ECM of MV in tissue culture media, and to a lesser extent in 3D matrices, were readily visualized using immunofluorescence techniques. MV in tissue culture media are lysed and protein content analyzed, but MV in 3D matrix first require removal of the supporting matrix, which can confound the analysis of MV ECM. Finally, MV can be preserved in cryoprotective media, whereby over 50% retain their baseline viability upon thawing. In summary, we find that MV isolated from human brains undergoing rapid autopsy are viable in standard tissue culture for up to 5days and the timeframe for experiments can be extended up to 11days by use of a supportive 3D matrix. Viable human MV allow for temporal and spatial analysis of relevant cellular and ECM components that have implications for microvascular function in neurodegenerative diseases, vascular brain injury, and neurotrauma.

Activated charcoal added to tissue culture media increases genotype-dependent biomass production in soybean

Due to its important participation in the agribusiness model worldwide, soybean actively drives national economies in producing countries. However, biotic and abiotic factors caused by pests and climate changes, respectively, can disrupt its productivity and consequently the business market. For this reason, the development of plants more tolerant to these negative environmental elements has been frequently one of the goals of scientific research. In the pipeline to obtain genetically improved plants, tissue culture protocols often represent a bottleneck, since the efficiency at this stage can be genotype-dependent. Therefore, the objective of this work was to evaluate the root regeneration process of two soybean genotypes (BRS 283 and BRS 537) in four different substrates (vermiculite, sand, medium containing activated charcoal and, control – MS medium and glucose). The rooting development was measured by the root’s length (cm²), dry mass (mg), volume (mm³), surface area (mm²), and diameter (mm). Results showed that in the activated charcoal medium, for both soybean genotypes, roots grew longer and presented a higher dry mass of roots, and root length when compared to vermiculite and sand substrates. We concluded that the efficiency of tissue culture is genotype-dependent since assayed genotypes presented phenotypical responses significantly different. The supplementation of tissue culture medium with active charcoal improved root growth for both genotypes. Therefore, it is likely that this medium can be also successfully applied to other soybean genotypes, or to other crops with similar tissue culture procedures to promote better rooting and plant establishment in further developmental stages.

Ontogenetic Changes in Blood Osmolality During the Postembryonic Development of Zebrafish (Danio rerio).

The zebrafish Danio rerio is a teleost model species widely used in developmental genetics, biomedical studies, toxicology, and drug screening. Despite the interest of this species in research, little is known through indirect observations about its blood osmolality, which is a key parameter for diverse experiments. In this study, we directly measured blood osmolality using nano-osmometry at different stages of zebrafish postembryonic development. We found that blood osmolality is close to 240 mOsm·kg−1 in early larvae. It progressively increased to ∼270 mOsm·kg−1 during the larval development before reaching ∼300 mOsm·kg−1 after metamorphosis in juveniles and later in adults. These ontogenetic changes in blood osmolality illustrate the physiological changes in osmoregulation associated with postembryonic development, including metamorphosis. These values are of practical interest for adjusting the osmolality of fixatives and cell and tissue culture media for research using zebrafish as a model.

Significant improvement of apple (Malus domestica Borkh.) transgenic plant production by pre-transformation with a Baby boom transcription factor.

BABY BOOM (BBM) is a member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family and its expression has been shown to improve herbaceous plant transformation and regeneration. However, this improvement has not been shown clearly for tree species. This study demonstrated that the efficiency of transgenic apple (Malus domestica Borkh.) plant production was dramatically increased by ectopic expression of the MdBBM1 gene. “Royal Gala” apple plants were first transformed with a CaMV35S-MdBBM1 construct (MBM) under kanamycin selection. These MBM transgenic plants exhibited enhanced shoot regeneration from leaf explants on tissue culture media, with most plants displaying a close-to-normal phenotype compared with CaMV35S-GUS transgenic plants when grown under greenhouse conditions, the exception being that some plants had slightly curly leaves. Thin leaf sections revealed the MBM plants produced more cells than the GUS plants, indicating that ectopic-expression of MdBBM1 enhanced cell division. Transcriptome analysis showed that mRNA levels for cell division activators and repressors linked to hormone (auxin, cytokinin and brassinosteroid) signalling pathways were enhanced and reduced, respectively, in the MBM plants compared with the GUS plants. Plants of eight independent MBM lines were compared with the GUS plants by re-transforming them with an herbicide-resistant gene construct. The number of transgenic plants produced per 100 leaf explants was 0–3% for the GUS plants, 3–8% for five MBM lines, and 20–30% for three MBM lines. Our results provided a solution for overcoming the barriers to transgenic plant production in apple, and possibly in other trees.

