A florigen-expressing subpopulation of companion cells expresses other small proteins and reveals a nitrogen-sensitive FT repressor.

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T (FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT-expressing cells in cotyledons and in true leaves differed transcriptionally. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

A Legionella effector kinase is activated by host inositol hexakisphosphate.

The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.

Pictorial Review and Basic Principles of Foot and Ankle Hardware Extraction.

UNLABELLED: This pictorial review presents basic principles of the types of hardware extraction commonly encountered in foot and ankle surgical practice. We review the indications, contraindications and complications of hardware removal including pain, intra-articular fixation, and carcinogenesis, as well as special considerations in pediatric patients and in the setting of infection. Figures are then used to describe the appropriate techniques for use of the screwdriver shafts, conical extraction screws, extraction bolts, hollow reamers, and other instruments found in most hardware extraction sets. LEVELS OF EVIDENCE: Therapeutic, Level V: Expert opinion.

Where art thou diabetic foot disease literature? A bibliometric inquiry into publication patterns.

The objective of the present investigation was to evaluate the published scientific data with respect to the diabetic foot. We specifically aimed to assess the quantity published and the specific location of the published reports. A standard PubMed(®) search was performed, and the returned abstracts were evaluated by a 2-person panel for their relevance to medical professionals working within the field of diabetic foot disease. We identified 1286 relevant studies published in 659 different journals in 2012. We also found a 6.94-fold increase in returned abstracts meeting our search criteria from January 1988 to December 2012. The results of our investigation provide unique information regarding the high volume and variety of published information pertaining to diabetic foot disease and perhaps highlights a need for multidisciplinary thinking with respect to publishing and data organization, in addition to patient care.

Can foot and ankle surgeons accurately estimate patient body mass index?

The body mass index (BMI) is an objective patient finding that has been established to have a negative effect on the development and outcomes of podiatric pathologic entities and interventions. The objective of the present investigation was to assess the ability of podiatric physicians to estimate the patient BMI from clinical and radiographic observation. For the clinical estimation of the patient BMI, podiatric specialists across 3 levels of experience (i.e., students, residents, and practicing clinicians) performed 294 estimations on 72 patients in 3 clinical situations (standing, sitting in a treatment chair, and lying in a hospital bed). It was more common to inaccurately estimate the patient BMI (77.9%) than it was to correctly estimate it (22.1%), with underestimations being the most common error (48.3%). The estimations were particularly inaccurate when the patients were in the common clinical situation of sitting in a treatment chair or lying in a hospital bed and with patients actually classified as obese. For the radiographic estimation of patient BMI, 150 consecutive lateral ankle radiographs were analyzed, with the ratio of the overlying soft tissue diameter to the underlying bone diameter calculated and compared. Positive, but weak, relationships were observed with these ratios. From these data, we have concluded that podiatric practitioners should perform an actual calculation of the patient BMI during the patient examination and medical decision-making process to fully appreciate the potential risks inherent to the treatment of obese patients.

Practice nurses and obesity: professional and practice-based factors affecting role adequacy and role legitimacy.

AIM: This qualitative study explored the professional and practice-based factors affecting the role legitimacy and adequacy of practice nurses in managing obese patients. BACKGROUND: There are strong clinical, financial and practical reasons for tackling obesity in UK general practice. Although practice nurses may seem to be in an ideal position to manage obesity, there remain questions about their role adequacy (sense of self-efficacy in responding to patients' problems) and role legitimacy (their perceived boundaries of professional responsibility and right to intervene). METHODS: Semi-structured face-to-face interviews were conducted with 22 practice nurses in Lambeth, Southwark and Lewisham in South London. Interviews were digitally recorded and transcribed. Key themes were identified following coding of the data. Findings Factors that positively affected nurses' role adequacy and legitimacy were: their belief that obesity management was part of their chronic disease management and health promotion remit; their confidence in their own communication skills and ability to build rapport with patients; having attended training and being supported to take extra time for obesity management. Factors negatively affecting their role legitimacy and adequacy were: their low awareness and use of guidance; lack of knowledge of referral options; limited knowledge and use of non-medical and non-persuasive approaches; perceived lack of expertise in motivating patients, as well as in nutrition, child obesity and assessment; belief that there were some contexts in which it was more appropriate to raise the issue than others; lack of culturally appropriate materials and language barriers; belief that they had limited impact on outcome and that the patient is responsible for lack of success. Other factors negatively affecting their role adequacy and legitimacy included their ambivalence about the effectiveness of the interventions offered; perceived lack of priority for obesity management within practices; lack of time; workload and lack of clarity on protocols and roles within the practice.

