Novel application of synchrotron x-ray computed tomography for ex-vivo imaging of subcutaneously injected polymeric microsphere suspension formulations.

PURPOSE: Subcutaneously or intramuscularly administered biodegradable microsphere formulations have been successfully exploited in the management of chronic conditions for over two decades, yet mechanistic understanding of the impact of formulation attributes on in vivo absorption rate from such systems is still in its infancy. METHODS: Suspension formulation physicochemical attributes may impact particulate deposition in subcutaneous (s.c.) tissue. Hence, the utility of synchrotron X-ray micro-computed tomography (µCT) for assessment of spatial distribution of suspension formulation components (PLG microspheres and vehicle) was evaluated in a porcine s.c. tissue model. Optical imaging of dyed vehicle and subsequent microscopic assessment of microsphere deposition was performed in parallel to compare the two approaches. RESULTS: Our findings demonstrate that synchrotron µCT can be applied to the assessment of microsphere and vehicle distribution in s.c. tissue, and that microspheres can also be visualised in the absence of contrast agent using this approach. The technique was deemed superior to optical imaging of macrotomy for the characterisation of microsphere deposition owing to its non-invasive nature and relatively rapid data acquisition time. CONCLUSIONS: The method outlined in this study provides a proof of concept feasibility for µCT application to determining the vehicle and suspended PLG microspheres fate following s.c. injection. A potential application for our findings is understanding the impact of injection, device and formulation variables on initial and temporal depot geometry in pre-clinical or ex-vivo models that can inform product design. Graphical abstract.

Celebrate Birth!-Chloe's Birth.

Leigh Anne shares the story not only of Chloe's birth but of the birth of her first daughter, Phoebe, and the loss of her second baby. She describes waiting for labor to start, and her frustration when arriving at the hospital to find, to her surprise, that she was not ready to push. With the support of her midwife, her husband, and her friend, Leigh Anne manages a hard labor, made long and difficult because of a compound presentation, and gives birth to her sweet baby girl.

Robust hematopoietic specification requires the ubiquitous Sp1 and Sp3 transcription factors.

BACKGROUND: Both tissue-specific and ubiquitously expressed transcription factors, such as Sp-family members, are required for correct development. However, the molecular details of how ubiquitous factors are involved in programming tissue-specific chromatin and thus participate in developmental processes are still unclear. We previously showed that embryonic stem cells lacking Sp1 DNA-binding activity (Sp1ΔDBD/ΔDBD cells) are able to differentiate into early blood progenitors despite the inability of Sp1 to bind chromatin without its DNA-binding domain. However, gene expression during differentiation becomes progressively deregulated, and terminal differentiation is severely compromised. RESULTS: Here, we studied the cooperation of Sp1 with its closest paralogue Sp3 in hematopoietic development and demonstrate that Sp1 and Sp3 binding sites largely overlap. The complete absence of either Sp1 or Sp3 or the presence of the Sp1 DNA-binding mutant has only a minor effect on the pattern of distal accessible chromatin sites and their transcription factor binding motif content, suggesting that these mutations do not affect tissue-specific chromatin programming. Sp3 cooperates with Sp1ΔDBD/ΔDBD to enable hematopoiesis, but is unable to do so in the complete absence of Sp1. Using single-cell gene expression analysis, we show that the lack of Sp1 DNA binding leads to a distortion of cell fate decision timing, indicating that stable chromatin binding of Sp1 is required to maintain robust differentiation trajectories. CONCLUSIONS: Our findings highlight the essential contribution of ubiquitous factors such as Sp1 to blood cell development. In contrast to tissue-specific transcription factors which are required to direct specific cell fates, loss of Sp1 leads to a widespread deregulation in timing and coordination of differentiation trajectories during hematopoietic specification.

A novel molten-salt electrochemical cell for investigating the reduction of uranium dioxide to uranium metal by lithium using in situ synchrotron radiation.

A novel electrochemical cell has been designed and built to allow for in situ energy-dispersive X-ray diffraction measurements to be made during reduction of UO2 to U metal in LiCl-KCl at 500°C. The electrochemical cell contains a recessed well at the bottom of the cell into which the working electrode sits, reducing the beam path for the X-rays through the molten-salt and maximizing the signal-to-noise ratio from the sample. Lithium metal was electrodeposited onto the UO2 working electrode by exposing the working electrode to more negative potentials than the Li deposition potential of the LiCl-KCl eutectic electrolyte. The Li metal acts as a reducing agent for the chemical reduction of UO2 to U, which appears to proceed to completion. All phases were fitted using Le Bail refinement. The cell is expected to be widely applicable to many studies involving molten-salt systems.

