Iron limitation of heterotrophic bacteria in the California Current System tracks relative availability of organic carbon and iron.

Iron is an essential nutrient for all microorganisms of the marine environment. Iron limitation of primary production has been well documented across a significant portion of the global surface ocean, but much less is known regarding the potential for iron limitation of the marine heterotrophic microbial community. In this work, we characterize the transcriptomic response of the heterotrophic bacterial community to iron additions in the California Current System, an eastern boundary upwelling system, to detect in situ iron stress of heterotrophic bacteria. Changes in gene expression in response to iron availability by heterotrophic bacteria were detected under conditions of high productivity when carbon limitation was relieved but when iron availability remained low. The ratio of particulate organic carbon to dissolved iron emerged as a biogeochemical proxy for iron limitation of heterotrophic bacteria in this system. Iron stress was characterized by high expression levels of iron transport pathways and decreased expression of iron-containing enzymes involved in carbon metabolism, where a majority of the heterotrophic bacterial iron requirement resides. Expression of iron stress biomarkers, as identified in the iron-addition experiments, was also detected insitu. These results suggest iron availability will impact the processing of organic matter by heterotrophic bacteria with potential consequences for the marine biological carbon pump.

Short-term acidification promotes diverse iron acquisition and conservation mechanisms in upwelling-associated phytoplankton.

Coastal upwelling regions are among the most productive marine ecosystems but may be threatened by amplified ocean acidification. Increased acidification is hypothesized to reduce iron bioavailability for phytoplankton thereby expanding iron limitation and impacting primary production. Here we show from community to molecular levels that phytoplankton in an upwelling region respond to short-term acidification exposure with iron uptake pathways and strategies that reduce cellular iron demand. A combined physiological and multi-omics approach was applied to trace metal clean incubations that introduced 1200 ppm CO2 for up to four days. Although variable, molecular-level responses indicate a prioritization of iron uptake pathways that are less hindered by acidification and reductions in iron utilization. Growth, nutrient uptake, and community compositions remained largely unaffected suggesting that these mechanisms may confer short-term resistance to acidification; however, we speculate that cellular iron demand is only temporarily satisfied, and longer-term acidification exposure without increased iron inputs may result in increased iron stress.

Protein-macrocycle polymorphism: crystal form IV of the Ralstonia solanacearum lectin-sulfonato-calix[8]arene complex.

Controlled protein assembly and crystallization is necessary as a means of generating diffraction-quality crystals as well as providing a basis for new types of biomaterials. Water-soluble calixarenes are useful mediators of protein crystallization. Recently, it was demonstrated that Ralstonia solanacearum lectin (RSL) co-crystallizes with anionic sulfonato-calix[8]arene (sclx8) in three space groups. Two of these co-crystals only grow at pH ≤ 4 where the protein is cationic, and the crystal packing is dominated by the calixarene. This paper describes a fourth RSL-sclx8 co-crystal, which was discovered while working with a cation-enriched mutant. Crystal form IV grows at high ionic strength in the pH range 5-6. While possessing some features in common with the previous forms, the new structure reveals alternative calixarene binding modes. The occurrence of C2-symmetric assemblies, with the calixarene at special positions, appears to be an important result for framework fabrication. Questions arise regarding crystal screening and exhaustive searching for polymorphs.

Cage versus sheet: Probing the Determinants of Protein - Cucurbit[7]uril Crystalline Architectures.

The donut-shaped cucurbit[n]urils (Qn) are a class of rigid macrocyclic receptor with protein recognition capabilities. Qn encapsulation of amino acid side chains can enable protein assembly. Recently, cucurbit[7]uril (Q7) has been applied as a molecular glue for organizing protein building blocks into crystalline architectures. Q7 co-crystallization with dimethylated Ralstonia solanacearum lectin (RSL*) has yielded novel crystalline architectures. Co-crystallization of RSL* and Q7 yields either cage- or sheet-like architectures which may be modulated via protein engineering. However, questions remain as to the factors dictating the formation of one architecture over another (cage versus sheet). Here, we make use of an engineered RSL*-Q7 system which co-crystallizes as the cage or sheet assembly with easily-distinguished crystal morphologies. Using this model system, we probe how the crystallization conditions dictate which crystalline architecture is adopted. Protein-ligand ratios and the sodium concentration were identified as key determinants for the growth of the cage versus sheet assemblies.

