Site-selective multi-porphyrin attachment enables the formation of a next-generation antibody-based photodynamic therapeutic

Herein we present a significant step towards next-generation antibody-based photodynamic therapeutics. Site-selective modification of a clinically relevant monoclonal antibody, with a serum-stable linker bearing a strained alkyne, allows for the controlled Cu-free “click” assembly of an in vitro active antibody-based PDT agent using a water soluble azide porpyhrin

Regioselective and stoichiometrically controlled conjugation of photodynamic sensitizers to a HER2 targeting antibody fragment

The rapidly increasing interest in the synthesis of antibody–drug conjugates as powerful targeted anticancer agents demonstrates the growing appreciation of the power of antibodies and antibody fragments as highly selective targeting moieties. This targeting ability is of particular interest in the area of photodynamic therapy, as the applicability of current clinical photosensitizers is limited by their relatively poor accumulation in target tissue in comparison to healthy tissue. Although synthesis of porphyrin–antibody conjugates has been previously demonstrated, existing work in this area has been hindered by the limitations of conventional antibody conjugation methods. This work describes the attachment of azide-functionalized, water-soluble porphyrins to a tratuzumab Fab fragment via a novel conjugation methodology. This method allows for the synthesis of a homogeneous product without the loss of structural stability associated with conventional methods of disulfide modification. Biological evaluation of the synthesized conjugates demonstrates excellent selectivity for a HER2 positive cell line over the control, with no dark toxicity observed in either case

TGFβ upregulates PAR-1 expression and signalling responses in A549 lung adenocarcinoma cells

The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an avβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2-and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin av and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer

Functionalized Cyclopentenones with Low Electrophilic Character as Anticancer Agents

In this study were synthesized non-Michael acceptor cyclopentenones (CP) from biomass derivative furfural as anticancer agents. Cyclic enones, both from natural sources and synthetic analogues, have been described as cytotoxic agents. Most of these agents were unsuccessful in becoming valuable therapeutic agents due to toxicity problems derived from unselective critical biomacromolecule alkylation. This may be caused by Michael addition to the enone system. Ab initio studies revealed that 2,4-substituted CPs are less prone to Michael additions, and as such were tested three families of those derivatives. We prepare the new CPs from furfural through a tandem furan ring opening/nazarov electrocyclization and further functionalization. Experimentally the 2,4-substituted CPs exhibited no reactivity towards sulphur nucleophiles, while maintaining cytotoxicity against HT-29, MCF-7, NCI-H460, HCT-116 and MDA-MB 231 cells lines. Moreover, the selected CP are non-toxic against healthy HEK 293T cell lines and present proper calculated drug-like properties

Potential new fluoroquinolone treatments for suspected bacterial keratitis.

Topical fluoroquinolones (FQs) are an established treatment for suspected microbial keratitis. An increased FQ resistance in some classes of bacterial pathogens is a concern. Some recently developed FQs have an extended spectrum of activity, making them a suitable alternative for topical ophthalmic use. For example, the new generation FQs, avarofloxacin, delafloxacin, finafloxacin, lascufloxacin, nadifloxacin, levonadifloxacin, nemonoxacin and zabofloxacin have good activity against the common ophthalmic pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae and several of the Enterobacteriaceae However, because there are no published ophthalmic break-point concentrations, the susceptibility of an isolated micro-organism to a topical FQ is extrapolated from systemic break-point data and wild type susceptibility. The purpose of this review is to compare the pharmacokinetics and pharmacodynamics of the FQs licensed for topical ophthalmic use with the same parameters for new generation FQs. We performed a literature review of the FQs approved for topical treatment and the new generation FQs licensed to treat systemic infections. We then compared the minimum inhibitory concentrations (MIC) of bacterial isolates and the published concentrations that FQs achieved in the cornea and aqueous. We also considered the potential suitability of new generation FQs for topical use based on their medicinal properties. Notably, we found significant variation in the reported corneal and aqueous FQ concentrations so that reliance on the reported mean concentration may not be appropriate, and the first quartile concentration may be more clinically relevant. The provision of the MIC for the microorganism together with the achieved lower (first) quartile concentration of a FQ in the cornea could inform management decisions such as whether to continue with the prescribed antimicrobial, increase the frequency of application, use a combination of antimicrobials or change treatment

Sequencing of \u3ci\u3eAspergillus nidulans\u3c/i\u3e and comparative analysis with \u3ci\u3eA. fumigatus\u3c/i\u3e and \u3ci\u3eA. oryzae\u3c/i\u3e

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso, and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation. Document includes all supplementary information (820 pages). Supplementary files are also attached below as Related files. THERE IS NO SUPPLEMENTARY FILE #7. PDF file size (with supplementary files included) is 10 Mbytes. An optimized version of the ARTICLE ONLY is attached as a Related File and is 1.9 Mbytes

Sequencing of \u3ci\u3eAspergillus nidulans\u3c/i\u3e and comparative analysis with \u3ci\u3eA. fumigatus\u3c/i\u3e and \u3ci\u3eA. oryzae\u3c/i\u3e

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso, and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation. Document includes all supplementary information (820 pages). Supplementary files are also attached below as Related files. THERE IS NO SUPPLEMENTARY FILE #7. PDF file size (with supplementary files included) is 10 Mbytes. An optimized version of the ARTICLE ONLY is attached as a Related File and is 1.9 Mbytes

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis

Aspects of free radical cyclisation

A model study was carried out to investigate the use of a novel 1,5 allylic abstraction-cyclisation reaction, in a proposed synthesis of the ring skeleton of the Pentalenolactone group of structures. The model study led to isolation of a product resulting from a stereospecific 6-exo-trig radical cyclisation. A new 1,5 allylic abstraction-cyclisation reaction has been developed, which has been used for the synthesis of 6,5 bicyclic ring systems, a spirocyclic ring system and cyclic ring systems. The analysis of results and possible implications are discussed. The development of a new synthetic method for the construction of both carbocyclic ring systems and oxygen containing heterocyclic ring systems was investigated. This method was based upon a 1,6 allylic abstraction-cyclisation reaction. The syntheses of the required precursors proved problematic but were eventually prepared in poor yield. Treatment of the various precursors under a variety of radical conditions did not lead to the any of the desired cyclisations. Some 1,6 and 1,7 diene systems were prepared and were found to undergo radical stannylation-cyclisation reactions, on treatment with tri-n-butyl and triphenyltin hydride. An interesting regiospecific cyclisation resulted from one of these investigations. Removal of the tin group from the cyclised products could not be effected.(DX889571)</p