Benthic macroinvertebrates along the Czech part of the Labe and lower section of the Vltava rivers from 1996–2005, with a particular focus on rare and alien species

In the Czech part of the Labe River and the lower part of the Vltava River, we examined if the benthic macroinvertebrate composition changed from 1996 to 2005 due to expected improvements in water quality resulting from socioeconomic changes in the Czech Republic since the 1990s. Special attention was given to rare and alien species. The four biological metrics used (Number of taxa, BMWP, Number of sensitive taxa, and Number of EPT taxa) demonstrated that there was indeed an improvement in water quality as well as a slight improvement of the Labe microhabitats during the investigated period. An increasing Number of taxa over time was observed at most sites. Two main concurrent ecological processes are recently in progress in the Labe: a recovery of native species and an expansion of alien species, some of which are considered invasive. The caddisfly Setodes punctatus and the beetle Pomatinus substriatus, considered as regionally extinct in the Czech Republic until 2005, were rediscovered during our investigations. Findings of the crustacean Hemimysis anomala (invasive) and the chironomids Stenochironomus sp. and Lipiniella sp. were the first records of these taxa in the Czech Republic.     

Mitochondrial Extrusion through the cytoplasmic vacuoles during cell death

Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.     

Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

Lignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.     

Dentinogenic potential of human adult dental pulp cells during the extended primary culture

Despite the frequent use of primary dental pulp cells in dental regenerative research, few systematic studies of stemness for osteogenic and dentinogenic differentiation of human adult pulp cells have been reported. To investigate the stemness of human adult dental pulp cells, pulp tissues were obtained from extracted third molars and used as a source of pulp cells. In FACS analysis and immunophenotyping, the general mesenchymal stem cell markers CD44, CD90, and CD146 were highly expressed in early passages of the pulp cell culture. The stem cell population was dramatically decreased in an expansion culture of human dental pulp cells. When pulp cells were treated with additives such as β-glycerophosphate, ascorbic acid, and dexamethasone, nodule formation was facilitated and mineralization occurred within 2 weeks. Expression of osteogenic markers such as alkaline phosphatase, osteocalcin, and osteonectin was relatively low in undifferentiated cells, but increased significantly under differentiation conditions in whole passages. Dentinogenic markers such as dentin sialophosphoprotein and dentin matrix protein-1 appeared to decrease in their expression with increasing passage number; however, peak levels of expression occurred at around passage 5. These data suggested that stem cells with differentiation potential might exist in the dental pulp primary culture, and that their phenotypes were changed during expansion culture over 8-9 passages. Under these conditions, a dentinogenic population of pulp cells occurred in limited early passages, whereas osteogenic cells occurred throughout the whole passage range.     

Carrier‐Selectivity‐Dependent Charge Recombination Dynamics in Organic Photovoltaic Cells with a Ferroelectric Blend Interlayer

Interfacial energetics determines the performance of organic photovoltaic (OPV) cells based on a thin film of organic semiconductor blends. Here, an approach to modulating the “carrier selectivity” at the charge collecting interfaces and the consequent variations in the nongeminate charge carrier recombination dynamics in OPV devices are demonstrated. A ferroelectric blend interfacial layer composed of a solution‐processable ferroelectric poly­mer and a wide bandgap semiconductor is introduced as a tunable electron selective layer in inverted OPV devices with non‐Ohmic contact electrodes. The direct rendering of dipole alignment within the ferroelectric blend layer is found to increase the carrier selectivity of the charge collecting interfaces up to two orders of magnitude. Transient photovoltaic analyses reveal that the increase of carrier selectivity significantly reduces the diffusion and recombination among minority carriers in the vicinity of the electrodes, giving rise to the 85% increased charge carrier lifetime. Furthermore, the carrier‐selective charge extraction leads to the constitution of the internal potential within the devices, even with energetically identical cathodes and anodes. With these carrier‐selectivity‐controlled interlayers, the devices based on various photoactive materials commonly display significant increments in the device performances, especially with the high fill factor of up to 0.76 under optimized conditions.     

