Carsten Theiss

Effects of thermal pretreatment on anaerobic digestion of Nannochloropsis salina biomass.

The marine microalga Nannochloropsis salina was investigated as feedstock for anaerobic digestion under batch and semi-continuous conditions for the first time. Biodegradability and methane yield were low under both digestion conditions. Thermal pretreatment prior to anaerobic digestion significantly increased the methane yield from 0.2 to 0.57 m(3) kg VS(-1) under batch conditions and from 0.13 to 0.27 m(3) kg VS(-1) in semi-continuous digestion. Still, the methane yield was limited with semi-continuous feeding due to volatile fatty acid (VFA) accumulation in the digester caused by high ammonium and salt concentrations in the feedstock. Despite VFA accumulation adaption of the microorganisms to the changing conditions and high buffer capacity resulted in steady methane production. A first energy balance considering the required heat for thermal pretreatment revealed significant benefit from the pretreatment. Conversely, the high energy demand for dewatering algal cultures is one major bottleneck for industrial-scale processing of microalgae.

Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane

The aim of the study was to determine if human limbal epithelial cells (HLEC) do not form gap junctions (GJ) during ex vivo expansion on preserved and intact human amniotic membrane (AM). Thereby, we attempt to evaluate if characteristic features of the limbal epithelial progenitor cells are preserved on AM. Primary human limbal (HLEC) and peripheral corneal (HPCEC) epithelial cells from limbal and peripheral corneal explants were cultured with SHEM either on intact AM or plastic. After 3-4 weeks, cell cultures were terminated and processed for immunofluorescence. In all cell cultures, formations of GJs were analyzed with a mouse monoclonal antibody to connexin 43 (Cx43) and a rabbit affinity purified antibody against connexin 26 (Cx26). Sections of human limbus and cornea served as positive control. Lucifer yellow (LY) known to be a GJ permeant dye was used to analyse functionality of GJ. Microinjection of LY into single cells was performed with a pressure microinjection device under visual control and with the aid of phase contrast optics. Dye spread of LY into adjacent cells indicating intercellular communication was compared between HLEC and HPCEC cultured either on AM or plastic. In vivo, a punctate pattern of Cx43 was typically found in basal and suprabasal corneal epithelial cells and labeling for Cx26 was observed in all cell layers of the human corneal epithelium, however, subpopulations of limbal basal epithelial cells lacked detectable fluorescence signals for both connexins. In HLEC cultured on AM, a scanty immunolabeling for Cx43 (12.6%) was noted, but HPCEC cultured on AM as well as HLEC cultured on plastic showed a higher labeling index (LI) for Cx43 (42.7 and 52.3%, respectively). A significant lower immunostaining for Cx26 was observed in HLEC cultured on AM (LI: 35.16%) in comparison to HLEC cultured on plastic (68.4%), as well as, HPCEC cultured either on AM or plastic (61% and 79.3%, respectively; p<0.001). Gap junctional communication was evidenced more frequently in HLEC cultured on plastic (51%, p<0.05) in contrast to HLEC cultures on AM, which exhibited a limited dye spread in 29.7% of injected cells. A significant difference in dye coupling was also evidenced between HPCEC on AM (52.9%; p<0.05) and HLEC on AM. Subpopulations of HLEC cultured on AM remain Cx43 and Cx26 negative and without functional GPs indicating that characteristic features of limbal epithelial progenitor cells might be preserved during ex-vivo expansion on AM. These data provide support to the use of the ex-vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.

The differential distribution of AMPA-receptor subunits in the tectofugal system of the pigeon

The tectofugal system of the pigeon was examined for the distribution of several glutamate-receptor subunits (AMPA Glu R1, Glu R2/3, Glu R4) and the calcium binding protein parvalbumin. With respect to the different antigens, a heterogeneous distribution was observed. Within the optic tectum, the Glu R1 like immunoreactivity was limited to the layers 2-5, 9, 10, and sparsely in layer 13, whereas the antibody to Glu R2/3 stained cell bodies in layers 9, 10, and very heavily in layer 13. In the rotundus only the Glu R4 antigen was expressed, while within the ectostriatal complex a large number of Glu R2/3 and a smaller contingent of Glu R4 positive neurons were stained. Quantitative analysis proved significant heterogeneities of these antigens in the mesencephalic as well as the diencephalic centre of the tectofugal pathway. The number of Glu R2/3 positive neurons undergoes a two-fold increase from the dorsal to the ventral lamina 13 of the optic tectum. Alterations in the amount of immunoreactive neurons were also observed within the rotundus, since the number of Glu R4 positive cells decreased from dorsal to ventral. Morphological differences and their correlation with functional specializations in visual information processing are discussed.