The Role of the Ubiquitously Expressed Transcription Factor Sp1 in Tissue-specific Transcriptional Regulation and in Disease.

Sp1 belongs to the 26 member strong Sp/KLF family of transcription factors. It is a paradigm for a ubiquitously expressed transcription factor and is involved in regulating the expression of genes associated with a wide range of cellular processes in mammalian cells. Sp1 can interact with a range of proteins, including other transcription factors, members of the transcription initiation complex and epigenetic regulators, enabling tight regulation of its target genes. In this review, we discuss the mechanisms involved in Sp1-mediated transcriptional regulation, as well as how a ubiquitous transcription factor can be involved in establishing a tissue-specific pattern of gene expression and mechanisms by which its activity may be regulated. We also consider the role of Sp1 in human diseases, such as cancer.

I12: the Joint Engineering, Environment and Processing (JEEP) beamline at Diamond Light Source.

I12 is the Joint Engineering, Environmental and Processing (JEEP) beamline, constructed during Phase II of the Diamond Light Source. I12 is located on a short (5 m) straight section of the Diamond storage ring and uses a 4.2 T superconducting wiggler to provide polychromatic and monochromatic X-rays in the energy range 50-150 keV. The beam energy enables good penetration through large or dense samples, combined with a large beam size (1 mrad horizontally × 0.3 mrad vertically). The beam characteristics permit the study of materials and processes inside environmental chambers without unacceptable attenuation of the beam and without the need to use sample sizes which are atypically small for the process under study. X-ray techniques available to users are radiography, tomography, energy-dispersive diffraction, monochromatic and white-beam two-dimensional diffraction/scattering and small-angle X-ray scattering. Since commencing operations in November 2009, I12 has established a broad user community in materials science and processing, chemical processing, biomedical engineering, civil engineering, environmental science, palaeontology and physics.

Transcription mediated insulation and interference direct gene cluster expression switches.

In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.

Structural characterisation of a layered double hydroxide nanosheet.

We report the atomic-scale structure of a Zn152Al-borate layered double hydroxide (LDH) nanosheet, as determined by reverse Monte Carlo (RMC) modelling of X-ray total scattering data. This study involves the extension of the RMC method to enable structural refinement of two-dimensional nanomaterials. The refined LDH models show the intra-layer geometry in this highly-exfoliated phase to be consistent with that observed in crystalline analogues, with the reciprocal-space scattering data suggesting a disordered arrangement of the Zn(2+) and Al(3+) cations within the nanosheet. The approach we develop is generalisable and so offers a method of characterising the structures of arbitrary nanosheet phases, including systems that support complex forms of disorder within the nanosheets themselves.

Clonal expansion within pneumococcal serotype 6C after use of seven-valent vaccine.

Streptococcus pneumoniae causes invasive infections, primarily at the extremes of life. A seven-valent conjugate vaccine (PCV7) is used to protect against invasive pneumococcal disease in children. Within three years of PCV7 introduction, we observed a fourfold increase in serotype 6C carriage, predominantly due to a single clone. We determined the whole-genome sequences of nineteen S. pneumoniae serotype 6C isolates, from both carriage (n = 15) and disease (n = 4) states, to investigate the emergence of serotype 6C in our population, focusing on a single multi-locus sequence type (MLST) clonal complex 395 (CC395). A phylogenetic network was constructed to identify different lineages, followed by analysis of variability in gene sets and sequences. Serotype 6C isolates from this single geographical site fell into four broad phylogenetically distinct lineages. Variation was seen in the 6C capsular locus and in sequences of genes encoding surface proteins. The largest clonal complex was characterised by the presence of lantibiotic synthesis locus. In our population, the 6C capsular locus has been introduced into multiple lineages by independent capsular switching events. However, rapid clonal expansion has occurred within a single MLST clonal complex. Worryingly, plasticity exists within current and potential vaccine-associated loci, a consideration for future vaccine use, target selection and design.