LNA-enhanced DNA FIT-probes for multicolour RNA imaging.

The simultaneous imaging of different RNA molecules in homogeneous solution is a challenge and requires optimisation to enable unambiguous staining of intracellular RNA targets. Our approach relies on single dye forced intercalation (FIT) probes, in which a visco-sensitive reporter of the thiazole orange (TO) family serves as a surrogate nucleobase and provides enhancements of fluorescence upon hybridisation. Previous FIT probes spanned the cyan and green emission range. Herein, we report for the first time chromophores for FIT probes that emit in the red range (above 600 nm). Such probes are valuable to overcome cellular auto-fluorescent background and enable multiplexed detection. In order to find suitable chromophores, we developed a submonomer approach that facilitated the rapid analysis of different TO family dyes in varied sequence positions. A carboxymethylated 4,4'-methine linked cyanine, which we named quinoline blue (QB), provided exceptional response characteristics at the 605 nm emission maximum. Exceeding previously reported base surrogates, the emission of the QB nucleotide intensified by up to 195-fold upon binding of complementary RNA. Owing to large extinction coefficients and quantum yields (up to ε = 129.000 L mol-1 cm-1 and Φ = 0.47, respectively) QB-FIT probes enable imaging of intracellular mRNA. A mixture of BO-, TO- and QB-containing FIT probes allowed the simultaneous detection of three different RNA targets in homogenous solution. TO- and QB-FIT probes were used to localize oskar mRNA and other polyadenylated mRNA molecules in developing oocytes from Drosphila melanogaster by means of wash-free fluorescent in situ hybridisation and super resolution microscopy (STED).

Total chemical synthesis of proteins without HPLC purification.

The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2-6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8-33% overall yield with 90-98% purity despite the omission of HPLC purification.

Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the ß2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By contrast, the experiments with a more rigid, duplex-spaced assembly revealed marked distance dependencies. Consequences for the function of adaptor proteins are discussed.

Acceleration of thiol additive-free native chemical ligation by intramolecular S → S acyl transfer.

Peptide-mercaptopropionylcysteine (MPA-Cys) thioesters show a surprisingly high reactivity in native chemical ligation (NCL) and allow thiol-additive free reactions. This facilitates sequential NCL reactions and ligation-desulfurization reactions in one-pot formats. The synthetic utility is demonstrated by the synthesis of a SH3 domain.

Concomitant chemoradiotherapy versus induction chemotherapy followed by chemoradiotherapy as definitive, first line treatment of squamous cell carcinoma of the head and neck. A retrospective single center analysis.

PURPOSE: Despite the lack of evidence to support its implementation in the clinical practice, induction chemotherapy (IC) before chemoradiotherapy (CRT) is often used in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN). We retrospectively examined the tolerability, feasibility, and clinical outcome of both concepts in a single center analysis. PATIENTS AND METHODS: In all, 83 patients were treated between 2007 and 2010 with IC + CRT (n = 42) or CRT alone (n = 41). IC consisted of docetaxel, cisplatin and 5-fluorouracil (TPF), or cisplatin and 5-fluorouracil (PF). All patients were scheduled to receive 2 cycles of PF during concurrent CRT. Adverse events were assessed according to the common toxicity criteria of adverse events (CTCAE v. 3.0). Associations were tested using the χ² test, and survival estimates were calculated according to Kaplan-Meier. RESULTS: The median follow-up was 30.35 months (range 2.66-61.25 months). At 2 years, the overall survival rate was significantly higher for primary CRT compared to IC + CRT group (74.8 % vs. 54 %, respectively; p = 0.041). Significantly more treatment-related overall grade 4 toxicities were documented in the IC + CRT group compared to the CRT group (42.9% vs. 9.8%; p = 0.001). Renal toxicity ≥ grade 2 occurred in 52.4 % vs. 7.3 % (p < 0.001), respectively. In all, 93 % of the patients with primary CRT compared to 71 % with IC + CRT received the planned full radiotherapy dose (p = 0.012). CONCLUSION: This is, to our knowledge, the largest retrospective study to compare IC + CRT with primary CRT. IC showed high acute toxicity, compromised the feasibility of concurrent CRT, and was associated with reduced overall survival rates compared to primary CRT. The lack of clinical benefit in conjunction with the increased toxicity does not support implementation of IC.

Tumour-infiltrating lymphocytes predict response to definitive chemoradiotherapy in head and neck cancer.

