In vivo lung morphometry with hyperpolarized 3He diffusion MRI in canines with induced emphysema: disease progression and comparison with computed tomography.

Despite a long history of development, diagnostic tools for in vivo regional assessment of lungs in patients with pulmonary emphysema are not yet readily available. Recently, a new imaging technique, in vivo lung morphometry, was introduced by our group. This technique is based on MRI measurements of diffusion of hyperpolarized (3)He gas in lung air spaces and provides quantitative in vivo tomographic information on lung microstructure at the level of the acinar airways. Compared with standard diffusivity measurements that strongly depend on pulse sequence parameters (mainly diffusion time), our approach evaluates a "hard number," the average acinar airway radius. For healthy dogs, we find here a mean acinar airway radius of approximately 0.3 mm compared with 0.36 mm in healthy humans. The purpose of the present study is the application of this technique for quantification of emphysema progression in dogs with experimentally induced disease. The diffusivity measurements and resulting acinar airway geometrical characteristics were correlated with the local lung density and local lung-specific air volume calculated from quantitative computed tomography data obtained on the same dogs. The results establish an important association between the two modalities. The observed sensitivity of our method to emphysema progression suggests that this technique has potential for the diagnosis of emphysema and tracking of disease progression or improvement via a pharmaceutical intervention.     

Visualization of Dynamic Sub-microsecond Changes in Membrane Potential

Direct observation of rapid membrane potential changes is critical to understand how complex neurological systems function. This knowledge is especially important when stimulation is achieved through an external stimulus meant to mimic a naturally occurring process. To enable exploration of this dynamic space, we developed an all-optical method for observing rapid changes in membrane potential at temporal resolutions of ～25 ns. By applying a single 600-ns electric pulse, we observed sub-microsecond, continuous membrane charging and discharging dynamics. Close agreement between the acquired results and an analytical membrane-charging model validates the utility of this technique. This tool will deepen our understanding of the role of membrane potential dynamics in the regulation of many biological and chemical processes within living systems.     

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

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Phosphorylation of NHERF1 S279 and S301 differentially regulates breast cancer cell phenotype and metastatic organotropism

Metastatic cancer cells are highly plastic for the expression of different tumor phenotype hallmarks and organotropism. This plasticity is highly regulated but the dynamics of the signaling processes orchestrating the shift from one cell phenotype and metastatic organ pattern to another are still largely unknown. The scaffolding protein NHERF1 has been shown to regulate the expression of different neoplastic phenotypes through its PDZ domains, which forms the mechanistic basis for metastatic organotropism. This reprogramming activity was postulated to be dependent on its differential phosphorylation patterns. Here, we show that NHERF1 phosphorylation on S279/S301 dictates several tumor phenotypes such as in vivo invasion, NHE1-mediated matrix digestion, growth and vasculogenic mimicry. Remarkably, injecting mice with cells having differential NHERF1 expression and phosphorylation drove a shift from the predominantly lung colonization (WT NHERF1) to predominately bone colonization (double S279A/S301A mutant), indicating that NHERF1 phosphorylation also acts as a signaling switch in metastatic organotropism.     

Drug development for the treatment of onchocerciasis: Population pharmacokinetic and adverse events modeling of emodepside

BackgroundTo accelerate the progress towards onchocerciasis elimination, a macrofilaricidal drug that kills the adult parasite is urgently needed. Emodepside has shown macrofilaricidal activity against a variety of nematodes and is currently under clinical development for the treatment of onchocerciasis. The aims of this study were i) to characterize the population pharmacokinetic properties of emodepside, ii) to link its exposure to adverse events in healthy volunteers, and iii) to propose an optimized dosing regimen for a planned phase II study in onchocerciasis patients.Methodology / Principal findingsPlasma concentration-time profiles and adverse event data were obtained from 142 subjects enrolled in three phase I studies, including a single-dose, and a multiple-dose, dose-escalation study as well as a relative bioavailability study. Nonlinear mixed-effects modeling was used to evaluate the population pharmacokinetic properties of emodepside. Logistic regression modeling was used to link exposure to drug-related treatment-emergent adverse events (TEAEs). Emodepside pharmacokinetics were well described by a transit-absorption model, followed by a 3-compartment disposition model. Body weight was included as an allometric function and both food and formulation had a significant impact on absorption rate and relative bioavailability. All drug-related TEAEs were transient, and mild or moderate in severity. An increase in peak plasma concentration was associated with an increase in the odds of experiencing a drug-related TEAE of interest.Conclusions/SignificancePharmacokinetic modeling and simulation was used to derive an optimized, body weight-based dosing regimen, which allows for achievement of extended emodepside exposures above target concentrations while maintaining acceptable tolerability margins.     

Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers

The two Flag/MaSp 2 silk proteins produced recombinantly were based on the basic consensus repeat of the dragline silk spidroin 2 protein (MaSp 2) from the Nephila clavipes orb weaving spider. However, the proline-containing pentapeptides juxtaposed to the polyalanine segments resembled those found in the flagelliform silk protein (Flag) composing the web spiral: (GPGGX(1) GPGGX(2))(2) with X(1) /X(2) = A/A or Y/S. Fibers were formed from protein films in aqueous solutions or extruded from resolubilized protein dopes in organic conditions when the Flag motif was (GPGGX(1) GPGGX(2))(2) with X(1) /X(2) = Y/S or A/A, respectively. Post-fiber processing involved similar drawing ratios (2-2.5×) before or after water-treatment. Structural (ssNMR and XRD) and morphological (SEM) changes in the fibers were compared to the mechanical properties of the fibers at each step. Nuclear magnetic resonance indicated that the fraction of β-sheet nanocrystals in the polyalanine regions formed upon extrusion, increased during stretching, and was maximized after water-treatment. X-ray diffraction showed that nanocrystallite orientation parallel to the fiber axis increased the ultimate strength and initial stiffness of the fibers. Water furthered nanocrystal orientation and three-dimensional growth while plasticizing the amorphous regions, thus producing tougher fibers due to increased extensibility. These fibers were highly hygroscopic and had similar internal network organization, thus similar range of mechanical properties that depended on their diameters. The overall structure of the consensus repeat of the silk-like protein dictated the mechanical properties of the fibers while protein molecular weight limited these same properties. Subtle structural motif re-design impacted protein self-assembly mechanisms and requirements for fiber formation.     

Pharmacokinetics and pharmacodynamics of intravenous artesunate during severe malaria treatment in Ugandan adults

ABSTRACT: BACKGROUND: Severe malaria is a medical emergency with high mortality. Prompt achievement of therapeutic concentrations of highly effective anti-malarial drugs reduces the risk of death. The aim of this study was to assess the pharmacokinetics and pharmacodynamics of intravenous artesunate in Ugandan adults with severe malaria. METHODS: Fourteen adults with severe falciparum malaria requiring parenteral therapy were treated with 2.4 mg/kg intravenous artesunate. Blood samples were collected after the initial dose and plasma concentrations of artesunate and dihydroartemisinin measured by solid-phase extraction and liquid chromatography-tandem mass spectrometry. The study was approved by the Makerere University Faculty of Medicine Research and Ethics Committee (Ref2010-015) and Uganda National Council of Science and Technology (HS605) and registered with ClinicalTrials.gov (NCT01122134). RESULTS: All study participants achieved prompt resolution of symptoms and complete parasite clearance with median (range) parasite clearance time of 17 (8-24) hours. Median (range) maximal artesunate concentration (Cmax) was 3260 (1020-164000) ng/mL, terminal elimination half-life (T1/2) was 0.25 (0.1-1.8) hours and total artesunate exposure (AUC) was 727 (290-111256) ngh/mL. Median (range) dihydroartemisinin Cmax was 3140 (1670-9530) ng/mL, with Tmax of 0.14 (0.6 - 6.07) hours and T1/2 of 1.31 (0.8-2.8) hours. Dihydroartemisinin AUC was 3492 (2183-6338) ngh/mL. None of the participants reported adverse events. CONCLUSIONS: Plasma concentrations of artesunate and dihydroartemisinin were achieved rapidly with rapid and complete symptom resolution and parasite clearance with no adverse events.     

DNA barcoding the native flowering plants and conifers of Wales

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification.     

PTEN loss defines a TGF-β-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis

We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-β is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-β signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-β antagonism. Tubule-specific induction of TGF-β led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-β depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-β antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.