The receptor architecture of the pigeons’ nidopallium caudolaterale: an avian analogue to the mammalian prefrontal cortex

The avian nidopallium caudolaterale is a multimodal area in the caudal telencephalon that is apparently not homologous to the mammalian prefrontal cortex but serves comparable functions. Here we analyzed binding-site densities of glutamatergic AMPA, NMDA and kainate receptors, GABAergic GABAA, muscarinic M1, M2 and nicotinic (nACh) receptors, noradrenergic α1 and α2, serotonergic 5-HT1A and dopaminergic D1-like receptors using quantitative in vitro receptor autoradiography. We compared the receptor architecture of the pigeons’ nidopallial structures, in particular the NCL, with cortical areas Fr2 and Cg1 in rats and prefrontal area BA10 in humans. Our results confirmed that the relative ratios of multiple receptor densities across different nidopallial structures (their “receptor fingerprints”) were very similar in shape; however, the absolute binding densities (the “size” of the fingerprints) differed significantly. This finding enables a delineation of the avian NCL from surrounding structures and a further parcellation into a medial and a lateral part as revealed by differences in densities of nACh, M2, kainate, and 5-HT1A receptors. Comparisons of the NCL with the rat and human frontal structures showed differences in the receptor distribution, particularly of the glutamate receptors, but also revealed highly conserved features like the identical densities of GABAA, M2, nACh and D1-like receptors. Assuming a convergent evolution of avian and mammalian prefrontal areas, our results support the hypothesis that specific neurochemical traits provide the molecular background for higher order processes such as executive functions. The differences in glutamate receptor distributions may reflect species-specific adaptations.

PEX14 is required for microtubule-based peroxisome motility in human cells

We have established a procedure for isolating native peroxisomal membrane protein complexes from cultured human cells. Protein-A-tagged peroxin 14 (PEX14), a central component of the peroxisomal protein translocation machinery was genomically expressed in Flp-In-293 cells and purified from digitonin-solubilized membranes. Size-exclusion chromatography revealed the existence of distinct multimeric PEX14 assemblies at the peroxisomal membrane. Using mass spectrometric analysis, almost all known human peroxins involved in protein import were identified as constituents of the PEX14 complexes. Unexpectedly, tubulin was discovered to be the major PEX14-associated protein, and direct binding of the proteins was demonstrated. Accordingly, peroxisomal remnants in PEX14-deficient cells have lost their ability to move along microtubules. In vivo and in vitro analyses indicate that the physical binding to tubulin is mediated by the conserved N-terminal domain of PEX14. Thus, human PEX14 is a multi-tasking protein that not only facilitates peroxisomal protein import but is also required for peroxisome motility by serving as membrane anchor for microtubules.

A ternary complex consisting of AICD, FE65, and TIP60 down-regulates Stathmin1

The ternary complex consisting of AICD/FE65/TIP60 is thought to play a role in gene expression and was suggested to have a crucial impact in Alzheimers disease. AICD is the intracellular subdomain of the amyloid precursor protein (APP) and able to bind the adapter protein FE65 and the histone acetyltransferase TIP60 setting up a nuclear dot-like phenotype. Within this work we readdressed the generation of the complex as a function of its compartments. Subsequently, we studied the proteome of AFT expressing cells vs. controls and identified Stathmin1 significantly down-regulated in AFT cells. Stathmin1 functions as an important regulatory protein of microtubule dynamics and was found associated with neurofibrillary tangles in brains of Alzheimers disease patients. We validated our results using an independent label-free mass spectrometry based method using the same cell culture model. In a reversal model with diminished APP expression, caused by simultaneous knock-down of all three members of the APP family, we further confirmed our results, as Stathmin1 was regulated in an opposite fashion. We hypothesize that AICD-dependent deregulation of Stathmin1 causes microtubule disorganization, which might play an important role for the pathophysiology of Alzheimers disease.

Impact of vegf on astrocytes: Analysis of gap junctional intercellular communication, proliferation, and motility

The purpose of the present study was to investigate the effects of vascular endothelial growth factor (VEGF) on gap junctional intercellular communication (GJIC), cell proliferation, and cell dynamics in primary astrocytes. VEGF is known as a dimeric polypeptide that potentially binds to two receptors, VEGFR‐1 and VEGFR‐2, however many effects are mediated by VEGFR‐2, for example, actin polymerization, forced cell migration, angiogenesis, and cell proliferation. Recently it has been shown that in case of hypoxia, ischemia or injury VEGF is upregulated to stimulate angiogenesis and cell proliferation. Besides this, VEGF reveals a potent therapeutical target for averting tumor vascularization, emerging in bevacizumab, the first humanized anti‐VEGF‐A antibody for treating recurrent Glioblastoma multiforme. To expand our knowledge about VEGF effects in glial cells, we cultivated rat astrocytes in medium containing VEGF for 1 and 2 days. To investigate the effects of VEGF on GJIC, we microinjected neurobiotin into a single cell and monitored dye‐spreading into adjacent cells. These experiments showed that VEGF significantly enhances astrocytic GJIC compared with controls. Cell proliferation measured by BrdU‐labeling also revealed a significant increase of astrocytic mitose rates subsequent to 1 day of VEGF exposure, whereas longer VEGF treatment for 2 days did not have additive effects. To study cell‐dynamics of astrocytes subsequent to VEGF treatment, we additionally transfected astrocytes with LifeAct‐RFP. Live‐cell imaging and quantitative analysis of these cells with aid of confocal laser scanning microscopy revealed higher process movement of VEGF‐treated astrocytes. In conclusion, VEGF strongly affects cell proliferation, GJIC, and motility in astrocytes.

