Performance of production of polyhydroxyalkanoates from food waste fermentation with Rhodopseudomonas palustris.

The use of waste as a carbon source can significantly reduce the cost of production of Polyhydroxyalkanoates (PHAs). In this study, an acidified hydrolysate solution derived from food waste (FW) was used as a carbon source for the synthesis of PHAs by Rhodopseudomonas palustris (R. palustris) and optimized the process parameters. The results showed that the PHAs yield reached 48.62% under optimal conditions (an incubation time of 30 days, volatile fatty acids (VFAs) in substrate concentration of 2202.21 mg⋅L-1, an initial pH of 8.0, and inoculum concentration of 15%). The fraction of VFAs affects the composition of PHAs, R. palustris first uses VFAs with an even number of carbons to synthesize poly(3-hydroxybutyrate)(3HB), and later uses VFAs with an odd number of carbons to synthesize poly-3-hydroxyvalerate (3HV). Pathways for the synthesis of PHAs by R. palustris were inferred. R. palustris is a strain with the potential to synthesize PHAs.

Multiscale prediction of patient-specific platelet function under flow.

During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A(2), recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y(1)/P2Y(12), TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y(1) inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

Enhancers of adeno-associated virus AAV2 transduction via high throughput siRNA screening.

Intracellular barriers to adeno-associated virus (AAV) transduction may limit gene delivery. We screened a short interfering RNA (siRNA) library targeting 5,520 genes to help identify pathways that modulate AAV transduction of human endothelium. In replicate screening, 50 pools (three siRNAs per gene) resulted in greater than eightfold reporter gene expression enhancement. Single siRNA confirmation tests demonstrated that at least one siRNA from each of the top 10 pools provided greater than twofold enhancement. Several siRNAs when used together resulted in additive effects and two of the most potent siRNA sequences were enhancers in cultured airway epithelium. However, enhanced transduction was not correlated with mRNA knockdown by quantitative real time PCR, indicating an off-target mechanism. In fact, four of the five most potent siRNAs contained a consensus hexamer region 5'-UGUUUC-3' at positions 2-7 of the antisense strand. The point mutation U4A within this region (but not mutations at positions 1 or 14) disrupted transduction enhancement, indicating a microRNA (miRNA)-like mechanism. Transcription profiling indicated that the hexamer also resulted in perturbation of the interferon pathway via reduced interferon-induced protein 44-like (IFI44L), interferon-inducible myxovirus resistance 1 (MX1), and interferon-induced protein with tetratricopeptide repeats (IFIT5) mRNAs. Direct interferon (α, ß, and ω) receptor 2 (IFNAR2) knockdown resulted in greater than twofold transduction enhancement. In addition to providing insight into AAV biology and enhanced transduction, the results demonstrate certain beneficial siRNA off-target effects.

Systems biology of coagulation initiation: kinetics of thrombin generation in resting and activated human blood.

Blood function defines bleeding and clotting risks and dictates approaches for clinical intervention. Independent of adding exogenous tissue factor (TF), human blood treated in vitro with corn trypsin inhibitor (CTI, to block Factor XIIa) will generate thrombin after an initiation time (T(i)) of 1 to 2 hours (depending on donor), while activation of platelets with the GPVI-activator convulxin reduces T(i) to ∼20 minutes. Since current kinetic models fail to generate thrombin in the absence of added TF, we implemented a Platelet-Plasma ODE model accounting for: the Hockin-Mann protease reaction network, thrombin-dependent display of platelet phosphatidylserine, VIIa function on activated platelets, XIIa and XIa generation and function, competitive thrombin substrates (fluorogenic detector and fibrinogen), and thrombin consumption during fibrin polymerization. The kinetic model consisting of 76 ordinary differential equations (76 species, 57 reactions, 105 kinetic parameters) predicted the clotting of resting and convulxin-activated human blood as well as predicted T(i) of human blood under 50 different initial conditions that titrated increasing levels of TF, Xa, Va, XIa, IXa, and VIIa. Experiments with combined anti-XI and anti-XII antibodies prevented thrombin production, demonstrating that a leak of XIIa past saturating amounts of CTI (and not "blood-borne TF" alone) was responsible for in vitro initiation without added TF. Clotting was not blocked by antibodies used individually against TF, VII/VIIa, P-selectin, GPIb, protein disulfide isomerase, cathepsin G, nor blocked by the ribosome inhibitor puromycin, the Clk1 kinase inhibitor Tg003, or inhibited VIIa (VIIai). This is the first model to predict the observed behavior of CTI-treated human blood, either resting or stimulated with platelet activators. CTI-treated human blood will clot in vitro due to the combined activity of XIIa and XIa, a process enhanced by platelet activators and which proceeds in the absence of any evidence for kinetically significant blood borne tissue factor.

