High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal,Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.     

Funktionelle Morphologie des Geruchsorgans und Histologie der Kopfanhänge der NasenmuräneRhinomuraena ambonensis (Teleostei,Anguilliformes)

Zusammenfassung 1. An drei, wahrscheinlich adulten Exemplaren der Nasenmuräne,Rhinomuraena ambonensis Barbour, wurden die funktionelle Morphologie des Geruchsorgans sowie dessen Histologie und die der bartelartigen Kopfanhänge untersucht.2. Die Ränder der vorderen Riechöffnungen sind zu trichterförmigen Hautlappen ausgewachsen. Die in ihrer Ausrichtung und Form durch Körperbewegungen kaum beeinflußbaren Trichter leiten auch im Ruhezustand des Tieres Wasser in die Riechhöhle, da ihre weite Öffnung im Strömungsbereich des Atemwassers liegt. Auf diese Weise erfolgt eine effektive Ventilation der Riechhöhle.3. Im mehrschichtigen Epithel der Trichter liegen Sekretzellen und vereinzelt Sinnesknospen; unmittelbar über den Epithelbildungszellen verlaufen Nervenfasern. Im Bindegewebe, das beidseitig von Epithel bedeckt ist, liegen Gefäße, Nervenbündel (Nervus trigeminus) und Pigmentzellen. Muskelgewebe fehlt. Neben dem Bindegewebe gibt es kein anderes Stützgewebe.4. Die beiden Riechhöhlen sind von extremer Ausdehnung. Die Zahl der lamellenartigen Riechfalten, die in zwei Doppelreihen nach dem 90°-Typ liegen, beträgt ca. 200. Zwischen den Innenrändern der Falten einer Doppelreihe, dorsal von der Mittelraphe, erstreckt sich eine zylindrische Rinne, die der Duftwasserpassage dient.5. Die Riechfalten sind bis auf die lateralen, dorsalen Teile und Teile der ventralen Faltenansätze kontinuierlich mit Riechepithel bedeckt. Der histologische Aufbau des Riechepithels entspricht dem des Aals. Die Rezeptorendichte schwankt zwischen 2–3×10⁴ pro mm². Im Verhältnis zur Riechhöhlenausdehnung ist die Riechfeldgröße erheblich.6. Tuscheexperimente zeigten, daß — ausgelöst durch die in die Längsrinne der Riechhöhle ragenden fingerförmigen Innenränder der Riechfalten — Mikroturbulenzen entstehen, die neben der Flimmerbewegung durch Kinocilien für die Mikroventilation des Faltensystems sorgen.7. Das auf der Symphyse des Oberkiefers zwischen den Trichterbasen sitzende Rostrum ist eine Hautprojektion, deren bindegewebiger Zentralteil ein mehrschichtiges Epithel trägt. Im Bindegewebe sind Gefäße, feine Nerven und Pigmentzellen eingebettet. Im Epithel finden sich geschmacksknospenähnliche Sinnesorgane. Prinzipiell haben die drei Mandibularbarteln den gleichen histologischen Aufbau wie das Rostrum. Auch ihr Epithel weist Sinnesknospen auf, die wie jene des Rostrums und der Trichter vielleicht als chemische Sinnesorgane beim Nahrungstest fungieren.8. Nach dem Ergebnis vorliegender Untersuchungen wirdRhinomuraena ambonensis in die Gruppe der makrosmatischen Knochenfische eingereiht.

