The Manipulation of Micro-organisms for the Production of Secondary Metabolites

Abstract

Toxicity testing with animals is expensive, ethically controversial, and not always predictive of the human response. Cell-based assays are regarded as an alternative. However, conventional two-dimensional cell cultures do not reproduce the tissue architecture in vivo, and do not forecast organ-specific toxicity. On the other hand, three-dimensional cultures emulate the biochemistry and mechanics of the microenvironment in tissues more closely. Therefore, they address the limitations of both animals and two-dimensional cultures, and provide more accurate data on the effects of short- and long-term exposure to toxicants. We provide an up-to-date overview on the use of three-dimensional cell cultures in toxicology. We anticipate that three-dimensional cultures will become invaluable to accomplish the 3R agenda (refinement, reduction, and replacement) for animal-based toxicity testing and will play a major role for the Registration, Evaluation and Authorisation of Chemicals in the European Union (REACH legislation).     

Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma: a prerequisite for adoptive cell transfer

Background aimsAdoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies.MethodsIn a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr⁵¹ release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion.ResultsTIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8⁺ and CD4⁺ subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells.ConclusionThe procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.     

An allied reprogramming,selection, expansion and differentiation platform for creating hiPSC on microcarriers

ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

We have developed an allied protocol for reprogramming, selecting, expanding and differentiating human pluripotent stem cells on Microcarriers (designated as RepMC). This method allows faster reprogramming, selecting 30‐50‐fold more candidates for characterization and also allows us to find high quality candidates that differentiate to cardiomyocytes and blood lineages. Mechanistically, this method appears to accelerate the induction, maturation and stabilization phases of reprogramming. Our findings help simplify the process of deriving and expanding iPSCs for therapeutic applications, offering a robust and scalable suspension platform for large‐scale generation of clinical grade iPSCs.     

Microbe–microbe interactions in mixed culture food fermentations

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Highlights► Microbial interactions in food fermentations are crucial for product functionality. ► Complex microbial consortia tolerate more variation in the environment. ► Knowledge of communities of fermenting microbes drives diversification of starter cultures. ► Use of complex starter cultures requires new propagation methodologies. ► Immobilised cell technology is a high potential solution for propagating complex cultures.     

Metabolism of non-phenolic β-O-4 lignin model compounds by the white-rot fungus Phlebia radiata

Summary The degradation of three non-phenolic -O-4 diarylpropane lignin model compounds was studied in cultures of the white-rot fungus Phlebia radiata. The degradation pattern of the model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) was also compared with that of Phanerochaete chrysosporium under conditions where both fungi were cultivated without agitation in an oxygen atmosphere. Compound I was readily degraded by both fungi, and qualitatively the degradation patterns were quite similar. The product, after C-C bond cleavage, was veratraldehyde (IV) which was almost stoichiometrically reduced to veratryl alcohol (V). However, large amounts of V were detected only in P. chrysosporium cultures. Experiments with the model compound 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (II) showed that in the presence of II, the total amount of veratryl compounds accounted for 15–33 m in standing cultures of Phlebia radiata. The model compound 1-(3,4-dimethoxyphenyl)-2-(4-methoxyphenoxy) propane-1,3-diol (III) was more readily degraded than I and II. The results indicated that, in P. radiata cultures, the acting enzymes were lignin peroxidases and IV reducing enzyme, while laccase was less important. Offprint requests to: A. Hatakka     

Infección múltiple fúngica en un paciente diabético

BackgroundMixed fungal infections although undervalued, are more common than mentioned in the scientific literature. These infections have a poor prognosis for the patient.ObjectivesWe present an unusual case of a 61-year-old diabetic male who had a rhino-orbito-sinusal zygomycosis in 2001. After surgical debridement of the infected parts, along with antifungal therapy with liposomal amphotericin B, the patient started improving. Several years later the patient was hospitalized due to a similar problem and was diagnosed of rhino-orbito-cerebral zygomycosis.MethodsIn both episodes, a histopathological examination and cultures were performed on the sinus lesions. Tissue sections were stained with haematoxylin and eosin, Giemsa, periodic acid-Schiff (PAS) and Grocott's methenamine silver, and cultures specific for fungi were performed.ResultsThe histopathology studies revealed the presence of bacteria, actinomyces and a mixed infection by at least four different fungi, all of them well differentiated by their morphology. Despite the rapid diagnosis the patient died due to spreading to the central nervous system.ConclusionsMixed infections by fungi are rare, but due to the high incidence of immunodeficiencies they could occur more often than reported. We would like to alert on the possibility of acquired mixed infection by fungi which have shown to be high aggressive and have a worse prognostic in patients with underlying diseases.     

