Statistical 3D morphology characterization of vaterite microspheres produced by engineered Escherichia coli.

Hollow vaterite microspheres are important materials for biomedical applications such as drug delivery and regenerative medicine owing to their biocompatibility, high specific surface area, and ability to encapsulate a large number of bioactive molecules and compounds. We demonstrated that hollow vaterite microspheres are produced by an Escherichia coli strain engineered with a urease gene cluster from the ureolytic bacteria Sporosarcina pasteurii in the presence of bovine serum albumin. We characterized the 3D nanoscale morphology of five biogenic hollow vaterite microspheres using 3D high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) tomography. Using automated high-throughput HAADF-STEM imaging across several sample tilt orientations, we show that the microspheres evolved from a smaller more ellipsoidal shape to a larger more spherical shape while the internal hollow core increased in size and remained relatively spherical, indicating that the microspheres produced by this engineered strain likely do not contain the bacteria. The statistical 3D morphology information demonstrates the potential for using biogenic calcium carbonate mineralization to produce hollow vaterite microspheres with controlled morphologies. STATEMENT OF SIGNIFICANCE: The nanoscale 3D structures of biomaterials determine their physical, chemical, and biological properties, however significant efforts are required to obtain a statistical understanding of the internal 3D morphology of materials without damaging the structures. In this study, we developed a non-destructive, automated technique that allows us to understand the nanoscale 3D morphology of many unique hollow vaterite microspheres beyond the spectroscopy methods that lack local information and microscopy methods that cannot interrogate the full 3D structure. The method allowed us to quantitatively correlate the external diameters and aspect ratios of vaterite microspheres with their hollow internal structures at the nanoscale. This work demonstrates the opportunity to use automated transmission electron microscopy to characterize nanoscale 3D morphologies of many biomaterials and validate the chemical and biological functionality of these materials.

Gene networks that compensate for crosstalk with crosstalk.

Crosstalk is a major challenge to engineering sophisticated synthetic gene networks. A common approach is to insulate signal-transduction pathways by minimizing molecular-level crosstalk between endogenous and synthetic genetic components, but this strategy can be difficult to apply in the context of complex, natural gene networks and unknown interactions. Here, we show that synthetic gene networks can be engineered to compensate for crosstalk by integrating pathway signals, rather than by pathway insulation. We demonstrate this principle using reactive oxygen species (ROS)-responsive gene circuits in Escherichia coli that exhibit concentration-dependent crosstalk with non-cognate ROS. We quantitatively map the degree of crosstalk and design gene circuits that introduce compensatory crosstalk at the gene network level. The resulting gene network exhibits reduced crosstalk in the sensing of the two different ROS. Our results suggest that simple network motifs that compensate for pathway crosstalk can be used by biological networks to accurately interpret environmental signals.

Artificial Repeat-Structured siRNA Precursors as Tunable Regulators for Saccharomyces cerevisiae.

RNA interference (RNAi) is widely used as a research tool for studying biological systems and implementing artificial genetic circuits that function by modulating RNA concentrations. Here we engineered Saccharomyces cerevisiae containing a heterologous Saccharomyces castelli RNAi system as a test-bed for RNAi-based circuits. Unlike prior approaches, we describe a strategy that leverages repeat-structured siRNA precursors with incrementally sized stems formed from 23 bp-repeats to achieve modular RNAi-based gene regulation. These enable repression strength to be tuned in a systematic manner by changing the size of the siRNA precursor hairpin stem, without modifying the number or sequence of target sites in the target RNA. We demonstrate that this hairpin-based regulation is able to target both cytoplasmic and nuclear localized RNAs and is stable over extended growth periods. This platform enables the targeting of cellular RNAs as a tunable regulatory layer for sophisticated gene circuits in Saccharomyces cerevisiae.

The Charles Drew program in Missouri: a description of a partnership among a blood center and several hospitals to address the care of patients with sickle cell disease.

Sickle cell disease (SCD) is an inherited blood disorder which can be complicated by stroke in infancy and childhood. The primary and secondary prevention of stroke in this patient population is regular RBC transfusion therapy at least every three weeks, but there is no consensus on the ideal RBC transfusion therapy. The Charles Drew Program, a partnership among a blood center and several hospitals affiliated with academic medical centers in Missouri, provides RBCs for the care of patients with SCD. There are three basic aims: the RBC components are phenotypically matched on three minor RBC antigens, the units are less than 7 days old, and each patient has a limited number of dedicated donors, so that the donor exposure is minimized. This report describes the operational phases of this program and summarizes its performance with respect to each of these aims.

Terpenols as substituents for the diastereoselective formation of enantiomerically pure triple lithium-bridged helicate type-coordination compounds.

The terpenols l(-)-borneol, (1S2S3S5R)-3-pinanol, (-)-menthol, and (-)-myrtenol are easily available chiral alcohols for the preparation of enantiomerically pure catechol esters -H2. Those ligands are used for the hierarchical assembly of triple lithium-bridged dinuclear titanium(iv) triscatecholate helicates Li[Li3()6Ti2]. In solution, the dimeric species are in a solvent dependent equilibrium with the monomer Li2[()3Ti]. The equilibrium is studied by (1)H NMR. CD spectroscopy indicates that the configuration at the complex units of the enantiomerically pure dimeric α-chiral derivatives Li[Li3()6Ti2] is opposite to the configuration of the monomers Li2[()3Ti]. For the γ-chiral complex Li2[()3Ti] only a de of 25% is observed and in this case no interpretation of the mechanism of stereocontrol is possible.

ABCA4 mutations and discordant ABCA4 alleles in patients and siblings with bull's-eye maculopathy.

AIM: To determine the frequency and nature of mutations in the gene ABCA4 in a cohort of patients with bull's-eye maculopathy (BEM). METHODS: A panel of 49 subjects (comprising 40 probands/families, 7 sibling pairs and a set of three sibs) with BEM, not attributable to toxic causes, was ascertained. Blood samples from each patient were used to extract genomic DNA, with subsequent mutation screening of the entire coding sequence of ABCA4, using single-strand conformational polymorphism (SSCP) analysis and direct sequencing. RESULTS: Fourteen probands (35%) were found to have a potentially disease-causing ABCA4 sequence variant on at least one allele. Three patients had a Gly1961Glu missense mutation, the most common variant in Stargardt disease (STGD), with 2 of these subjects having a macular dystrophy (MD) phenotype and a second ABCA4 variant previously associated with STGD. The second most common STGD mutation, Ala1038Val, was seen in one patient with cone-rod dystrophy (CORD). Five novel ABCA4 variants were detected. Two sibships were identified with a similar intra-familial phenotype but discordant ABCA4 variants. CONCLUSIONS: Variations in the ABCA4 gene are common in BEM. Two sibships showed discordant ABCA4 variants. One of these sibships illustrates that ABCA4 variants can be identified in families that have another molecular cause for their disease, due to the high prevalence of ABCA4 disease alleles in the population. The discordance evident in the second sibship may yet also be a chance finding in families with macular disease of another genetic cause, or it may represent a complex mode of inheritance determined/modified by the combination of ABCA4 alleles.