Faculty Bibliography

ECF transporters are a family of active transporters for vitamins. They are composed of four subunits: a membrane-embedded substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT) and two ATP-binding-cassette ATPases (EcfA and EcfA'). We have investigated the mechanism of the ECF transporter for riboflavin from the pathogen Listeria monocytogenes, LmECF-RibU. Using structural and biochemical approaches, we found that ATP binding to the EcfAA' ATPases drives a conformational change that dissociates the S subunit from the EcfAA'T ECF module. Upon release from the ECF module, the RibU S subunit then binds the riboflavin transport substrate. We also find that S subunits for distinct substrates compete for the ATP-bound state of the ECF module. Our results explain how ECF transporters capture the transport substrate and reproduce the in vivo observations on S-subunit competition for which the family was named.

BACKGROUND: Cell-assisted lipotransfer has shown much promise as a technique to improve fat graft take. However, the concentration of stromal vascular fraction cells required to optimally enhance fat graft retention remains unknown. METHODS: Human lipoaspirate was processed for both fat transfer and harvest of stromal vascular fraction (SVF) cells. Cells were then mixed back with fat at varying concentrations ranging from 10,000 to 10 million cells per 200 mul of fat. Fat graft volume retention was assessed via CT scanning over 8 weeks, and then fat grafts were explanted and compared histologically for overall architecture and vascularity. RESULTS: Maximum fat graft retention was seen at a concentration of 10,000 cells per 200 mul of fat. The addition of higher number of cells negatively impacted fat graft retention, with supplementation of 10 million cells producing the lowest final volumes, lower than fat alone. Interestingly, fat grafts supplemented with 10,000 cells showed significantly increased vascularity and decreased inflammation, while fat grafts supplemented with 10 million cells showed significant lipodegeneration compared to fat alone CONCLUSIONS:: Our study demonstrates dose dependence in the number of SVF cells that can be added to a fat graft to enhance retention. While cell-assisted lipotransfer may help promote graft survival, this effect may need to be balanced with the increased metabolic load of added cells that may compete with adipocytes for nutrients during the post-graft period.

BACKGROUND AND PURPOSE: To improve oral delivery of AmB by achieving increased oral bioavailability and synergistically enhanced antileishmanial activity using copaiba oil (Cop) through nano-emulsified carrier system (CopNEC). EXPERIMENTAL APPROACH: The AmB encapsulated nano-emulsified carrier (CopNEC-AmB) comprised of Cop, d-alpha Tocopheryl Polyethylene Glycol 1000 Succinate and phosphatidylcholine was prepared by high pressure homogenization method. Stability study of CopNEC-AmB was carried out in simulated gastric fluid and simulated intestinal fluid. The CopNEC-AmB and plain AmB were compared for in vitro antileishmanial activity, their pharmacokinetics, organ distribution and toxicity profiling. RESULTS: The optimized CopNEC-AmB has globule size of 127+/-21 nm, PDI 0.11+/-0.02, and zeta potential (-)38.5+/-2.7 with encapsulation efficiency 91.9+/-1.4 %w/w. The high resolution transmission electron microscopy illustrates spherical particle geometry with homogeny in their sizes. The optimized CopNEC-AmB was found to be stable in gastro-intestinal fluids showing insignificant changes in globule size and encapsulation efficiency. The AUC0-48 value of CopNEC-AmB in rats was significantly improved showing 7.2 folds higher oral bioavailability than free drug. The in vitro antileishmanial activity of CopNEC-AmB was significantly higher (p<0.05) than the free drug because copaiba oil synergistically enhanced chemotherapy of leishmaniasis by causing drastic changes in morphology of Leishmania parasite and rupture of the plasma membrane with loss of their contents. The CopNEC-AmB showed significantly lesser hemolytic toxicity and cell cytotoxicity, and absence of any changes in histopathology of kidney tissues as compared to free drug. CONCLUSION: This prototype CopNEC formulation showed improved bioavailability and nontoxic synergistic antileishmanial chemotherapy of AmB due to copaiba oil against leishmaniasis.

