Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Partial nephrectomy in a patient with dwarfism.

We describe the case of a 50-year-old male with achondroplastic dwarfism who presents with a renal mass in his left kidney concerning for renal cell carcinoma. The patient successfully underwent a robotic partial nephrectomy, which revealed a T1a renal cell carcinoma. The tumor was excised successfully without any intraoperative complications demonstrating that a robotic partial nephrectomy is technically both safe and effective in patients with achondroplastic dwarfism.

Posterior Malleolar Fractures Associated With Tibial Shaft Fractures and Sequence of Fixation.

OBJECTIVES: The purpose of this study was to evaluate posterior malleolar injuries associated with nailed tibial fractures and to determine the quality of reduction based on the sequence of fixation in associated fracture patterns. DESIGN: Retrospective cohort study. PATIENTS: 1113 tibia fractures treated with an intramedullary nail at 3 level I trauma centers. INTERVENTION: Tibial shaft fractures with posterior malleolar injury were analyzed regarding type of fracture, mechanism of injury, energy of injury, fracture characteristic, surgical characteristics including sequence of fixation, obvious intraoperative displacement of the posterior malleolar fragment, and the quality of reduction. One group ("malleolus-first") consisted of patients in whom the posterior malleolus was fixed before tibial nailing and the other group ("tibia-first") included patients in whom tibial nailing was done before posterior malleolus fixation. OUTCOMES MEASURED: Intraoperative displacement, quality of reduction. RESULTS: Ninety-six of 1113 (9%) nailed tibial shaft fracture patients had a concomitant posterior malleolus fracture (9%). Of the 96 posterior malleolar fracture patients, 70 patients were operatively treated (73%). In the malleolus-first group (54 patients), intraoperative displacement of the posterior malleolar fragment was observed in 1 patient, and 1 case of poor reduction of the posterior malleolar fragment was observed (2%). In the tibia-first group (16 patients), obvious intraoperative displacement of the posterior malleolar fragment was observed in 5 patients (31%), and poor reduction of the posterior malleolar fragment was observed in 7 patients (44%). These percentages of patients with poor quality of reduction were statistically significantly different (p ≤ 0.01). CONCLUSION: Many low-energy tibia fractures with a spiral configuration do have an associated posterior malleolus fracture. In order to avoid intraoperative displacement and poor reduction, we recommend fixation of the posterior malleolar fragment before nailing of the tibia in associated fracture pattern. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Quinine binding by the cocaine-binding aptamer. Thermodynamic and hydrodynamic analysis of high-affinity binding of an off-target ligand.

The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.

Co-repressor release but not ligand binding is a prerequisite for transcription activation by human retinoid acid receptor alpha ligand-binding domain.

Nuclear hormone receptors coordinately regulate the activity of genetic networks through the recruitment of transcriptional co-regulators, including co-repressors and co-activators. Allosteric modulation of the ligand-binding domain by hormonal activators shifts the co-factor binding preference by defined structural changes in overlapping docking sites. We report here that mutations at conserved residues within the docking motif of the retinoic acid receptor alpha cause defects in dimerization, co-regulator association, and transcriptional regulation. Furthermore, although a minimal co-repressor receptor interaction domain is sufficient for receptor binding, flanking sequences appear to stabilize this interaction without interfering with ligand sensitivity. However, ligand sensitivity is changed by the K262A mutation, which requires much higher concentrations of all-trans-retinoic acid to promote co-repressor dissociation. Consequently, K262A functions as a dominant-negative mutant at low concentrations of all-trans-retinoic acid. As a result, transcriptional activation is mechanistically linked to co-repressor release.

Isolation and characterization of mammalian HDAC10, a novel histone deacetylase.

Acetylation of histone core particles plays an important role in modulating chromatin structure and gene expression. The acetylation status of the histone tails is determined by two opposing enzymatic activities, histone acetyltransferases and histone deacetylases (HDACs). Here we describe the isolation and characterization of HDAC10, a novel class II histone deacetylase. Molecular cloning and Northern blot analyses reveal that the HDAC10 transcript is widely expressed and subjected to alternative splicing. HDAC10 is both nuclear and cytoplasmic, a feature reminiscent of HDACs 4, 5, and 7. Distinct from other family members, HDAC10 harbors an amino-terminal catalytic domain and a carboxyl pseudo-repeat that shares significant homology with its catalytic domain. Mutational analysis reveals that transcriptional repression by HDAC10 requires its intrinsic histone deacetylase activity. Taken together, HDAC10 represents a distinct HDAC that may play a role in transcription regulation.