A protein functionalization platform based on selective reactions at methionine residues.

Nature has a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a marked increase in their functional diversity1. Inspired by this, chemical tools have been developed for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations that are suitable for effective protein functionalization is limited, because the stringent demands inherent to biological systems preclude the applicability of many potential processes2. These chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near-perfect conversion2-7. Although many bioconjugation methods exist at cysteine, lysine and tyrosine, a method targeting a less-explored amino acid would considerably expand the protein functionalization toolbox. Here we report the development of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the electrophilic reactivity of a bespoke hypervalent iodine reagent, the S-Me group in the side chain of methionine can be targeted. The bioconjugation reaction is fast, selective, operates at low-micromolar concentrations and is complementary to existing bioconjugation strategies. Moreover, it produces a protein conjugate that is itself a high-energy intermediate with reactive properties and can serve as a platform for the development of secondary, visible-light-mediated bioorthogonal protein functionalization processes. The merger of these approaches provides a versatile platform for the development of distinct transformations that deliver information-rich protein conjugates directly from the native biomacromolecules

Active surveillance of Q fever in human and animal population of Cyprus

BACKGROUND: A long-term active surveillance of Q fever was conducted in Cyprus organized in two phases. METHODS: Following serological tests and identification of seropositive humans and animals for C. burnetii in two villages (VIL1 and VIL2), all seronegative individuals were followed up for one year on a monthly basis by trained physicians to detect possible seroconversion for Q fever. In the second phase of the study, active surveillance for one year was conducted in the entire Cyprus. Physicians were following specific case definition criteria for Q fever. Standardized questionnaires, a geographical information system on a regional level, Immunofluorescence Assay (IFA) examinations and shell vial technique were used. RESULTS: Eighty-one seronegative humans and 239 seronegative animals from both villages participated in the first phase surveillance period of Q fever. Despite the small number of confirmed clinical cases (2 humans and 1 goat), a significant percentage of new seropositives for C. burnetii (44.4% of human participants and 13.8% of animals) was detected at the end of the year. During the second phase of surveillance, 82 humans, 100 goats, and 76 sheep were considered suspected cases of Q fever. However, only 9 human, 8 goat, and 4 sheep cases were serologically confirmed, while C. burnetii was isolated from three human and two animal samples. The human incidence rate was estimated at 1.2 per 100,000 population per year. CONCLUSION: A small number of confirmed clinical cases of Q fever were observed despite the high seroprevalence for C. burnetii in human and animal population of Cyprus. Most of the cases in the local population of Cyprus appear to be subclinical. Moreover further studies should investigate the role of ticks in the epidemiology of Q fever and their relation to human seropositivity

Natural killer cells are crucial for the efficacy of Icon (factor VII/human IgG1 Fc) immunotherapy in human tongue cancer

<p>Abstract</p> <p>Background</p> <p>Icon is a novel, dual neovascular- and cancer cell-targeting immunotherapeutic agent and has shown efficacy in the treatment of cancer, wet form macular degeneration and endometriosis. However, its underlying mechanism remains to be investigated. The objective of this study is to elucidate the mechanism of Icon immunotherapy in cancer using a squamous carcinoma human tongue cancer line TCA8113 <it>in vitro </it>and <it>in vivo </it>in severe combined immunodeficiency (SCID) mice.</p> <p>Results</p> <p>We showed that Icon, as a chimeric factor VII and human IgG1 Fc immunoconjugate, could separately induce murine natural killer (NK) cells and activate complement to kill TCA8113 cancer cells <it>in vitro </it>via antibody dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, Icon-NK ADCC had a significantly stronger effect than that of Icon-CDC. Moreover, Icon could completely eradicate established human tongue tumour xenografts <it>in vivo </it>in the CB-17 strain of SCID mice that have functional NK cells at a normal level, whereas it was less effective in SCID/Beige mice that do not have functional NK cells.</p> <p>Conclusions</p> <p>We conclude that NK cells are crucial for the efficacy of Icon immunotherapy in the treatment of cancer. The results also suggest that impaired NK level/activity could contribute to the resistance to therapeutic antibodies that are currently under investigation in preclinical and clinical studies.</p

Tuning Curvature and Stability of Monoolein Bilayers by Designer Lipid-Like Peptide Surfactants