Platelet derived growth factor BB is a ligand for dermatan sulfate chain(s) of small matrix proteoglycans from normal and fibrosis affected fascia

Structural requirements of the short isoform of platelet derived growth factor BB (PDGF-BB) to bind dermatan sulfate (DS)/chondroitin sulfate (CS) are unknown. Meanwhile the interaction may be important for tissue repair and fibrosis which involve both high activity of PDGF-BB and matrix accumulation of DS. We examined by the solid phase assay the growth factor binding to DS chains of small proteoglycans from various fasciae as well as to standard CSs. Before the assay a structural analysis of DSs and CSs was accomplished involving the evaluation of their epimerization and/or sulfation patterns. In addition, in vivo acceptors for PDGF-BB in fibrosis affected fascia were detected. PDGF-BB binding sites on DSs/CSs are located in long chain sections with the same type of hexuronate isomer however without any apparent preference to glucuronate or iduronate residues. Alternatively, the interaction seems to involve two shorter DS chain sections assembling disaccharides with the same type of hexuronate isomer which are separated by disaccharide(s) with another hexuronate one. Moreover, DS/CS affinity to the growth factor most probably depends on an accumulation of di-2,4-O-sulfated disaccharides in binding site while the presence of 6-O-sulfated N-acetyl-galactosamine residues rather attenuates the binding. All examined fascia DSs and standard CSs showed significant PDGF-BB binding capability with the highest affinity found for normal palmar fascia decorin DS. In fibrosis affected palmar fascia DS/CS proteoglycans are able to form with PDGF-BB supramolecular complexes also including other matrix components such as type III collagen and fibronectin which bind the growth factor covalently. Our results suggest that DS chains of fascia matrix small PGs may regulate PDGF-BB availability leading to restriction of fibrosis associated with Dupuytren's disease or to control of normal fascia repair.     

Pyrazolone-fused combretastatins and their precursors: synthesis,cytotoxicity, antitubulin activity and molecular modeling studies

A series of pyrazolone-fused combretastatins and precursors were synthesized and their cytotoxicity as well as antitubulin potential was evaluated. The hydrazide 9f and the pyrazolone-fused combretastatins 12a, 12b and 12c were highly cytotoxic against various tumor cell lines including cisplatin resistant cells. The same compounds were also the best inhibitors of tubulin polymerization. Molecular modeling results showed that they bind the colchicine binding site at the tubulin heterodimer. The hydrazide 9f arrested HeLa cells in the G2/M phase of the cell cycle and strongly affected cell shape and microtubule network.     

Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons

Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain-type neural precursor (NP) cells that proliferate in vitro by specific mitogens. We investigated the potential of these NP cells derived from hES cells (hES-NP) for the large-scale generation of human dopamine (DA) neurons for functional analyses and therapeutic applications. To address this, hES-NP cells were expanded in vitro for 1.5 months with six passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES-NP cell number was increased by > 2 × 10⁴-folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity toward neuronal cells, exhibiting in vitro pre-synaptic DA neuronal functionality. Furthermore, the hES-NP cells can be cryopreserved without losing their proliferative and developmental potential. Upon transplantation into a Parkinson's disease rat model, the multi-passaged hES-NP cells survived, integrated into the host striatum, and differentiated toward the neuronal cells expressing DA phenotypes. A significant reduction in the amphetamine-induced rotation score of Parkinson's disease rats was observed by the cell transplantation. Taken together, these findings indicate that hES-NP cell expansion is exploitable for a large-scale generation of experimental and transplantable DA neurons of human-origin.     