Evaluating the Use of Blood Cultures in the Management of Children Hospitalized for Community-Acquired Pneumonia

BackgroundBlood cultures are often recommended for the evaluation of community-acquired pneumonia (CAP). However, institutions vary in their use of blood cultures, and blood cultures have unclear utility in CAP management in hospitalized children.ObjectiveTo identify clinical factors associated with obtaining blood cultures in children hospitalized with CAP, and to estimate the association between blood culture obtainment and hospital length of stay (LOS).MethodsWe performed a multicenter retrospective cohort study of children admitted with a diagnosis of CAP to any of four pediatric hospitals in the United States from January 1, 2011-December 31, 2012. Demographics, medical history, diagnostic testing, and clinical outcomes were abstracted via manual chart review. Multivariable logistic regression evaluated patient and clinical factors for associations with obtaining blood cultures. Propensity score-matched Kaplan-Meier analysis compared patients with and without blood cultures for hospital LOS.ResultsSix hundred fourteen charts met inclusion criteria; 390 children had blood cultures obtained. Of children with blood cultures, six (1.5%) were positive for a pathogen and nine (2.3%) grew a contaminant. Factors associated with blood culture obtainment included presenting with symptoms of systemic inflammatory response syndrome (OR 1.78, 95% CI 1.10–2.89), receiving intravenous hydration (OR 3.94, 95% CI 3.22–4.83), receiving antibiotics before admission (OR 1.49, 95% CI 1.17–1.89), hospital admission from the ED (OR 1.65, 95% CI 1.05–2.60), and having health insurance (OR 0.42, 95% CI 0.30–0.60). In propensity score-matched analysis, patients with blood cultures had median 0.8 days longer LOS (2.0 vs 1.2 days, P < .0001) without increased odds of readmission (OR 0.94, 95% CI 0.45–1.97) or death (P = .25).ConclusionsObtaining blood cultures in children hospitalized with CAP rarely identifies a causative pathogen and is associated with increased LOS. Our results highlight the need to refine the role of obtaining blood cultures in children hospitalized with CAP.     

Psoralea Esculent A as a Prairie Resource: An Ethnographic Appraisal

Abstract

The role of the prairie turnip, Psoralea esculenta Pursh, in the economy of Prairie and Plains cultures is examined through ethnographic and early eyewitness accounts. Results suggest that these economies were more reliant on vegetal resources than is often conceded, a conclusion in keeping with recent studies of hunter-gatherers. The concept of the upland prairies as an insignificant vegetal resource zone for prehistoric groups is questioned.     

Use of bacteria for rapid,pH-neutral,hydrolysis of the model hydrophobic carboxylic acid ester p-nitrophenyl picolinate

Abstract

Caulobacter crescentus, Escherichia coli and Bacillus subtilis cultures promote the hydrolysis of the model ester p-nitrophenyl picolinate (PNPP) at neutral pH with high efficiency. Hydrolysis is related to cell concentration, while the interaction of PNPP with both bacterial cells and their extracellular molecules is required for a maximum rate of PNPP hydrolysis in C. crescentus cultures. Furthermore, C. crescentus cultures hydrolyse PNPP at concentrations useful in synthetic chemistry.     

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell–multilayered periosteal sheets in vitro

Background aimsCultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell–extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges.MethodsPeriosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively.ResultsReal-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium–expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets.ConclusionsIntegrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.     