Solid-state NMR - a complementary technique for protein framework characterization.

Protein frameworks are an emerging class of biomaterial with medical and technological applications. Frameworks are studied mainly by X-ray diffraction or scattering techniques. Complementary strategies are required. Here, we report solid-state NMR analyses of a microcrystalline protein-macrocycle framework and the rehydrated freeze-dried protein. This methodology may aid the characterization of low-crystallinity frameworks.

Protein Frameworks with Thiacalixarene and Zinc.

Controlled protein assembly provides a means to generate biomaterials. Synthetic macrocycles such as the water-soluble sulfonato-calix[n]arenes are useful mediators of protein assembly. Sulfonato-thiacalix[4]arene (tsclx 4 ), with its metal-binding capacity, affords the potential for simultaneous macrocycle- and metal-mediated protein assembly. Here, we describe the tsclx 4 -/Zn-directed assembly of two proteins: cationic α-helical cytochrome c (cyt c) and neutral ß-propeller Ralstonia solanacearum lectin (RSL). Two co-crystal forms were obtained with cyt c, each involving multinuclear zinc sites supported by the cone conformation of tsclx 4 . The tsclx 4 /Zn cluster acted as an assembly node via both lysine encapsulation and metal-mediated protein-protein contacts. In the case of RSL, tsclx 4 adopted the 1,2-alternate conformation and supported a dinuclear zinc site with concomitant encapsulation and metal-binding of two histidine side chains. These results, together with the knowledge of thiacalixarene/metal nanoclusters, suggest promising applications for thiacalixarenes in biomaterials and MOF fabrication.

Comparison of four methods to -determine cage width in tibial tuberosity advancement.

INTRODUCTION: The objective of this study was to test a method for determining the width of the tibial tuberosity advancement (TTA) cage without the need for extension of the stifle joint, while producing preoperative radiographs. TTA cage size was determined by applying 4 different methods using radiograph images in mediolateral direction of fully extended stifles of dogs (n=43), with and without naturally occurring cranial cruciate ligament disease: parallel shift (PS) method, common tangent (CT) method, «2,1¼ method (cage size = 2,1 × tibia plateau length - tibial tuberosity width), and margo cranialis (MC) method (cage size = length of MC/6*1,75). Two new methods, «2,1¼, and MC were compared to the existing CT and PS methods. All 4 methods resulted in reliable cage sizes. Intraclass correlation coefficients showed an excellent reliability of the CT and PS to the «2,1¼ method and a good reliability in regards to the MC method. In conclusion, TTA cage size of the tibia anatomy alone can be determined on radiographs without the need of full extension of the stifle joint. Based on the results, two different methods of cage size measurement are recommended in order to increase diagnostic accuracy and to prevent the insertion of suboptimal cages.