This study reports the effect of loading four different charged designer lipid-like short anionic and cationic peptide surfactants on the fully hydrated monoolein (MO)-based Pn3m phase (Q224). The studied peptide surfactants comprise seven amino acid residues, namely A6D, DA6, A6K, and KA6. D (aspartic acid) bears two negative charges, K (lysine) bears one positive charge, and A (alanine) constitutes the hydrophobic tail. To elucidate the impact of these peptide surfactants, the ternary MO/peptide/water system has been investigated using small-angle X-ray scattering (SAXS), within a certain range of peptide concentrations (R≤0.2) and temperatures (25 to 70°C). We demonstrate that the bilayer curvature and the stability are modulated by: i) the peptide/lipid molar ratio, ii) the peptide molecular structure (the degree of hydrophobicity, the type of the hydrophilic amino acid, and the headgroup location), and iii) the temperature. The anionic peptide surfactants, A6D and DA6, exhibit the strongest surface activity. At low peptide concentrations (R = 0.01), the Pn3m structure is still preserved, but its lattice increases due to the strong electrostatic repulsion between the negatively charged peptide molecules, which are incorporated into the interface. This means that the anionic peptides have the effect of enlarging the water channels and thus they serve to enhance the accommodation of positively charged water-soluble active molecules in the Pn3m phase. At higher peptide concentration (R = 0.10), the lipid bilayers are destabilized and the structural transition from the Pn3m to the inverted hexagonal phase (H2) is induced. For the cationic peptides, our study illustrates how even minor modifications, such as changing the location of the headgroup (A6K vs. KA6), affects significantly the peptide's effectiveness. Only KA6 displays a propensity to promote the formation of H2, which suggests that KA6 molecules have a higher degree of incorporation in the interface than those of A6K

C-type lectin-like domains in Fugu rubripes

BACKGROUND: Members of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. Three genome-level studies on human, C. elegans and D. melanogaster reported previously demonstrated almost complete divergence among invertebrate and mammalian families of CTLD-containing proteins (CTLDcps). RESULTS: We have performed an analysis of CTLD family composition in Fugu rubripes using the draft genome sequence. The results show that all but two groups of CTLDcps identified in mammals are also found in fish, and that most of the groups have the same members as in mammals. We failed to detect representatives for CTLD groups V (NK cell receptors) and VII (lithostathine), while the DC-SIGN subgroup of group II is overrepresented in Fugu. Several new CTLD-containing genes, highly conserved between Fugu and human, were discovered using the Fugu genome sequence as a reference, including a CSPG family member and an SCP-domain-containing soluble protein. A distinct group of soluble dual-CTLD proteins has been identified, which may be the first reported CTLDcp group shared by invertebrates and vertebrates. We show that CTLDcp-encoding genes are selectively duplicated in Fugu, in a manner that suggests an ancient large-scale duplication event. We have verified 32 gene structures and predicted 63 new ones, and make our annotations available through a distributed annotation system (DAS) server and their sequences as additional files with this paper. CONCLUSIONS: The vertebrate CTLDcp family was essentially formed early in vertebrate evolution and is completely different from the invertebrate families. Comparison of fish and mammalian genomes revealed three groups of CTLDcps and several new members of the known groups, which are highly conserved between fish and mammals, but were not identified in the study using only mammalian genomes. Despite limitations of the draft sequence, the Fugu rubripes genome is a powerful instrument for gene discovery and vertebrate evolutionary analysis. The composition of the CTLDcp superfamily in fish and mammals suggests that large-scale duplication events played an important role in the evolution of vertebrates

Rationale for targeting the ErbB family of receptors in patients with advanced squamous cell carcinoma

Squamous cell carcinoma (SCC) of the lung represents around 30% of all non-small-cell lung cancers. Treatment options for nonsquamous histology have increased in recent years following the development of pemetrexed chemotherapy and the identification of activating EGFR mutations and ALK rearrangements as targets for effective noncytotoxic agents. By contrast, until recently the development of new therapies for SCC has lagged behind. However, the identification of important genetic events driving SCC, including a greater understanding of the role of the ErbB receptor family in SCC pathogenesis, as well as recent immunotherapy advances, have led to new treatment options for SCC

Ultrastructural and carbohydrate histochemical study of the Vater-Pacini corpuscles in the digital pads of the North American raccoon (Procyon lotor), with special regard to basic function

Numerous and very large Vater-Pacini corpuscles were observed in the forefoot digital pads of the North American raccoon (Procyon lotor). In addition to ultrastructure, the distribution and selectivity of complex glycoconjugates in this sensory corpuscle type were examined by carbohydrate histochemical techniques, in particular lectin histochemistry. The Vater-Pacini corpuscles present showed the typical lamellar structure known for mammals and contained high amounts of neutral and acidic glycoconjugates with various saccharide residues (α-l-fucose, β-d-galactose, sialic acid) in a specific intracorpuscular distribution pattern, including variations between the outer lamellae and the inner core. The results obtained are discussed with regard to possible functions of the Vater-Pacini corpuscles found in the raccoon forefoot pads. The corpuscular glycoconjugate components may furnish a high and differentiated viscoelasticity for rapid pressure transmission within the large Vater-Pacini corpuscles. Thus, the digital pads of the forepaws can be considered as part of a specific mechanoreceptor system related to excellent object manipulation properties in this mammalian species