Zero-Background Small-Molecule Sensors for Near-IR Fluorescent Imaging of Biomacromolecular Targets in Cells.

In this study, we report a general approach to the design of a new generation of small-molecule sensors that produce a zero background but are brightly fluorescent in the near-IR spectral range upon selective interaction with a biomolecular target. We developed a fluorescence turn-on/-off mechanism based on the aggregation/deaggregation of phthalocyanine chromophores. As a proof of concept, we designed, prepared, and characterized sensors for in-cell visualization of epidermal growth factor receptor (EGFR) tyrosine kinase. We established a structure/bioavailability correlation, determined conditions for the optimal sensor uptake and imaging, and demonstrated binding specificity and applications over a wide range of treatment options involving live and fixed cells. The new approach enables high-contrast imaging and requires no in-cell chemical assembly or postexposure manipulations (i.e., washes). The general design principles demonstrated in this work can be extended toward sensors and imaging agents for other biomolecular targets.

Adult Height, 22q11.2 Deletion Extent, and Short Stature in 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11.2DS) manifests as a wide range of medical conditions across a number of systems. Pediatric growth deficiency with some catch-up growth is reported, but there are few studies of final adult height. We aimed to investigate how final adult height in 22q11.2DS compared with general population norms, and to examine predictors of short stature in in a cohort of 397 adults with 22q11.2DS (aged 17.6-76.3 years) with confirmed typical 22q11.2 microdeletion (overlapping the LCR22A to LCR22B region). We defined short stature as <3rd percentile using population norms. For the subset (n = 314, 79.1%) with 22q11.2 deletion extent, we used a binomial logistic regression model to predict short stature in 22q11.2DS, accounting for effects of sex, age, ancestry, major congenital heart disease (CHD), moderate-to-severe intellectual disability (ID), and 22q11.2 deletion extent. Adult height in 22q11.2DS showed a normal distribution but with a shift to the left, compared with population norms. Those with short stature represented 22.7% of the 22q11.2DS sample, 7.6-fold greater than population expectations (p < 0.0001). In the regression model, moderate-to-severe ID, major CHD, and the common LCR22A-LCR22D (A-D) deletion were significant independent risk factors for short stature while accounting for other factors (model p = 0.0004). The results suggest that the 22q11.2 microdeletion has a significant effect on final adult height distribution, and on short stature with effects appearing to arise from reduced gene dosage involving both the proximal and distal sub-regions of the A-D region. Future studies involving larger sample sizes with proximal nested 22q11.2 deletions, longitudinal lifetime data, parental heights, and genotype data will be valuable.

Essential role of calcium in extending RTX adhesins to their target.

RTX adhesins are long, multi-domain proteins present on the outer membrane of many Gram-negative bacteria. From this vantage point, adhesins use their distal ligand-binding domains for surface attachment leading to biofilm formation. To expand the reach of the ligand-binding domains, RTX adhesins maintain a central extender region of multiple tandem repeats, which makes up most of the proteins' large molecular weight. Alignments of the 10-15-kDa extender domains show low sequence identity between adhesins. Here we have produced and structurally characterized protein constructs of four tandem repeats (tetra-tandemers) from two different RTX adhesins. In comparing the tetra-tandemers to each other and already solved structures from Marinomonas primoryensis and Salmonella enterica, the extender domains fold as diverse beta-sandwich structures with widely differing calcium contents. However, all the tetra-tandemers have at least one calcium ion coordinated in the linker region between beta-sandwich domains whose role appears to be the rigidification of the extender region to help the adhesin extend its reach.

Correction: Structure and functional analysis of a bacterial adhesin sugar-binding domain.

[This corrects the article DOI: 10.1371/journal.pone.0220045.].

Structure and functional analysis of a bacterial adhesin sugar-binding domain.

Bacterial adhesins attach their hosts to surfaces through one or more ligand-binding domains. In RTX adhesins, which are localized to the outer membrane of many Gram-negative bacteria via the type I secretion system, we see several examples of a putative sugar-binding domain. Here we have recombinantly expressed one such ~20-kDa domain from the ~340-kDa adhesin found in Marinobacter hydrocarbonoclasticus, an oil-degrading bacterium. The sugar-binding domain was purified from E. coli with a yield of 100 mg/L of culture. Circular dichroism analysis showed that the protein was rich in beta-structure, was moderately heat resistant, and required Ca2+ for proper folding. A crystal structure was obtained in Ca2+ at 1.2-Å resolution, which showed the presence of three Ca2+ ions, two of which were needed for structural integrity and one for binding sugars. Glucose was soaked into the crystal, where it bound to the sugar's two vicinal hydroxyl groups attached to the first and second (C1 and C2) carbons in the pyranose ring. This attraction to glucose caused the protein to bind certain polysaccharide-based column matrices and was used in a simple competitive binding assay to assess the relative affinity of sugars for the protein's ligand-binding site. Fucose, glucose and N-acetylglucosamine bound most tightly, and N-acetylgalactosamine hardly bound at all. Isothermal titration calorimetry was used to determine specific binding affinities, which lie in the 100-µM range. Glycan arrays were tested to expand the range of ligand sugars assayed, and showed that MhPA14 bound preferentially to branched polymers containing terminal sugars highlighted as strong binders in the competitive binding assay. Some of these binders have vicinal hydroxyl groups attached to the C3 and C4 carbons that are sterically equivalent to those presented by the C1 and C2 carbons of glucose.

Staphylococcus aureus heme and siderophore-iron acquisition pathways.

Staphylococcus aureus is a versatile opportunistic human pathogen. Infection by this bacterium requires uptake of iron from the human host, but iron is highly restricted in this environment. Staphylococcus aureus iron sufficiency is achieved primarily through uptake of heme and high-affinity iron chelators, known as siderophores. Two siderophores (staphyloferrins) are produced and secreted by S. aureus into the extracellular environment to capture iron. Staphylococcus aureus expresses specific uptake systems for staphyloferrins and more general uptake systems for siderophores produced by other microorganisms. The S. aureus heme uptake system uses highly-specific cell surface receptors to extract heme from hemoglobin and hemoglobin-haptoglobin complexes for transport into the cytoplasm where it is degraded to liberate iron. Initially thought to be independent systems, recent findings indicate that these iron uptake pathways intersect. IruO is a reductase that releases iron from heme and some ferric-siderophores. Moreover, multifunctional SbnI produces a precursor for staphyloferrin B biosynthesis, and also binds heme to regulate expression of the staphyloferrin B biosynthesis pathway. Intersection of the S. aureus iron uptake pathways is hypothesized to be important for rapid adaptation to available iron sources. Components of the heme and siderophore uptake systems are currently being targeted in the development of therapeutics against S. aureus.

Backbone 1H, 13C, and 15N NMR resonance assignments of the Krüppel-like factor 4 activation domain.

Krüppel-like factor 4 (KLF4) is a transcription factor involved in diverse biological processes, including development, cellular differentiation and proliferation, and maintenance of tissue homeostasis. KLF4 has also been associated with disease states, such as cardiovascular disease and several cancers. KLF4 contains an activation domain, repression domain, and a structurally characterized C-terminal zinc finger domain that mediates its binding to DNA. The structurally uncharacterized KLF4 activation domain is critical for transactivation by KLF4 and mediates its binding to the transcriptional coactivator CBP/p300. Here, we report the 1H, 15N, 13CO, 13Cα and 13Cß NMR chemical shift assignments of KLF41-130, which contains the KLF4 activation domain. Narrow chemical shift dispersion in the 1H dimension of the 1H-15N HSQC spectrum suggests that the KLF41-130 fragment is intrinsically disordered.