INTRODUCTION: L'objectif de cette étude était de tester une méthode pour déterminer la largeur de la cage d'avancement de la tubérosité tibiale (TTA) sans avoir besoin d'extension du grasset, tout en réalisant des radiographies préopératoires. La taille de la cage TTA a été déterminée en appliquant 4 méthodes différentes, utilisant des images radiographiques dans l'axe médiolatéral des grassets complètement étendus de chiens (n ​​= 43), avec et sans pathologie naturelle du ligament croisé antérieur: méthode du décalage parallèle (PS), méthode de la tangente commune (CT), méthode «2,1¼ (taille de la cage = 2,1 × longueur du plateau du tibia ­ largeur de la tubérosité tibiale) et méthode de la margo cranialis (MC) (taille de la cage = longueur de MC/6*1,75). Deux nouvelles méthodes, «2,1¼ et MC ont été comparées aux méthodes CT et PS existantes. Les 4 méthodes ont abouti à des tailles de cage fiables. Les coefficients de corrélation intra classe ont montré une excellente fiabilité du CT et du PS avec la méthode «2,1¼ et une bonne fiabilité vis-à-vis de la méthode MC. En conclusion, la taille de la cage TTA de l'anatomie du tibia seule peut être déterminée sur des radiographies sans qu'il soit nécessaire d'étendre complètement l'articulation du grasset. Sur la base des résultats, deux méthodes différentes de mesure de la taille des cages sont recommandées afin d'augmenter la précision du diagnostic et d'empêcher la mise en place de cages sous-optimales.

Segregated Protein-Cucurbit[7]uril Crystalline Architectures via Modulatory Peptide Tectons.

One approach to protein assembly involves water-soluble supramolecular receptors that act like glues. Bionanoarchitectures directed by these scaffolds are often system-specific, with few studies investigating their customization. Herein, the modulation of cucurbituril-mediated protein assemblies through the inclusion of peptide tectons is described. Three peptides of varying length and structural order were N-terminally appended to RSL, a ß-propeller building block. Each fusion protein was incorporated into crystalline architectures mediated by cucurbit[7]uril (Q7). A trimeric coiled-coil served as a spacer within a Q7-directed sheet assembly of RSL, giving rise to a layered material of varying porosity. Within the spacer layers, the coiled-coils were dynamic. This result prompted consideration of intrinsically disordered peptides (IDPs) as modulatory tectons. Similar to the coiled-coil, a mussel adhesion peptide (Mefp) also acted as a spacer between protein-Q7 sheets. In contrast, the fusion of a nucleoporin peptide (Nup) to RSL did not recapitulate the sheet assembly. Instead, a Q7-directed cage was adopted, within which disordered Nup peptides were partially "captured" by Q7 receptors. IDP capture occurred by macrocycle recognition of an intrapeptide Phe-Gly motif in which the benzyl group was encapsulated by Q7. The modularity of these protein-cucurbituril architectures adds a new dimension to macrocycle-mediated protein assembly. Segregated protein crystals, with alternating layers of high and low porosity, could provide a basis for new types of materials.

Noncovalent Protein-Pseudorotaxane Assembly Incorporating an Extended Arm Calix[8]arene with α-Helical Recognition Properties.

Water-soluble, anionic calix[n]arenes are useful receptors for protein recognition and assembly. For example, sulfonato-calix[8]arene (sclx 8 ) can encapsulate proteins and direct their assembly into porous frameworks. In this work, we turned our attention to an "extended arm" calixarene with 16 phenyl rings. We hypothesized that this larger receptor would have increased capacity for protein masking/encapsulation. A cocrystal structure of p-benzyl-sulfonato-calix[8]arene (b-sclx 8 ) and cytochrome c (cyt c) revealed a surprising assembly. A pseudorotaxane comprising a stack of three b-sclx 8 molecules threaded by polyethylene glycol (PEG) was bound to the protein. The trimeric b-sclx 8 stack, a tubelike structure with a highly charged surface, mediated assembly via a new mode of protein recognition. The calixarene stack presents four hydrophobic grooves, each of which binds to one cyt c by accommodating the N-terminal α-helix. This unprecedented binding mode suggests new possibilities for supramolecular protein chemistry.

Facile Fabrication of Protein-Macrocycle Frameworks.