Low CO2 Levels Are Detrimental for In Vitro Plantlets through Disturbance of Photosynthetic Functionality and Accumulation of Reactive Oxygen Species

Photosynthesis of plantlets in tissue culture containers is not considered important, compared to photosynthesis of ex vitro plants, due to the exogenous source of carbohydrates present in tissue culture media. However, CO2 starvation can generate a burst of reactive oxygen species (ROS). We examined this phenomenon in tissue culture, since CO2 levels may become very low during the light period. The research was carried out with lily scales, regenerating adventitious bulblets, and with Arabidopsis seedlings. CO2 starvation was achieved by placing a small vial of concentrated KOH solution in the culture container. CO2 removal reduced the growth of regenerated lily bulblets by 33% or 23%, with or without sucrose in the medium, respectively. In Arabidopsis seedlings, CO2 removal decreased growth by 50% or 78% in the presence or absence of sucrose in the medium, respectively. Therefore, the addition of sucrose as a replacement for photosynthesis resulted in only partial recovery of growth. Staining with nitroblue tetrazolium (NBT) showed little to no ROS in ex vitro growing seedlings, while abundant ROS were detected in seedlings grown under in vitro CO2 starvation. Seedlings grown under normal tissue culture conditions (no CO2 withdrawal) showed low levels of ROS. In lily tissue culture, CO2 starvation decreased the maximum quantum efficiency of photosystem II (Fv/Fm) from 0.69 to 0.60, and in Arabidopsis from 0.76 to 0.62. Fv/Fm of ex vitro lily and Arabidopsis seedlings was 0.77 and 0.79, respectively. This is indicative of a disturbance in photosynthesis functionality and the occurrence of in vitro stress under reduced CO2 concentrations. We conclude that poor growth, in the absence of CO2, was partly due to strongly reduced photosynthesis, while the detrimental effects were most likely due to a burst of ROS.

Generation of an inactivated vaccine for avian pathogenic Escherichia coli using microarrays: A more rational approach to inactivated vaccine design.

Background:Escherichia coli remains a major pathogen of poultry. Most vaccines are inactivated and produced empirically. Although inactivated Salmonella vaccines have been produced by culture under conditions of Fe deprivation, no vaccines have been produced which are likely to express all the proteins expressed during infection of antigen-presenting cells.Aim:The aim was to produce a more protective inactivated vaccine by culturing the avian E. coli in a synthetic medium that resembled the environment of the phagolysosome.Methods:Global gene expression in a pathogenic avian O78:K80 strain of E. coli, harvested from infected avian macrophage-like HD11 cells, was compared by microarray with bacteria cultured in a tissue culture medium. A liquid synthetic medium was produced based on the environmental conditions identified to which the bacteria were exposed intracellularly. A bacterin was produced from this strain and its protective ability was assessed in chickens.Results:The changes in E. coli gene expression observed included the use of different electron acceptors and carbon sources such as ethanolamine, β-glucosides, galactonate, dicarboxylic acids, and amino acids, up-regulation of genes associated with Fe and Mn uptake, and up-regulation of type-1 and curli fimbriae, other adhesion genes and down-regulation of sialic acid synthesis genes. The bacterin produced in the synthetic medium was statistically more protective than a bacterin prepared from bacteria cultured in the nutrient broth when tested in vaccinated chickens challenged with a different virulent E. coli O78:K80 strain.Conclusion:The approach of using gene expression to produce synthetic media for the generation of more effective bacterins could be used for a number of intracellular bacteria pathogens including Enteroinvasive E. coli, Salmonella, and the Pasteurella/Riemerella/Mannheimia group of organisms.