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Functional morphology of the olfactory organ and histology of the head appendages of the moray eelRhinomuraena ambonensis (Teleostei, Anguilliformes)

In three individuals of the moray eelRhinomuraena ambonensis (Barbour), the functional morphology and histology of the olfactory organ as well as the histology of the different head appendages were investigated. The edges of the anterior nares are extremely extended, forming a funnel-like wide opening through which the odour water passes. Even when the fish does not move, an effective ventilation of the olfactory chambers is maintained by a permanent water current towards the funnel opening induced by the peristaltic movements of the musculature of the gill chamber (principle ofBernoulli). The histological structure of the head appendages (funnels, rostrum and mandibulary barbels, which are all skin projections) is described. The epithelia of all these appendages bear sensory organs which are similar to taste buds. The two olfactory organs are of extremely large size. Each chamber contains about 100 olfactory lamellae which are arranged in two lines (90°-type;Holl 1965). A cylindrical cavity extends between the inner edges of the olfactory lamellae through which the water runs to the posterior nare. Interlamellar microventilation is produced by numerous turbulences which are caused by the edges of the olfactory lamellae and by the kinocilia of the olfactory epithelium. The histological structure of the olfactory epithelium is similar to that ofAnguilla anguilla. The different results demonstrate thatRhinomuraena ambonensis is probably a macrosmatic fish.

     

Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.     

Surface functionalization allowing repetitive use of optical sensors for real‐time detection of antibody‐bacteria interaction

In this study, sensor surface functionalization allowing the repetitive use of a sensing device was evaluated for antibody‐based detection of living bacteria using an optical planar Bragg grating sensor. To achieve regenerable immobilization of bacteria specific antibodies, the heterobifunctional cross‐linker N‐succinimidyl 3‐(2‐pyridyldithio) propionate (SPDP) was linked to an aminosilanized sensor surface and subsequently reduced to expose sulfhydryl groups enabling the covalent conjugation of SPDP‐activated antibodies via disulfide bonds. The immobilization of a capture antibody specific for Staphylococcus aureus on the sensor surface as well as specific binding of S. aureus could be monitored, highlighting the applicability of optical sensors for the specific detection of large biological structures. Reusability of bacteria saturated sensors was successfully demonstrated by cleaving the antibody along with bound bacteria through reduction of disulfide bonds and subsequent re‐functionalization with activated antibody, resulting in comparable sensitivity towards S. aureus.

     

Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform

Background

Genetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation.

Results

We developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). Two uAA, propargyl-L-lysine (Plk) and (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Alk), were incorporated at the same site into eGFP co-expressing the native PylRS/tRNA^Pyl _CUA pair originating from Methanosarcina barkeri in E. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelized E. coli cultivations was correlated to (i) uAA concentration and the (ii) time of uAA addition to the expression medium as well as to induction parameters including the (iii) time and (iv) amount of IPTG supplementation. The online measurements of the HTMS were consolidated by end point-detection using standard enzyme-linked immunosorbent procedures.

Conclusion

The developed HTMS is powerful tool for parallelized and rapid screening. In light of uAA integration, future applications may include parallelized screening of different PylRS/tRNA^Pyl _CUA pairs as well as further optimization of culture conditions.

     

Electron microscopic study of the origin and formation of Reissner's fiber in the subcommissural organ of Cebus apella (Primates,Platyrrhini)

Summary Two kinds of secretion are formed in cells of the subcommissural organ (SCO) of Cebus apella. The light secretion is found in saccules originating from the endoplasmic reticulum. This secretion is stored in the peripheral portion of the cells and is not involved in formation of Reissner's fiber (RF). In close association with the Golgi complex, electron-dense granules are developed, containing a finely granular substance. These granules accumulate beneath the apical plasmalemma of the cell. Their content is discharged into the third ventricle, where it occurs in the form of a thin layer of secretion. This material appears to constitute the RF at the level of the entrance to the mesencephalic aqueduct.     

Apical protuberances of supporting cells in the regio olfactoria of the mole,Talpa europaea,Linnaeus, 1758 (Insectivora,Talpidae)

Summary The regio olfactoria of the mole, Talpa europaea, was studied by scanning and transmission electron microscopy. Peculiar structural differentiations, i.e. ovoid-shaped, balloon-like protuberances were found on the surface of the supporting cells. The apical portion of these protuberances contained finely dispersed granular material, whereas in their central part vesicular extensions of the smooth endoplasmic reticulum were observed.