Correlation of cell cycle kinetics with p63 expression in human limbal epithelial cells expanded on intact human amniotic membrane.

Aim: To analyse the correlation of p63 expression and cell cycle kinetics of human limbal epithelial cells (HLECs) expanded on amniotic membrane (AM) or plastic. Methods: Primary HLECs were cultured either on cryopreserved intact AM or plastic dishes for 2 weeks. Cells were labelled with 5 μM 5-bromo-2′-deoxyuridine (BrdU) for 3 days, followed by an interval of either 7 or 14 days in BrdU-free medium. The expression of p63 and BrdU labelling was detected by immunocytochemistry. Results: More cells on AM (56%) than on plastic (24%) retained their BrdU label after the 7-day interval (p < 0.001). The difference was even more pronounced after 14 days (20 and 2%, p < 0.001). All BrdU-labelled cells were also p63 positive. 2.5-fold more cells on AM (54%) than on plastic (21%) were BrdU positive/p63 positive after 7 days (p < 0.001). It increased to a 9-fold difference after 14 days (p < 0.001). The BrdU label was lost more quickly than the p63 expression during the observation period, indicating that p63 expression was not confined to stem cells but existed also in transient amplifying cells. Conclusions: The combination of p63 expression and BrdU label retention is a better criterion to characterize stemness than either marker alone. AM as a culture substrate preserves stemness better than plastic.

Decreased keratocyte death after laser-assisted subepithelial keratectomy and photorefractive keratectomy in rabbits.

Purpose: To compare keratocyte loss in the corneal stroma after laser‐assisted subepithelial keratectomy (LASEK) and photorefractive keratectomy (PRK) in rabbits. Setting: Department of Ophthalmology, University of Essen, Essen, and the Institute of Anatomy, University of Bochum, Bochum, Germany. Methods: Laser‐assisted subepithelial keratectomy and PRK were performed in rabbits and studied 1, 3, 10, and 20 days after surgery. Excimer photoablation was done unilaterally with a 6.0 mm ablation zone and an 80 μm depth, equivalent to −6.0 diopters. Keratocyte death was analyzed using DNA fragmentation‐detecting terminal deoxynucleotidyl transferase deoxy‐UTR‐nick end labeling (TUNEL) assay and transmission electron microscopy. Results: Numerous TUNEL‐positive keratocytes occurred 1 day after PRK; the number decreased significantly after 3 days. After LASEK, significantly fewer TUNEL‐positive keratocytes were noted at the early time points (P<.001 at 1 day; P≤.05 at 3 days). At 10 days, the number of TUNEL‐positive keratocytes decreased in both groups but remained significantly higher after PRK than after LASEK (P<.001). Twenty days after both procedures, no significant signs of keratocyte death were found in the corneal stroma. Transmission electron microscopy revealed few apoptotic keratocytes after LASEK. After PRK, apoptotic keratocytes, characterized by chromatin condensation, apoptotic bodies, and cell shrinkage, were scattered in the stroma. The ultrastructural findings confirmed the results obtained with the TUNEL assay. Conclusions: Laser‐assisted subepithelial keratectomy induced significantly less apoptotic keratocyte death than PRK and promoted wound healing in the acute phase after photoablation. This procedure may offer the possibility of treating higher myopia with a decreased risk for developing wound healing‐related complications known to occur after PRK.

Distribution of BDNF, NT-3, trkB and trkC in the developing retino-tectal system of the pigeon (Columba livia)

The distribution of the neurotrophins BDNF and NT-3 as well as their corresponding high-affinity receptors trkB and trkC was characterized by immunohistochemistry in the developing retino-tectal system of the pigeon. These neurotrophins are known to be important for survival and development of neuronal tissues, but also for activity-dependent neuronal plasticity. In pigeons visual asymmetry is established at the morphological and behavioral level due to a natural asymmetrical light input before hatch, which is followed by a posthatch period of consolidation with unbiased light stimulation. Since the retino-tectal system is the crucial entity of these events, we studied the retinal and the tectal distribution of these neurotrophins and their receptors during retino-tectal formation, to analyze the developmental sequences to which these neurotrophins are tuned. Here we demonstrate that in altricial pigeons no retinal immunolabeling of BDNF, NT-3 or their receptors could be detected before hatch, although a prominent tectal labeling pattern throughout most layers was evident. After hatch, both neurotrophins and their receptors showed a dramatic increase of retinal and tectal distribution. While the tectal and retinal protein synthesis of NT-3 vanished after 2 weeks, that of BDNF could still be revealed in adults. Therefore, the establishment of the retino-tectal system does not seem to depend on these neurotrophins before hatch, although they are probably utilized to shape the intratectal wiring pattern. In contrast, BDNF and NT-3 could play a prominent role in posthatch retino-tectal plasticity, as the consolidation of tectal asymmetries requires posthatch modifications of tectal circuits and proceeds within the first two posthatching weeks. These data are comparable with the distribution of neurotrophins in the retino-tectal system of chicks, although the onset of neurotrophin synthesis seems to be earlier in precocial chicks.