Preloaded descemet membrane endothelial keratoplasty grafts with endothelium outward: a validation study of the DMEK rapid device

AbstractPurposeTo validate a device (DMEK Rapid, Geuder AG, Germany) for preloading and transporting DMEK grafts with endothelium facing outwards.MethodsStripped DMEK tissues were divided into 2 groups: internal transportation simulations (study A) and cross‐country transport study (Study B). Study A was designed to investigate the influence of 2 types of media, tissue culture medium (TCM; n = 12) or transport medium (TM; n = 12), on DMEK tissues preloaded in the DMEK Rapid device. The tissues were placed on the laboratory rocker for 4 days at room temperature. For Study B, DMEK tissues (n = 9), preloaded with TM were shipped from Italy to the UK and analysed within 72 hr. For both studies, endothelial cell loss (ECL – trypan blue staining), morphology (alizarin red staining), viability (live/dead staining) and expression of biomarker (ZO‐1 immunostaining) were performed.ResultsPost‐stripping ECL was 7.5 ± 66.2% in TCM group, compared to 9.1 ± 64.9 from the TM group (p = 0.54). Post‐simulation ECL was 14.3 ± 63.8% from the TCM group and 11.5 ± 64.6% from the TM group (p = 0.08). Alizarin red (n = 4 in each group) showed the low polymorphism in both media groups. Weka segmentation of the live/dead analysis showed 78.83 ± 62.78% viability in TCM group and 80.83 ± 63.48% viable cells in TM group (p = 0.60). ZO‐1 immunostaining (n = 4 in each group) showed the presence of tight junctions in both the groups. In study B (n = 9), post‐stripping ECL was 10.1 ± 64.5% and 3.3 ± 66.6% after transporting the tissues from Italy to the UK (p = 0.05). Alizarin red (n = 3) showed proper tissue morphology and viability of 85.16 ± 65.33% was detected (n = 3). ZO‐1 (n = 3) was expressed in all tested grafts.ConclusionsPreloading DMEK tissue with endothelium outwards and transported within 72 hr using the DMEK Rapid device is safe and can be pursued for clinical application.

HD2 overexpression induces dwarfism and elevated histone deacetylase activities in transgenic Ananas comosus variety MD2

Plant tissue culture has served as one of the most efficient biotechnological tools to mass produce high quality major crops. Nevertheless, the use of this technique can result in the occurrence of somaclonal variation, which may be brought upon by genetic and/or epigenetic modulations. The epigenetic modulation of plant growth, particularly on the importance of histone deacetylation/acetylation on genome stability, transcriptional regulation, as well as plant development, has been reported in several species such as Arabidopsis, rice, and maize. However, the information on its role in regulating the growth of major crops such as pineapple is still lacking. Previously, we had reported that supplementation of high cytokinin in the tissue culture media and exposure to salinity and abscisic acid (ABA) resulted in the production of somaclonal variants that exhibited dwarf phenotypes, and that these occurrences were associated with epigenetic modulation via the involvement of histone deacetylases (HDACs). Thus, in the present study, we aimed to further elucidate and verify the involvement of histone deacetylation in the occurrence of dwarfism in tissue culture-derived pineapple plants, through HD2 gene overexpression. AtHD2 gene from Arabidopsis thaliana was synthesized and expressed in phenotypically normal Ananas comosus. Transformation efficiency was also studied, and application of 200-µM acetosyringone was observed to be more efficient than 500 µM vanillin by 10%. The transgenic plants were observed to exhibit dwarfed phenotypes and showed significantly high HDAC enzyme activities. Ploidy evaluation of the transgenic plants showed no ploidy change had occurred in the samples and remained as 2n. The relative expression level of AtHD2 was also higher (3.2-fold up-regulation) in the transgenic plants compared to the wild type plants. Collectively, these results indicate that HDACs indeed played an essential role in the response of A. comosus towards abiotic stress, and that this phenomenon is epigenetically regulated.