Outer membrane vesicles as a platform for the discovery of antibodies to bacterial pathogens.

Bacterial outer membrane vesicles (OMVs) are nanosized spheroidal particles shed by gram-negative bacteria that contain biomolecules derived from the periplasmic space, the bacterial outer membrane, and possibly other compartments. OMVs can be purified from bacterial culture supernatants, and by genetically manipulating the bacterial cells that produce them, they can be engineered to harbor cargoes and/or display molecules of interest on their surfaces including antigens that are immunogenic in mammals. Since OMV bilayer-embedded components presumably maintain their native structures, OMVs may represent highly useful tools for generating antibodies to bacterial outer membrane targets.

Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6.

Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) among other malignancies. The MUC16-specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging.

Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering.

Next-generation DNA sequencing (NGS) technologies have made it possible to interrogate antibody repertoires to unprecedented depths, typically via sequencing of cDNAs encoding immunoglobulin variable domains. In the absence of heavy-light chain pairing, the variable domains of heavy chain-only antibodies (HCAbs), referred to as single-domain antibodies (sdAbs), are uniquely amenable to NGS analyses. In this chapter, we provide simple and rapid protocols for producing and sequencing multiplexed immunoglobulin variable domain (VH, V or V) amplicons derived from a variety of sources using the Illumina MiSeq platform.

Isolation and characterization of a VHH targeting the Acinetobacter baumannii cell surface protein CsuA/B.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A.

A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria.

The measurement of physicochemical parameters in living cells can provide information on individual cellular organelles, helping us to understand subcellular function in health and disease. While organelle-specific chemical probes have allowed qualitative evaluation of microenvironmental variations, the simultaneous quantification of mitochondrial local microviscosity (η ) and micropolarity (ϵ ), along with concurrent structural variations, has remained an unmet need. Herein, we describe a new multifunctional mitochondrial probe (MMP) for simultaneous monitoring of η and ϵ by fluorescence lifetime and emission intensity recordings, respectively.

Delivery and Release of Small-Molecule Probes in Mitochondria Using Traceless Linkers.

Mitochondria-penetrating peptides (MPPs) are specific targeting vectors for the localization of small molecules to the mitochondrial matrix. Mitochondrial targeting of small molecules has enabled the development of a number of potential therapeutics and chemical probes. However, the need for covalent conjugation of small molecules to MPPs can negatively affect the activity of the appended cargo against its cellular target.

Peptide-Mediated Delivery of Chemical Probes and Therapeutics to Mitochondria.

Mitochondria are organelles with critical roles in key processes within eukaryotic cells, and their dysfunction is linked with numerous diseases including neurodegenerative disorders and cancer. Pharmacological manipulation of mitochondrial function is therefore important both for basic science research and eventually, clinical medicine. However, in comparison to other organelles, mitochondria are difficult to access due to their hydrophobic and dense double membrane system as well as their negative membrane potential.

Mitochondrial Chemical Biology: New Probes Elucidate the Secrets of the Powerhouse of the Cell.

Mitochondria are energy-producing organelles with essential functions in cell biology, and mitochondrial dysfunction is linked to a wide range of human diseases. Efforts to better understand mitochondrial biology have been limited by the lack of tools for manipulating and detecting processes occurring within the organelle. Here, we highlight recent significant advances in mitochondrial chemical biology that have produced new tools and techniques for studying mitochondria.

Molecular vehicles for mitochondrial chemical biology and drug delivery.

The mitochondria within human cells play a major role in a variety of critical processes involved in cell survival and death. An understanding of mitochondrial involvement in various human diseases has generated an appreciable amount of interest in exploring this organelle as a potential drug target. As a result, a number of strategies to probe and combat mitochondria-associated diseases have emerged.

Rapid identification of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Compared to DNA sequence analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) correctly identified 100% of Cryptococcus species, distinguishing the notable pathogens Cryptococcus neoformans and C. gattii. Identification was greatly enhanced by supplementing a commercial spectral library with additional entries to account for subspecies variability.