1H NMR investigation of thermally triggered insulin release from poly(N-isopropylacrylamide) microgels.

We describe investigations of insulin release from thermoresponsive microgels using variable temperature (1)H NMR. Microgel particles composed of poly(N-isopropylacrylamide) were loaded with the peptide via a swelling technique, and this method was compared to simple equilibrium partitioning. Variable temperature (1)H NMR studies suggest that the swelling loading method results in enhanced entrapment of the peptide versus equilibrium partitioning. A centrifugation-loading assay supports this finding. Pseudo-temperature jump (1)H NMR measurements suggest that the insulin release rate is partially decoupled from microgel collapse. These types of direct release investigations could prove to be useful methods in the future design of controlled macromolecule drug delivery devices.

Phase transition behavior, protein adsorption, and cell adhesion resistance of poly(ethylene glycol) cross-linked microgel particles.

Thermoresponsive poly(N-isopropylacrylamide) (pNIPAm) microgel particles cross-linked with various concentrations of PEG diacrylates of 3 different PEG chain lengths were synthesized via free-radical precipitation polymerization in order to investigate the phase transition and protein adsorption behavior as the hydrophilicity of the network is increased. Photon correlation spectroscopy (PCS) reveals that, as the concentration of PEG cross-linker incorporated into the particles is increased, an increase in the temperature and breadth of the phase transition occurs. Qualitative differences in particle density using isopycnic centrifugation confirm that higher PEG concentrations result in denser networks. The efficient incorporation of PEG cross-linker was confirmed with (1)H NMR, and variable temperature NMR studies suggest that, in the deswollen state, the longer PEG cross-links protrude from the dense globular network. This behavior apparently manifests itself as a decrease in nonspecific protein adsorption with increasing PEG length and content. Furthermore, when electrostatically attached to a glass surface, the particles containing the longer chain lengths exhibited enhanced nonfouling behavior and were resistant to cell adhesion in serum-containing media. The excellent performance of these particulate films and the simplicity with which they are assembled suggests that they may be applicable in a wide range of applications where nonfouling coatings are required.

Doxorubicin uptake and release from microgel thin films.

We report investigations on the thermally regulated uptake and release of the chemotherapeutic drug doxorubicin from microgel thin films. A spin coating, layer-by-layer (scLbL) assembly approach was used to prepare thin films composed of thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-AAc) microgels by alternatively exposing a 3-aminopropyltrimethoxysilane (APTMS) functionalized glass substrate to polyanionic pNIPAm-AAc microgels and polycationic poly(allylamine hydrochloride) (PAH). Using this method, 10, 20, and 30 microgel layer films were constructed with uniform layer buildup, as confirmed by quartz crystal microgravimetry (QCM). The films were subsequently loaded with doxorubicin by cycling the temperature of the film in an aqueous doxorubicin solution between 25 and 50 degrees C. Release characteristics were then examined using UV-vis spectroscopy, which revealed temperature-dependent release properties.

Thermally modulated insulin release from microgel thin films.

We describe investigations of thermally triggered insulin release from poly(N-isopropylacrylamide-co-acrylic acid) microgel thin films prepared by layer-by-layer (LbL) polyelectrolyte assembly. The thermoresponsivity of these films was confirmed using light scattering techniques. Simultaneous monitoring of film collapse and insulin release kinetics shows that deswelling of the films is partially decoupled from macromolecule release and that release is mainly governed by partitioning effects. We hypothesize, however, that film thermoresponsivity plays an important role in that subjection to many thermal cycles enables the embedded peptide to solubilize and subsequently partition through film layers. Direct pulsatile and extended release studies confirm the capability of these films to release bursts of insulin over many cycles, and confirm that the magnitude of the release can be controlled based on film thickness. These insulin-impregnated films are extremely stable with the potential to release constant pulses of peptide for more than 1 month at a time.