Chemical constituents isolated from Paeonia lactiflora roots and their neuroprotective activity against oxidative stress in vitro

The methanolic extract of Paeonia lactiflora roots significantly protected primary cultures of rat cortical cells exposed to oxidative stress induced by H₂O₂. Seven monoterpenes, paeonilactone-B (1), paeonilactone-C (2), paeoniflorigenone (3), benzoylpaeoniflorin (4), paeoniflorin (5), oxypaeoniflorin (6) and albiflorin (7), were isolated by bioactivity-guided fractionation and further separation using chromatographic techniques. Among them, compounds 2 and 4 significantly protected primary cultures of rat cortical cells against H₂O₂-induced neurotoxicity.     

A Retrospective Evaluation of Critical Care Blood Culture Yield – Do Support Services Contribute to the “Weekend Effect”?

BackgroundThe “weekend effect” describes an increase in adverse outcomes for patients admitted at the weekend. Critical care units have moved to higher intensity working patterns to address this with some improved outcomes. However, support services have persisted with traditional working patterns. Blood cultures are an essential diagnostic tool for patients with sepsis but yield is dependent on sampling technique and processing. We therefore used blood culture yield as a surrogate for the quality of support service provision.We hypothesized that blood culture yields would be lower over the weekend as a consequence of reduced support services.MethodsWe performed a retrospective observational study examining 1575 blood culture samples in a university hospital critical care unit over a one-year period.ResultsPatients with positive cultures had, on average, higher APACHE II scores (p = 0.015), longer durations of stay (p = 0.03), required more renal replacement therapy (p<0.001) and had higher mortality (p = 0.024). Blood culture yield decreased with repeated sampling with an increased proportion of contaminants. Blood cultures were 26.7% less likely to be positive if taken at the weekend (p = 0.0402). This effect size is the equivalent to the impact of sampling before and after antibiotic administration.ConclusionsOur study demonstrates that blood culture yield is lower at the weekend. This is likely caused by delays or errors in incubation and processing, reflecting the reduced provision of support services at the weekend. Reorganization of services to address the “weekend effect” should acknowledge the interdependent nature of healthcare service delivery.     

Ricerche Sulla Cultura Sommersa di Cellule Singole di Piante Superiori

Abstract

Research on submerged culture of single cells of higher plants. — The author describes a method which allows to obtain submerged cultures of single cells of Phaseolus vulgaris and Nicotiana tabacum. The medium composition in macroelements in the culture on agar appears to effect to a great extent the ability of tissues to dissociate into single cells in the subsequent liquid culture. In this respect Heller's solution results to be more suitable than Gautheret's and Hildebrandt and Ri-ker's.

Cells are grown at 24 [ddot]C in 300 ml flasks containing 60 ml of broth on a rotary shaker at 220 rpm.

To prevent contaminations some antibacterial agents were added to cultures of Phaseolus vulgaris. Among these Penicillin and Neomycin were not tossic at 20 and 5 ppm concentrations respectively.

The presence of septa, which are observed also in largely vacuolate cells, seems to confirm the ability of single cells to divide.

The optimum 2,4-D concentration for growth decreases from 6 × 10^-8 to 6 × 10^-8 during successive liquid cultures, each of them being inoculated with on amount of the previous one. This fact, showing the adaptation of liquid cultures to decreasing concentrations of the growth hormone, is in agreement with previous observations in solid cultures by several authors.     

A xeno-free culture method that enhances Wharton's jelly mesenchymal stromal cell culture efficiency over traditional animal serum–supplemented cultures