Precisely defined protein aggregates, as exemplified by crystals, have applications in functional materials. Consequently, engineered protein assembly is a rapidly growing field. Anionic calix[n]arenes are useful scaffolds that can mold to cationic proteins and induce oligomerization and assembly. Here, we describe protein-calixarene composites obtained via cocrystallization of commercially available sulfonato-calix[8]arene (sclx8) with the symmetric and "neutral" protein RSL. Cocrystallization occurred across a wide range of conditions and protein charge states, from pH 2.2-9.5, resulting in three crystal forms. Cationization of the protein surface at pH ∼ 4 drives calixarene complexation and yielded two types of porous frameworks with pore diameters >3 nm. Both types of framework provide evidence of protein encapsulation by the calixarene. Calixarene-masked proteins act as nodes within the frameworks, displaying octahedral-type coordination in one case. The other framework formed millimeter-scale crystals within hours, without the need for precipitants or specialized equipment. NMR experiments revealed macrocycle-modulated side chain pKa values and suggested a mechanism for pH-triggered assembly. The same low pH framework was generated at high pH with a permanently cationic arginine-enriched RSL variant. Finally, in addition to protein framework fabrication, sclx8 enables de novo structure determination.

Protein recognition by cucurbit[6]uril: high affinity N-terminal complexation.

The donut-shaped cucurbit[n]urils (Qn, n = 6-8) are rigid macrocyclic receptors with widespread use in protein recognition. To date, most applications have centred on the encapsulation of N-terminal aromatic residues by Q7 or Q8. Less attention has been placed on Q6, which can recognize lysine side chains due to its high affinity for alkylamines. In this work, we investigated protein-Q6 complexation by using NMR spectroscopy. Attempts to crystallize protein-Q6 complexes were thwarted by the crystallization of Q6. We studied four proteins that vary in size, net charge, and lysine content. In addition to Q6 interactions with specific Lys or dimethylated Lys residues, we report striking evidence for N-terminal recognition. High affinity (micromolar) binding occurred with the N-terminal Met-Lys motif present in one of the four model proteins. Engineering this feature into another model protein yielded a similar high affinity site. We also present evidence for Q8 binding at this N-terminal feature. These data expand the cucurbituril toolkit for protein sensing.

Engineered assembly of a protein-cucurbituril biohybrid.

A crystalline biohybrid with a 4 : 1 protein : cucurbituril mass ratio is presented. This result was achieved by engineering additional cucurbit[7]uril (Q7) binding sites into a ß-propeller protein. In contrast to the parent protein, Q7-controlled assembly of the engineered variant occurred in solution, as evidenced by NMR and SAXS measurements.

Measuring the Impact of PEGylation on a Protein-Polysaccharide Interaction.

PEGylation is the most widely used half-life extension strategy for protein therapeutics. While it imparts a range of attractive attributes PEGylation can impede protein binding and reduce efficacy. A model system to probe the effects of PEGylation on protein binding has practical applications. Here, we present a system based on complex formation between a hexavalent lectin (RSL) and the globular polysaccharide Ficoll PM70 (a type of glycocluster). Mutants of the lectin were used to generate conjugates with 3, 6, or 12 PEG (1 kDa) chains. Using NMR spectroscopy we monitored how the degree of PEGylation impacted the lectin-Ficoll interaction. The binding propensity was observed to decrease with increasing polymer density. Apparently, the extended PEG chains sterically impede the lectin-Ficoll binding. This deduction was supported by molecular dynamics simulations of the protein-polymer conjugates. The implications for protein-surface interactions are discussed.

Academic engaged time of students with low-incidence disabilities in general education classes.

Academic engaged time of 6 students with low-incidence disabilities enrolled in a general elementary classrooms for reading or math and at least one other subject was compared to that of 6 students without disabilities from the same classes and 6 students without disabilities from different classes that were not inclusive. Three dependent measures used were frequency of observation intervals that students were engaged in academic responding, task management, and competing behaviors. Results showed no significant differences in academic responding and task management behaviors of students with and without disabilities enrolled in general education classes, significant differences between these groups on frequency of competing behaviors, no significant differences between students without disabilities on academic responding and task management, and significant differences between students without disabilities on frequency of competing behaviors.