BACKGROUND: We aimed to investigate the prognostic value of tumour-infiltrating lymphocytes' (TILs) expression in pretreatment specimens from patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT). METHODS: The prevalence of CD3+, CD8+, CD4+ and FOXP3+ TILs was assessed using immunohistochemistry in tumour tissue obtained from 101 patients before CRT and was correlated with clinicopathological characteristics as well as local failure-free- (LFFS), distant metastases free- (DMFS), progression-free (PFS) and overall survival (OS). Survival curves were measured using the Kaplan-Meier method, and differences in survival between the groups were estimated using the log-rank test. Prognostic effects of TIL subset density were determined using the Cox regression analysis. RESULTS: With a mean follow-up of 25 months (range, 2.3-63 months), OS at 2 years was 57.4% for the entire cohort. Patients with high immunohistochemical CD3 and CD8 expression had significantly increased OS (P=0.024 and P=0.028), PFS (P=0.044 and P=0.047) and DMFS (P=0.021 and P=0.026) but not LFFS (P=0.90 and P=0.104) in multivariate analysis that included predictive clinicopathologic factors, such as age, sex, T-stage, N-stage, tumour grading and localisation. Neither CD4 nor FOXP3 expression showed significance for the clinical outcome. The lower N-stage was associated with improved OS in the multivariate analysis (P=0.049). CONCLUSION: The positive correlation between a high number of infiltrating CD3+ and CD8+ cells and clinical outcome indicates that TILs may have a beneficial role in HNSCC patients and may serve as a biomarker to identify patients likely to benefit from definitive CRT.

Role of wound macrophages in skin flap loss or survival in an experimental diabetes model.

BACKGROUND: Tightly controlled wound inflammation is a central determinant of skin flap survival in healthy mice. This study investigated inflammatory response patterns in caudally pedicled skin flaps in diabetic mice during severely impaired conditions of necrotic skin flap tissue loss. METHODS: Skin flap biopsies were analysed by RNase protection assay, quantitative real-time polymerase chain reaction, immunohistochemistry, enzyme-linked immunosorbent assay and immunoblotting. RESULTS: Skin flaps were characterized by the necrotic loss of tissue starting from distal areas of the flaps in diabetic mice. Decay of epidermal and dermal structures within skin flap tissue was paralleled by an immune cell-mediated expression of chemokines (macrophage inflammatory protein 2, macrophage chemoattractant protein 1), cyclo-oxygenase (COX) 2 and inducible nitric oxide synthase (iNOS). Distal regions of necrotic skin flap tissue were infiltrated by excess numbers of neutrophils and macrophages, and the latter were polarized towards a proinflammatory state as they expressed COX-2 and iNOS. Experimental depletion of inflammatory macrophages inhibited necrotic destruction of the distal skin flap tissue in diabetic mice despite the persistence of neutrophil infiltration and inflammation. CONCLUSION: Wound macrophages play a pivotal role in determining the survival or loss of skin flap tissue under disturbed wound healing conditions in obese diabetic mice.

Resorbable collagen membrane in surgical repair of fistula following palatoplasty in nonsyndromic cleft palate.

Treatment of palato-nasal fistula following primary palatoplasty in patients with nonsyndromic cleft palate is often complicated by recurrence. The authors have tested the feasibility of a surgical technique adding a resorbable collagen membrane at the bony edge of the fistula and report the outcome in the first 14 patients in an open, non-comparative, preliminary investigation. The procedure was well tolerated by all patients, with no relapses during follow up ranging from 4 to 12 months.

[Ultrasonic bone cutting in oral surgery: a review of 60 cases]. / Ultraschallbasiertes Knochenschneiden in der Oralchirurgie: eine Ubersicht anhand von 60 Patientenfällen.