Discovery of potent small-molecule inhibitors of multidrug-resistant Plasmodium falciparum using a novel miniaturized high-throughput luciferase-based assay.

Malaria is a global health problem that causes significant mortality and morbidity, with more than 1 million deaths per year caused by Plasmodium falciparum. Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites, which necessitates the discovery of new drugs. To identify new antimalarials, we developed an automated 384-well plate screening assay using P. falciparum parasites that stably express cytoplasmic firefly luciferase. After initial optimization, we tested two different types of compound libraries: known bioactive collections (Library of Pharmacologically Active Compounds [LOPAC] and the library from the National Institute of Neurological Disorders and Stroke [NINDS]) and a library of uncharacterized compounds (ChemBridge). A total of 12,320 compounds were screened at 5.5 microM. Selecting only compounds that reduced parasite growth by 85% resulted in 33 hits from the combined bioactive collection and 130 hits from the ChemBridge library. Fifteen novel drug-like compounds from the bioactive collection were found to be active against P. falciparum. Twelve new chemical scaffolds were found from the ChemBridge hits, the most potent of which was a series based on the 1,4-naphthoquinone scaffold, which is structurally similar to the FDA-approved antimalarial atovaquone. However, in contrast to atovaquone, which acts to inhibit the bc(1) complex and block the electron transport chain in parasite mitochondria, we have determined that our new 1,4-napthoquinones act in a novel, non-bc(1)-dependent mechanism and remain potent against atovaquone- and chloroquine-resistant parasites. Ultimately, this study may provide new probes to understand the molecular details of the malaria life cycle and to identify new antimalarials.

A small-molecule oxocarbazate inhibitor of human cathepsin L blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney 293T cells.

A tetrahydroquinoline oxocarbazate (PubChem CID 23631927) was tested as an inhibitor of human cathepsin L (EC 3.4.22.15) and as an entry blocker of severe acute respiratory syndrome (SARS) coronavirus and Ebola pseudotype virus. In the cathepsin L inhibition assay, the oxocarbazate caused a time-dependent 17-fold drop in IC(50) from 6.9 nM (no preincubation) to 0.4 nM (4-h preincubation). Slowly reversible inhibition was demonstrated in a dilution assay. A transient kinetic analysis using a single-step competitive inhibition model provided rate constants of k(on) = 153,000 M(-1)s(-1) and k(off) = 4.40 x 10(-5) s(-1) (K(i) = 0.29 nM). The compound also displayed cathepsin L/B selectivity of >700-fold and was nontoxic to human aortic endothelial cells at 100 muM. The oxocarbazate and a related thiocarbazate (PubChem CID 16725315) were tested in a SARS coronavirus (CoV) and Ebola virus-pseudotype infection assay with the oxocarbazate but not the thiocarbazate, demonstrating activity in blocking both SARS-CoV (IC(50) = 273 +/- 49 nM) and Ebola virus (IC(50) = 193 +/- 39 nM) entry into human embryonic kidney 293T cells. To trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity-based probe biotin-Lys-C5 alkyl linker-Tyr-Leu-epoxide (DCG-04) was used to label the active site of cysteine proteases in 293T lysates. The reduction in active cathepsin L in inhibitor-treated cells correlated well with the observed potency of inhibitors observed in the virus pseudotype infection assay. Overall, the oxocarbazate CID 23631927 was a subnanomolar, slow-binding, reversible inhibitor of human cathepsin L that blocked SARS-CoV and Ebola pseudotype virus entry in human cells.

