Anti-protease and Immunomodulatory Activities of Bacteria Associated with Caribbean Sponges

Marine sponges and their associated bacteria have been proven to be a rich source of novel secondary metabolites with therapeutic usefulness in cancer, infection, and autoimmunity. In this study, 79 strains belonging to 20 genera of the order Actinomycetales and seven strains belonging to two genera of the order Sphingomonadales were cultivated from 18 different Caribbean sponges and identified by 16S rRNA gene sequencing. Seven of these strains are likely to represent novel species. Crude extracts from selected strains were found to exhibit protease inhibition against cathepsins B and L, rhodesain, and falcipain-2 as well as immunomodulatory activities such as induction of cytokine release by human peripheral blood mononuclear cells. These results highlight the significance of marine sponge-associated bacteria to produce bioactive secondary metabolites with therapeutic potential in the treatment of infectious diseases and disorders of the immune system.     

High-Throughput Genotyping of Green Algal Mutants Reveals Random Distribution of Mutagenic Insertion Sites and Endonucleolytic Cleavage of Transforming DNA

A high-throughput genetic screening platform in a single-celled photosynthetic eukaryote would be a transformative addition to the plant biology toolbox. Here, we present ChlaMmeSeq (Chlamydomonas MmeI-based insertion site Sequencing), a tool for simultaneous mapping of tens of thousands of mutagenic insertion sites in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. We first validated ChlaMmeSeq by in-depth characterization of individual insertion sites. We then applied ChlaMmeSeq to a mutant pool and mapped 11,478 insertions, covering 39% of annotated protein coding genes. We observe that insertions are distributed in a manner largely indistinguishable from random, indicating that mutants in nearly all genes can be obtained efficiently. The data reveal that sequence-specific endonucleolytic activities cleave the transforming DNA and allow us to propose a simple model to explain the origin of the poorly understood exogenous sequences that sometimes surround insertion sites. ChlaMmeSeq is quantitatively reproducible, enabling its use for pooled enrichment screens and for the generation of indexed mutant libraries. Additionally, ChlaMmeSeq allows genotyping of hits from Chlamydomonas screens on an unprecedented scale, opening the door to comprehensive identification of genes with roles in photosynthesis, algal lipid metabolism, the algal carbon-concentrating mechanism, phototaxis, the biogenesis and function of cilia, and other processes for which C. reinhardtii is a leading model system.     

Analysis of putative protomer crosstalk in the trimeric transporter BetP: The heterotrimer approach

The homotrimeric, secondary active betaine carrier BetP from Corynebacterium glutamicum is a model system for stress-regulated transport in bacteria. Its activity responds to hyperosmotic stress and it harbors two different functions, transport catalysis (betaine uptake) and stimulus sensing, resp. activity regulation. Structural information from 2D and 3D crystals as well as functional analysis of monomerized BetP suggested the presence of conformational crosstalk between the individual protomers. To study whether the oligomeric state is functionally significant on a mechanistic level we generated heterooligomeric complexes of BetP in which single protomers within the trimer can be addressed. By testing dominant negative effects in a trimer of one active protomer combined with two protomers in which transport and regulation were abolished, we provide experimental evidence for the absence of functionally significant conformational crosstalk between the protomers on the level of both transport and regulation. This is supported by experiments using mutant forms of putative interacting signal donor and acceptor domains of individual BetP protomers. This result has important consequences for oligomeric transport proteins in general and BetP in particular.     

A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.     

Equine bronchial epithelial cells differentiate into ciliated and mucus producing cells in vitro

We describe a method for creating differentiated equine bronchial epithelial cell cultures that can be used for in vitro studies including airway disease mechanisms and pathogen–host interactions. Our method is based on the culturing of human tracheobronchial epithelial cells at an air–liquid interface (ALI) in specific serum-free, hormone-supplemented medium. Bronchial epithelial cells are isolated and grown on T-Clear® insert membranes. Within 2 to 3 wk, cells differentiate into ciliated and mucus producing cells as demonstrated by confocal and electron microscopy. Furthermore, the demonstration of the two major gel-forming mucin species, Muc5ac and Muc5b, in our bronchial epithelial cell culture system validates this method for studies of respiratory tract disease of the horse.     

Catheterless Long-Term Ambulatory Urodynamic Measurement Using a Novel Three-Device System

Aims

Long-term urodynamics are required because bladder-emptying disorders are often not clearly revealed by conventional urodynamics. Patients with severe clinical overactive bladder symptoms, for instance, often show normal results. This may be due to the short evaluation time and psychological factors that complicate conventional urodynamics. This study aimed to develop an ambulatory three-component urodynamic measurement system that is easy to operate, registers urodynamic parameters for several days, and has no negative impact on the patient.

Methods

We developed an intravesical capsule combined with a hand-held device to register voiding desire and micturition, and an alarm pad device that detects urine loss. Recently, the intravesical capsule and its proven function were detailed in the literature. Here, we present detailed in vitro results using a female bladder model. The flexible capsule was C-shaped to minimize the risk of expulsion from the bladder during micturition. Results of biocompatibility evaluation of the intravesical capsule, which is called Wille Capsule (WiCa) are described.

Results

The WiCa with an oval nose and a maximum outer diameter of 5.5 mm was easily inserted through a 25-French cystoscope. Removing the WiCa by grasping the nose using the female model with bladder was easily conducted. Expulsion of the WiCa during voiding was avoided through a novel C-shaped device design. Based on in vitro cytotoxicity studies, the capsule is a promising and safe device.

Conclusion

Our novel system is an innovative minimally-invasive tool for accurate long-term urodynamic measurement, and does not require inserting a transurethral catheter.