BACKGROUND: Dalbavancin is a lipoglycopeptide antibiotic with Gram-positive activity and novel pharmacokinetic (PK) properties that result in a prolonged terminal half-life of 15.5 days in adults. Once weekly dosing in adults in phase 3 studies of complicated skin and skin structure infections documented dalbavancin exposures associated with clinical and microbiologic efficacy. PK properties have not been examined in children. The primary objective of this open-label, multicenter single-dose phase 1 study was to characterize the PK of dalbavancin in hospitalized pediatric subjects 12-17 years of age. METHODS: A single dose of 1000 mg of dalbavancin (the standard adult dose) was administered as a 30-minute intravenous infusion to subjects weighing 60 kg or greater and 15 mg/kg for subjects weighing <60 kg. A noncompartmental PK analysis was performed. RESULTS: The apparent terminal t1/2 was approximately 9 days and was similar for dalbavancin dosages of 1000 mg and 15 mg/kg. Median dalbavancin plasma exposures (Cmax and AUCinf) when administered as 1000 mg to subjects weighing 60 kg or greater were similar to those when dalbavancin was administered at 15 mg/kg to subjects weighing <60 kg and slightly lower than exposures in adults given 1000 mg in prior PK and treatment studies. Single dose dalbavancin was well tolerated. CONCLUSIONS: Given dalbavancin exposures documented in children 12-17 years of age, and recognized dose proportionality, appropriate dosing can be modeled for pediatric phase 3 trials in acute bacterial skin and skin structure infections, to achieve the same exposure that is reported to be safe and effective in adults.

Aromatase inhibitors (AIs) are effective drugs that reduce or eliminate hormone sensitive breast cancer. However, despite their efficacy, resistance to these drugs can occur in some patients. The INrf2 (Keap1):Nrf2 complex serves as a sensor of drug/radiation-induced oxidative/electrophilic stress. INrf2 constitutively suppresses Nrf2 by functioning as an adapter protein for the Cul3/Rbx1-mediated ubiquitination/degradation of Nrf2. Upon stress, Nrf2 dissociates from INrf2, is stabilized, translocates to the nucleus, and coordinately induces a battery of cytoprotective gene expression. Current studies investigated the role of Nrf2 in AI resistance. RT-PCR and immunoblot assays showed that AI-resistant breast cancer LTLTCa and AnaR cells express lower INrf2 and higher Nrf2 protein levels, as compared to drug sensitive MCF-7Ca and AC1 cells, respectively. The increase in Nrf2 was due to lower ubiquitination/degradation of Nrf2 in AI-resistant cells. Higher Nrf2-mediated levels of biotransformation enzymes, drug-transporters and anti-apoptotic proteins contributed to reduced efficacy of drugs and aversion to apoptosis that led to drug resistance. shRNA inhibition of Nrf2 in LTLTCa (LTLTCa-Nrf2KD) cells reduced resistance and sensitized cells to AI exemestane. Interestingly, LTLTCa-Nrf2KD cells also showed reduced levels of aldehyde dehydrogenase, a marker of Tumor-Initiating Cells and significantly decreased mammosphere formation, as compared to LTLTCa-Vector control cells. The results together suggest that persistent AI treatment down-regulated INrf2 leading to higher expression of Nrf2 and Nrf2 regulated cytoprotective proteins that resulted in increased AI drug resistance. These findings provide a rationale for the development of Nrf2 inhibitors to overcome resistance and increase efficacy of AI.

Transforming growth factor-beta (TGF-beta) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-beta signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-beta1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein-protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-beta1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-beta1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFbeta-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients.