The novel miR-9500 regulates the proliferation and migration of human lung cancer cells by targeting Akt1

MicroRNAs have crucial roles in lung cancer cell development. They regulate cell growth, proliferation and migration by mediating the expression of tumor suppressor genes and oncogenes. We identified and characterized the novel miR-9500 in human lung cancer cells. The miR-9500 forms a stem-loop structure and is conserved in other mammals. The expression levels of miR-9500 were reduced in lung cancer cells and lung cancer tissues compared with normal tissues, as verified by TaqMan miRNA assays. It was confirmed that the putative target gene, Akt1, was directly suppressed by miR-9500, as demonstrated by a luciferase reporter assay. The miR-9500 significantly repressed the protein expression levels of Akt1, as demonstrated via western blot, but did not affect the corresponding mRNA levels. Akt1 has an important role in lung carcinogenesis, and depletion of Akt1 has been shown to have antiproliferative and anti-migratory effects in previous studies. In the current study, the overexpression of miR-9500 inhibited cell proliferation and the expression of cell cycle-related proteins. Likewise, the overexpression of miR-9500 impeded cell migration in human lung cancer cells. In an in vivo assay, miR-9500 significantly suppressed Fluc expression compared with NC and ASO-miR-9500, suggesting that cell proliferation was inhibited in nude mice. Likewise, miR-9500 repressed tumorigenesis and metastasis by targeting Akt1. These data indicate that miR-9500 might be applicable for lung cancer therapy.MicroRNAs (miRNAs) are small, non-coding RNAs, 18–25 nucleotides (nt) in length that regulate gene expression by binding to the 3′-untranslated region (UTR) of their target genes,^{1, 2} and these RNAs are processed from introns, exons or intergenic regions.³ First, miRNAs are transcribed by RNA polymerase II into primary miRNA (pri-miRNA) molecules that contain several thousand nucleotides. The pri-miRNAs are then sequentially processed by a microprocessor, such as Drosha RNase III endonuclease and DiGeorge syndrome region gene 8 protein (DGCR8), to form ∼70 nt-stem-loop intermediates known as miRNA precursors (pre-miRNAs).^{4, 5} The pre-miRNAs are then exported from the nucleus into the cytoplasm via Exportin-5 (EXP5), with its cofactor Ran-GTP; in the cytoplasm, these pre-miRNAs are processed into 18–25 nt mature miRNA duplexes by the RNase III endonuclease Dicer.^{6, 7} The mature miRNA duplexes, along with the Argonaute proteins, are integrated as single-stranded RNAs into an RNA-induced silencing complex, which induces either the cleavage or the translational inhibition of the targeted mRNAs.^{8, 9, 10} miRNAs have been implicated in a variety of biological processes associated with cancer development, including cell proliferation and invasion,¹¹ and miRNA expression is deregulated in many forms of cancer.¹²Cancer is a major public health problem worldwide. Lung cancer represents one of the most predominant types of cancer, with high mortality rates in both men and women. Epithelial lung cancer can be categorized into one of two types: small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). NSCLC accounts for ∼80% of lung cancer cases, and these cases can be further categorized as adenocarcinoma (40%), squamous cell carcinoma (30–35%), and large cell carcinoma (5–15%). NSCLC has a 5-year survival rate of only 16%.^{13, 14, 15} Current studies have shown that miRNAs are deregulated in various cancers, including NSCLC, and may act as oncogenes or tumor suppressor genes.¹⁶ For example, the Let-7 family,¹⁷ miR-15a/16,¹⁸ miR-17-92,¹⁹ miR-107 and miR-185,²⁰ are deregulated in lung cancer.Some studies have reported that phosphatidylinositol 3-kinase (PI3K) signaling is activated in human cancers^{21, 22} and has an important role in the progression of NSCLC. The PI3K pathway modulates several cellular mechanisms, such as cell survival, proliferation, migration and motility, and thereby significantly affects the growth of tumors.^{23, 24} The primary regulator of the PI3K pathway is Akt, a protein kinase B that mediates cell survival, cell death,²⁵ cell growth, cell migration and angiogenesis.^{26, 27, 28} The silencing of the Akt1 gene has been shown to inhibit the proliferation of gastric cancer cells both in vitro and in vivo.²⁹ Other studies have shown that aberrant AKT activation has a critical role in tumorigenesis.³⁰In this study, we identified small RNAs in lung cancer cells. To analyze a novel miRNA signature, we examined the structure and sequence of the small RNAs, analyzed the expression patterns of the novel miRNAs in lung cancer tissues and assessed the miRNA target genes. Our data revealed that miR-9500 regulates certain human lung cancer cell functions, including cell growth, proliferation, and migration.