PURPOSE: Surgical reconstruction of bony defects in the oral cavity can often be challenging since thin and fragile bony structures are especially prone to fracture cased by bulky cutting tips or the application of significant pressure by conventional mechanical instruments. The risk of accidental damage to adjacent soft tissue structures, such as nerves, by a dental drill or saw is also extremely high. The use of modulated ultrasound (piezosurgery) makes it possible to overcome such complications as a result of the precise and minimally invasive surgery technique which is limited to mineralized hard tissue. MATERIALS AND METHODS: In 60 patients (38 male, 22 female) a piezosurgery device was used for different bone augmentation procedures before dental implant placement. The instrument uses modulated ultrasound (25 - 30 kHz) and the amplitude of the working tip ranges from 60 mum to 200 mum. The device was employed for sinus floor elevation (25), alveolar ridge augmentation using an autogenous block graft (25), alveolar ridge splitting (5) or lateralization of the alveolar nerve (5). Physiological sodium chloride was used as a cooling solution. For all osteotomies mode boosted burst c and pump 5 were used. RESULTS: Piezoelectric osteotomy permitted micrometric selective cutting and a clear surgical site due to the cavitation effect created by the cooling solution and the oscillating tip. No excessive bleeding was encountered. The risk of accidental soft tissue harm, such as perforating the sinus membrane or damaging adjacent nerves, was definitely lower than in the case of a conventional bur. No serious complications were encountered in the postoperative wound healing process after 2, 14, 30 and 90 days. However, the surgical procedures were time-consuming. CONCLUSION: Piezosurgery is an advantageous osteotomy technique for delicate structures in the oral and maxillofacial region. With respect to osteotomies of thin and fragile bones, the application of ultrasound is superior to other mechanical instruments because of the extremely precise and virtually arbitrary cut geometries, easy handling, efficient bone ablation and minimal accidental damage to adjacent soft tissue structures.

Convergent strategies for the attachment of fluorescing reporter groups to peptide nucleic acids in solution and on solid phase.

The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation probes that can report the presence of a particular DNA sequence. This paper describes convergent methods for the solution- and solid-phase synthesis of multiply labelled PNA oligomers. The solid-phase synthesis of protected PNA enabled the selective attachment of fluorescent labels at the C-terminal end (3' in DNA) which demonstrated that further manipulations on protected PNA fragments are feasible. For the conjugation to internal sites, a method is introduced that allows for the on-resin assembly of modified monomers thereby omitting the need to synthesise an entire monomer in solution. Furthermore, it is shown that the application of a highly orthogonal protecting group strategy in combination with chemoselective conjugation reactions provides access to a rapid and automatable solid-phase synthesis of dual labelled PNA probes. Real-time measurements of nucleic acid hybridisation were possible by taking advantage of the fluorescence resonance energy transfer (FRET) between suitably appended fluorophoric groups. Analogously to DNA-based molecular beacons, the dual labelled PNA probes were only weakly fluorescing in the single-stranded state. Hybridisation to a complementary oligonucleotide, however, induced a structural reorganisation and conferred a vivid fluorescence enhancement.

Sequence fidelity of a template-directed PNA-ligation reaction.

The ligation method and an appended duplex-stabilizing dye affect both yield and sequence selectivity of a template-controlled PNA-ligation. The highest selectivity was obtained with a peptide condensation that formed an abasic site.

Synthesis of a triply phosphorylated pentapeptide from human tau-protein.

Two different strategies for the synthesis of a triply phosphorylated pentapeptide are described. In both cases a monophosphorylated selectively N-deprotected tripeptide is employed as C-terminal fragment. Coupling of this building block with a C-terminally unmasked bis-phosphorylated seryl-dipeptide unexpectedly failed due to decomposition of this peptide upon activation with different coupling reagents. Instead stepwise N-terminal elongation of the peptide chain with serine derivatives and subsequent O-phosphorylation of the serine OH-groups was successful. These results indicate that assembly of multiply phosphorylated peptides from preformed multiply phosphorylated phosphopeptide building blocks in general may be problematic and that a stepwise elongation of the amino acid chain may be preferable.

Glycopeptide synthesis and the effects of glycosylation on protein structure and activity.

Despite the omnipresence of protein glycosylation in nature, little is known about how the attachment of carbohydrates affects peptide and protein activity. One reason is the lack of a straightforward method to access biologically relevant glycopeptides and glycoproteins. The isolation of homogeneous glycopeptides from natural sources is complicated by the heterogeneity of naturally occuring glycoproteins. It is chemical and chemoenzymatic synthesis that is meeting the challenge to solve this availability problem, thus playing a key role for the advancement of glycobiology. The current art of glycopeptide synthesis, albeit far from being routine, has reached a level of maturity that allows for the access to homogeneous and pure material for biological and medicinal research. Even the ambitious goal of the total synthesis of an entire glycoprotein is within reach. It is demonstrated that with the help of synthetic glycopeptides the effects of glycosylation on protein structure and function can be studied in molecular detail. For example, in immunology, synthetic (tumour-specific) glycopeptides can be used as immunogens to elicit a tumour-cell-specific immune response. Again, synthetic glycopeptides are an invaluable tool to determine the fine specificity of the immune response that can be mediated by both carbohydrate-specific B and T cells. Furthermore, selected examples for the use of synthetic glycopeptides as ligands of carbohydrate-binding proteins and as enzyme substrates or inhibitors are presented.

