Phenotypic landscape of a bacterial cell

The explosion of sequence information in bacteria makes developing high-throughput, cost-effective approaches to matching genes with phenotypes imperative. Using E. coli as proof of principle, we show that combining large-scale chemical genomics with quantitative fitness measurements provides a high-quality data set rich in discovery. Probing growth profiles of a mutant library in hundreds of conditions in parallel yielded > 10,000 phenotypes that allowed us to study gene essentiality, discover leads for gene function and drug action, and understand higher-order organization of the bacterial chromosome. We highlight new information derived from the study, including insights into a gene involved in multiple antibiotic resistance and the synergy between a broadly used combinatory antibiotic therapy, trimethoprim and sulfonamides. This data set, publicly available at http://ecoliwiki.net/tools/chemgen/, is a valuable resource for both the microbiological and bioinformatic communities, as it provides high-confidence associations between hundreds of annotated and uncharacterized genes as well as inferences about the mode of action of several poorly understood drugs.     

Environmental Factors Associated with the Eukaryotic Microbial Diversity of Ulleungdo Volcanic Island in South Korea

Microbiology - Ulleungdo is a volcanic island with an oceanic climate seasonally affected by warm and cold currents. The freshwater ecosystems of Ulleungdo Island act as an ecological indicator...     

Zebrafish transgenic Enhancer TRAP line database (ZETRAP)

Background  

The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution.     

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50–200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex “work-in-progress” MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.     

Inactivation of aminated horseradish peroxidase by interaction with S-Sepharose

Horseradish peroxidase which had been aminated by periodate oxidation and reductive amination was purified by cation-exchange chromatography on S-Sepharose. Instead of the expected single peak of aminated enzyme, two distinct peaks of protein were eluted from the column. Evaluation of the protein in each of the two distributions showed that peak number 1 had spectral properties and specific activity similar to those of native enzyme. Distribution number 2 had a threefold reduction in the extinction in the Soret region at 404 nm and was completely devoid of enzymatic activity. This inactivation was caused by a specific interaction between the aminated peroxidase and the S-Sepharose matrix, resulting in a displacement of the heme prosthetic group out of its native orientation. The inactivation of the aminated peroxidase was found to be dependent on time, pH, and the support matrix itself. These results indicate that the S-Sepharose and Mono-S resins are not interchangeable, despite the chemical similarities of the two resins.     

The kinetics and mechanism of shear inactivation of lipase from Candida cylindracea

Shearing experiments were conducted in a stirred tank reactor with 0.1% lipase solutions of Candida cylindracea. Inactivation of the lipase solutions were observed at various shear rates from 50 to 150 s(-1) after continuous shearing for ca. 30-240 min under optimal pH and temperature conditions. However, there was no shear stress denaturation of the lipase when it was subjected to shear stresses of 0.72-109.2 kg/m/s(2) and shear rate of 100 s(-1). In the presence of polypropylene glycol, the rate of denaturation of the lipase decreased by 93%. When the lipase solution was filled to the brim, the rate of denaturation of the lipase decreased by 97% compared to that when reactor was half-filled. The rate of denaturation of the lipase decreased by 61% when probes in the fermentor were removed. There was no significant difference in the rate of denaturation of the lipase under ambient conditions compared with that in the absence of oxygen, or in the absence of free metal ions. Recovery of lipase activity from the first hour of shearing was observed at a shear rate of 150 s(-1). The native lipase and the lipase which had recovered its activity showed similar pH profiles, temperature profiles, and activation energies. Temperature was found to have no effect in the rate of shear-induced denaturation of the lipase in the range 20 to 30 degrees C during shearing at 100 s (-1)and optimal pH. Above 30 degrees C, the rate of denaturation of the lipase increased drastically as a function of temperature. The significance of the findings in the de sign of reactor systems for hydrolysis or esterification of oils by lipase will be discussed.     

Plant biomechanics and resilience to environmental changes are controlled by specific lignin chemistries in each vascular cell type and morphotype

The biopolymer lignin is deposited in the cell walls of vascular cells and is essential for long-distance water conduction and structural support in plants. Different vascular cell types contain distinct and conserved lignin chemistries, each with specific aromatic and aliphatic substitutions. Yet, the biological role of this conserved and specific lignin chemistry in each cell type remains unclear. Here, we investigated the roles of this lignin biochemical specificity for cellular functions by producing single cell analyses for three cell morphotypes of tracheary elements, which all allow sap conduction but differ in their morphology. We determined that specific lignin chemistries accumulate in each cell type. Moreover, lignin accumulated dynamically, increasing in quantity and changing in composition, to alter the cell wall biomechanics during cell maturation. For similar aromatic substitutions, residues with alcohol aliphatic functions increased stiffness whereas aldehydes increased flexibility of the cell wall. Modifying this lignin biochemical specificity and the sequence of its formation impaired the cell wall biomechanics of each morphotype and consequently hindered sap conduction and drought recovery. Together, our results demonstrate that each sap-conducting vascular cell type distinctly controls their lignin biochemistry to adjust their biomechanics and hydraulic properties to face developmental and environmental constraints.

During the development of each vascular cell, specific lignin chemistries control their biomechanics and water conduction properties to face environmental changes.

IN A NUTSHELL Background: Lignin comprises multiple cell wall–localized aromatic polymers that are essential for vascular plants to conduct water and strengthen their organs. It has long been thought that lignin was randomly and nonspecifically assembled to provide mechanical strengthening and waterproofing to cells by filling-up the empty spaces in the cell walls. However, the different cell types and morphotypes forming the different sap-conducting pipes and their cell wall layers (inner vs. outer layer) exhibit specific lignin chemistries that are conserved among plant species. We, therefore, investigated the function of these specific lignin chemistries at the cell and cell wall layer levels for the different sap-conducting pipes in plants. Question: What is the function of a specific lignin chemistry for the different plant sap-conducting pipe cells? Can changes in the lignin chemistry of sap-conducting cells affect their hydraulic capacity when facing environmental conditions such as drought? Findings: We answered these questions by changing lignin levels and composition, using drugs to block lignin formation, and/or genetic engineering to switch off genes, in three complementary systems: (1) isolated cells grown in test tubes that we can trigger to become sap conduits, (2) annual plants, and (3) hardwood trees. We show that lignin chemistry is specific to each cell morphotype and changes during cell maturation, modifying the amount of lignin, the chemical composition of lignin units, and the position of these units in the longer polymer. These specific lignin chemistries are required for the proper function of each cell morphotype to properly conduct the sap and strengthen plant organs. Modifying the amount, the composition, and the time when specific units with distinct chemistry are incorporated in lignin of each cell morphotype has dramatic effects, causing defects in sap conduit hydraulic and biomechanical properties. The ratio between the different chemical units of lignin needs to be fine-tuned to adjust plant sap conduction and mechanical strengthening. Thus, changes in the proportion of lignin units with distinct chemistries confer different hydraulic and mechanical properties enabling plants to better resist and/or recover from drought. We also revealed that increases in the proportion of lignin units with aldehyde modulate plant pipe hydraulic and mechanical properties. Next steps: We are now working to identify and understand the molecular mechanisms that control the formation of specific lignin chemistries in distinct sites and times during the development of the different cell wall layers in each cell type and morphotype.