Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging

A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.     

Complement protein C1q inhibits antibody-dependent enhancement of flavivirus infection in an IgG subclass-specific manner

Severe dengue virus infection can occur in humans with pre-existing antibodies against the virus. This observation led to the hypothesis that a subneutralizing antibody level in vivo can increase viral burden and cause more severe disease. Indeed, antibody-dependent enhancement of infection (ADE) in vitro has been described for multiple viruses, including the flaviviruses dengue virus and West Nile virus. Here, we demonstrate that the complement component C1q restricts ADE by anti-flavivirus IgG antibodies in an IgG subclass-specific manner in cell culture and in mice. IgG subclasses that avidly bind C1q induced minimal ADE in the presence of C1q. These findings add a layer of complexity for the analysis of humoral immunity and flavivirus infection.     

Archaeological and Contemporary Evidence Indicates Low Sea Otter Prevalence on the Pacific Northwest Coast During the Late Holocene

Ecosystems - The historic extirpation and subsequent recovery of sea otters (Enhydra lutris) have profoundly changed coastal social-ecological systems across the northeastern Pacific. Today, the...     

Purification of a conjugated polysaccharide vaccine using tangential flow diafiltration

Conjugated vaccines prepared from the capsular polysaccharide of Streptococcus pneumoniae can provide immunization against invasive pneumococcal disease, meningitis, and otitis media. One of the critical steps in the production of these vaccines is the removal of free (unreacted) polysaccharides from the protein-polysaccharide conjugate. Experimental studies were performed to evaluate the effects of membrane pore size, filtrate flux, and solution conditions on the transmission of both the conjugate and free polysaccharide through different ultrafiltration membranes. Conjugate purification was done using diafiltration performed in a linearly-scalable tangential flow filtration cassette. More than 98% of the free polysaccharide was removed within a 5-diavolume diafiltration process, which is a significant improvement over previously reported results for purification of similar conjugated vaccines. These results clearly demonstrate the opportunities for using ultrafiltration/diafiltration for the final purification of conjugated vaccine products.     

Spontaneous pulmonary adenocarcinoma and subcutaneous cavernous hemangiomas arising in a squirrel monkey (Saimiri sciureus)

Benign and malignant pulmonary tumors have been reported in both Old World and New World monkeys but are uncommon. Hemangiomas are also rarely reported in nonhuman primates. Here we present a case of two primary neoplasms (a papillary adenocarcinoma of bronchioloalveolar origin and multiple cavernous subcutaneous hemangiomas) arising in an aged squirrel monkey (Saimiri sciureus).