Background aimsMesenchymal stromal cell (MSC) transplantation holds great promise for use in medical therapies. Several key features of MSCs, including efficient cell growth, generation of sufficient cell numbers and safety, as determined by teratoma formation, make MSCs an ideal candidate for clinical use. However, MSCs derived under standard culture conditions, co-cultured with animal by-products, are inappropriate for therapy because of the risks of graft rejection and animal virus transmission to humans. Alternative serum sources have been sought for stem cell production.MethodsWe demonstrate for the first time that human serum from umbilical cord blood (hUCS) is an effective co-culture reagent for MSC production from Wharton's jelly MSCs (WJMSCs). Ten umbilical cords were used to generate parallel cultures of WJMSC lines under medium supplemented with hUCS and embryonic stem cell-qualified fetal bovine serum. The WJMSC lines from each medium were analyzed and compared with regard to cell line derivation, proliferation ability and characteristic stability.ResultsThe phenotypic characteristics of WJMSC derived under either medium showed no differences. WJMSC lines derived under hUCS medium displayed comparable primary culture cell outgrowth, lineage differentiation capacity and cell recovery after cryopreservation compared with supplementation with embryonic stem cell-qualified fetal bovine serum medium. However, superior cell proliferation rates and retention of in vitro propagation (>22 passages) were observed in WJMSC cultures supplemented with hUCS. Additionally, more robust population doubling times were observed in hUCS-supplemented cultures.ConclusionsWe conclude that hUCS is an efficient and effective serum source for animal product–free WJMSC line production and can generate MSC lines that may be appropriate for therapeutic use.     

Vegetative growth and reproduction of Fossombronia brasiliensis Steph.: the influence of photoperiod,temperature and inorganic nitrogen source

Abstract

Monoclonal cultures of Fossombronia brasiliensis were grown with different photoperiods, temperatures and inorganic nitrogen sources. The subsequent vegetative growth and production of gametangia is described. A quantitative analysis of the numbers of antheridia and archegonia and their relative proportions is given. At 18°C, F. brasiliensis was found to be a short-day plant, with a critical night length of between six and twelve hours, while at 10°C it exhibited quantitative SD plant properties. Plants produced more male gametangia at 18°C and more female gametangia at 10°C. Nitrogen as nitrate consistently produced more gametangia than the ammonium source.     

Formation of Ni- and Zn-Sulfides in Cultures of Sulfate-Reducing Bacteria

The purpose of this study was to characterize Ni- and Zn-sulfides precipitated in sulfate-reducing bacterial cultures. Fe-free media containing 58 mM SO ₄ ^2? were amended with Ni and Zn chloride followed by inoculation. Precipitates were sampled from cultures after two weeks of incubation at 22, 45, and 60 ^° C. Abiotic controls were prepared by reacting bacteria-free liquid media with Na ₂ S solutions under otherwise identical conditions. Precipitates were collected anaerobically, freeze-dried and analyzed by x-ray diffraction (XRD), scanning electron microscopy, and for total Ni, Zn, and S. In Ni-containing media, biogenic sulfide precipitates were mostly heazelwoodite (Ni ₃ S ₂ ), whereas abiotic precipitates were mixed heazelwoodite and vaesite (NiS ₂ ). The biogenic Ni-precipitates were better crystalline than the corresponding abiotic samples. Sphalerite (ZnS) was identified by XRD in precipitates sampled from Zn-containing media. Scanning electron microscopy revealed disordered morphological features for the sulfides, which occurred mostly as aggregates of fine particles in biogenic samples, whereas abiotic precipitates contained more plate- and needle-like structures.     

Esporotricosis linfangítica bilateral y simultánea

BackgroundSporotrichosis is the most frequent subcutaneous mycoses in Jalisco, Mexico. The forms of transmission described in the literature are from bites of different animals and injuries due to utensils.AimsWe present an unusual case with bilateral and lymphangitic cutaneous lesions in the upper limbs caused by a pocket gopher bite (Geomys bursarius).MethodsMycology studies were performed on the arm lesions, including Gram and Ziehl Neelsen stains, direct exam, Sabouraud and mycobiotic cultures at temperatures of 25–28 °C.ResultsGram and Ziehl Neelsen stains were negative. Sporothrix schenckii grew in the culture plates. Treatment with saturated potassium iodide solution was prescribed and four months later complete remissions of the lesions were achieved, and the control cultures were negative.ConclusionsThe most common clinical presentations of sporotrichosis are the fixed and lymphangitic forms. Bilateral lymphangitic sporotrichosis is rare.     

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

Background aimsMesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation.MethodsMSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells.ResultsHuman L-MSC cultures were typically CD34⁻, CD45⁻ and HLA-DR⁻ and CD73⁺, CD90⁺, CD105⁺ and HLA-ABC⁺. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation.ConclusionsL-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.