Chemically Modified Antisense Oligonucleotides-Recent Improvements of RNA Binding and Ribonuclease H Recruitment.

The incorporation of rationally designed sugar or nucleobase modifications into oligonucleotides gives remarkable improvements of the antisense activity. For example, the cytosine analogue 1 recognizes both the Watson-Crick and the Hoogsteen site of a guanine nucleotide 2, which dramatically enhances the RNA-binding affinity and selectivity.

A Convergent Strategy for the Modification of Peptide Nucleic Acids: Novel Mismatch-Specific PNA-Hybridization Probes.

Coupling of nonnatural nucleobases to the orthogonally protected backbone 1 on the solid phase provided access to novel peptide nucleic acid (PNA) conjugates 2, which are difficult to synthesize by standard routes. Hybridization probes containing the thiazolorange dye might allow DNA sequence analysis in real time. B-CH(2)CO=modified nucleobase, fluorescent dye, etc; Boc, Fmoc=protecting groups.

A sequencing strategy for the localization of O-glycosylation sites of MUC1 tandem repeats by PSD-MALDI mass spectrometry.

It is demonstrated with glycopeptides of the polymorphic epithelial mucin (MUC1) that post-source decay matrix-assisted laser desorption ionization (PSD-MALDI) is a fast, highly sensitive, and reproducible method for the localization of O-glycosylation sites by reflectron time-of-flight (TOF) mass spectrometry. We have analyzed GalNAc-carrying peptides of up to 25 amino acids, and could distinguish even neighboring glycosylation sites. This method was also able to localize and characterize disaccharides (e.g., the Thomsen-Friedenreich disaccharide) on MUC1 derived peptides. PSD-MALDI-MS fragment ion patterns were recorded in the positive ion mode from the synthetic peptide TAP25 [(T1aAPPAHGVT9S10APDT14RPAPGS20) T1bAPPA], an overlapping sequence of MUC1 tandem repeats, which was glycosylated with GaINAc in vitro. The glycosylation sites found were either Thr9 or Thr1b in the monoglycosylated, Thr9 and Thr1b in the diglycosylated, and Thr9, Thr1b, and Ser20 in the triglycosylated peptide. A single PSD-MALDI-MS spectrum of the underivatized and uncleaved di- or triglycosylated TAP25 peptide was sufficient to identify the glycosylation sites, thereby distinguishing six potential, partly adjacent, glycosylation sites. The monoglycosylated fraction was found to consist of a mixture of two glycosylated species with the same molecular weight. This was shown by the analysis of proteolytic digests. PSD-MALDI-MS of the resulting peptides right out of the digestion probe was sufficient to identify the Gal-NAc-glycosylation sites as either Thr9 or Thr1b, respectively. Beyond the methodical aspects the results revealed that in vitro glycosylation of the TAP25 peptide with a transferase system from human milk differs from that obtained with a breast cancer cell transferase system.

Stimulated elastase release from human leukocytes: influence of anti-asthmatic, anti-inflammatory and calcium antagonist drugs in vitro.

In the present study the effects of drugs, with different modes of action, on FMLP-stimulated release of elastase from human leukocytes were investigated in vitro. Anti-asthmatic and anti-allergic drugs were compared to well-known anti-inflammatory and anti-histamine agents. The anti-asthmatic/anti-allergic compounds azelastine, astemizole and oxatomide, and the 5-lipoxygenase inhibitor NDGA, were able to suppress the release of elastase from human leukocytes in concentrations between 10 and 100 microM. NSAIDs such as indomethacin, diclofenac and piroxicam and the glucocorticoids dexamethasone and hydrocortisone showed little or no activity. The histamine H1 antagonists mepyramine and ketotifen and the calcium antagonists verapamil, nifedipine and TMB-8 were also ineffective in suppressing FMLP-induced elastase release. Reduction in elastase release by azelastine, that accumulates in lung tissue during long-term treatment in animals, may contribute to its anti-inflammatory and anti-allergic effects which are thought to be central to its use in asthma therapy.