Male olive (Papio anubis) and hamadryas (P. hamadryas) baboons have distinctive sociobehavioral and physical characteristics. In the Awash National Park, Ethiopia, a hybrid population at the contact zone between these two species, exhibits heterogeneous sociobehavioral and physical characteristics. The ambiguity of the hybrid social environment and disruption of parental stress genotypes may be sources of physiological stress for hybrids. We examined levels of chronic stress among males of the three populations and tested the prediction that chronic cortisol levels would be higher among the hybrids. Animals were captured, sampled, and released during the wet season, and a hair sample was taken for assay. Cortisol was extracted from 182 hair samples with methanol and quantified by ELISA. We included age, age class, rainfall variation, and species affiliation in models examining variation in hair cortisol levels. Species and age significantly contributed to models explaining variation in hair cortisol. Infant hypercortisolism was observed in all three groups, and a decline in cortisol through juvenile and adolescent stages, with a subsequent rise in adulthood. This rise occurred earliest in hamadryas, corroborating other evidence of the precocious development of hamadryas baboons. As expected, hybrids had significantly elevated hair cortisol compared with olive baboons and hamadryas, irrespective of age, except for very young animals. Infant hypercortisolism was also less pronounced among hybrids. Species differences and age-related differences in cortisol levels suggest a dysregulated cortisol phenotype in hybrids, and possibly reflect some form of hybrid disadvantage. More work will be required to disentangle the effects of genetic factors and the social environment.

Recent studies in humans and in genetic mouse models have identified Slit- and NTRK-like family (Slitrks) as candidate genes for neuropsychiatric disorders. All Slitrk isotypes are highly expressed in the CNS, where they mediate neurite outgrowth, synaptogenesis, and neuronal survival. However, the molecular mechanisms underlying these functions are not known. Here, we report that Slitrk5 modulates brain-derived neurotrophic factor (BDNF)-dependent biological responses through direct interaction with TrkB receptors. Under basal conditions, Slitrk5 interacts primarily with a transsynaptic binding partner, protein tyrosine phosphatase delta (PTPdelta); however, upon BDNF stimulation, Slitrk5 shifts to cis-interactions with TrkB. In the absence of Slitrk5, TrkB has a reduced rate of ligand-dependent recycling and altered responsiveness to BDNF treatment. Structured illumination microscopy revealed that Slitrk5 mediates optimal targeting of TrkB receptors to Rab11-positive recycling endosomes through recruitment of a Rab11 effector protein, Rab11-FIP3. Thus, Slitrk5 acts as a TrkB co-receptor that mediates its BDNF-dependent trafficking and signaling.

Extracellular signalling molecules control many biological processes, but the influence of tissue architecture on the local concentrations of these factors is unclear. Here we examine this issue in the Drosophila egg chamber, where two anterior cells secrete Unpaired (Upd) to activate Signal transducer and activator of transcription (STAT) signalling in the epithelium. High STAT signalling promotes cell motility. Genetic analysis shows that all cells near the Upd source can respond. However, using upright imaging, we show surprising asymmetries in STAT activation patterns, suggesting that some cells experience different Upd levels than predicted by their location. We develop a three-dimensional mathematical model to characterize the spatio-temporal distribution of the activator. Simulations show that irregular tissue domains can produce asymmetric distributions of Upd, consistent with results in vivo. Mutant analysis substantiates this idea. We conclude that cellular landscape can heavily influence the effect of diffusible activators and should be more widely considered.

By the end of 2014, an estimated 8.5 million men had undergone voluntary medical male circumcision (VMMC) for HIV prevention in 14 priority countries in eastern and southern Africa, representing more than 40% of the global target. However, demand, especially among men most at risk for HIV infection, remains a barrier to realizing the program's full scale and potential impact. We analyzed current demand generation interventions for VMMC by reviewing the available literature and reporting on field visits to programs in 7 priority countries. We present our findings and recommendations using a framework with 4 components: insight development; intervention design; implementation and coordination to achieve scale; and measurement, learning, and evaluation. Most program strategies lacked comprehensive insight development; formative research usually comprised general acceptability studies. Demand generation interventions varied across the countries, from advocacy with community leaders and community mobilization to use of interpersonal communication, mid- and mass media, and new technologies. Some shortcomings in intervention design included using general instead of tailored messaging, focusing solely on the HIV preventive benefits of VMMC, and rolling out individual interventions to address specific barriers rather than a holistic package. Interventions have often been scaled-up without first being evaluated for effectiveness and cost-effectiveness. We recommend national programs create coordinated demand generation interventions, based on insights from multiple disciplines, tailored to the needs and aspirations of defined subsets of the target population, rather than focused exclusively on HIV prevention goals. Programs should implement a comprehensive intervention package with multiple messages and channels, strengthened through continuous monitoring. These insights may be broadly applicable to other programs where voluntary behavior change is essential to achieving public health benefits.