Kinetic characterization and molecular docking of a novel, potent, and selective slow-binding inhibitor of human cathepsin L.

A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.15) with an IC(50) of 56 nM, was developed after a 57,821-compound screen of the National Institutes of Health Molecular Libraries Small Molecule Repository. After a 4-h preincubation with cathepsin L, this compound became even more potent, demonstrating an IC(50) of 1.0 nM. The thiocarbazate was determined to be a slow-binding and slowly reversible competitive inhibitor. Through a transient kinetic analysis for single-step reversibility, inhibition rate constants were k(on) = 24,000 M(-1)s(-1) and k(off) = 2.2 x 10(-5) s(-1) (K(i) = 0.89 nM). Molecular docking studies were undertaken using the experimentally derived X-ray crystal structure of papain/CLIK-148 (1cvz. pdb). These studies revealed critical hydrogen bonding patterns of the thiocarbazate with key active site residues in papain. The thiocarbazate displayed 7- to 151-fold greater selectivity toward cathepsin L than papain and cathepsins B, K, V, and S with no activity against cathepsin G. The inhibitor demonstrated a lack of toxicity in human aortic endothelial cells and zebrafish. In addition, the thiocarbazate inhibited in vitro propagation of malaria parasite Plasmodium falciparum with an IC(50) of 15.4 microM and inhibited Leishmania major with an IC(50) of 12.5 microM.

Hemodynamic regulation of inflammation at the endothelial-neutrophil interface.

Arterial shear stress can regulate endothelial phenotype. The potential for anti-inflammatory effects of shear stress on TNFalpha-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm(2) blocked by >80% the induction by 5 ng/mL TNFalpha of interleukin-8 (IL-8) and IL-6 secretion (50 and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 microM nitro-L-arginine methylester, L-NAME). Exposure of TNFalpha-stimulated HAEC to arterial shear stress for 5 h also reduced by 60% (p < 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm(2). Also, neutrophil rolling lengths at 1 dyne/cm(2) were longer when TNFalpha-stimulated HAEC were presheared for 5 h at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFkappaB, shear stress caused a marked 40-fold induction of luciferase in TNFalpha-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress. Hemodynamic force exerts anti-inflammatory effects on cytokine-activated endothelium by attenuation of cytokine expression and neutrophil firm arrest.

Shear stress causes nuclear localization of endothelial glucocorticoid receptor and expression from the GRE promoter.

We tested the hypothesis that steady laminar shear stress activates the glucocorticoid receptor (GR) and its transcriptional signaling pathway in an effort to investigate the potential involvement of GR in shear stress-induced antiatherosclerosis actions in the vasculature. In both bovine aortic endothelial cells (BAECs) and NIH3T3 cells expressing GFP-GR chimeric protein, wall shear stress of 10 or 25 dynes/cm2 caused a marked nuclear localization of GFP-GR within 1 hour to an extent comparable to induction with 25 micromol/L dexamethasone. The shear mediated nuclear localization of GFP-GR was significantly reduced by 25 micromol/L of the MEK1 inhibitor (PD098059) or the PI 3-kinase inhibitor (LY294002). Also, Western blots demonstrated translocation of endogenous GR into nucleus of sheared BAECs. Promoter construct studies using glucocorticoid response element (GRE)-driven expression of secreted alkaline phosphatase (SEAP) indicated that BAECs exposed to shear stress of 10 and 25 dynes/cm2 for 8 hours produced >9-fold more SEAP (n=6; P<0.005) than control cells, a level comparable to that observed with dexamethasone. Shear stress enhanced SEAP expression at 6 hours was reduced 50% (n=5; P<0.005) by MEK1/2 or PI 3-kinase inhibitors, but not by the NO inhibitor, L-NAME. Finally, in human internal mammary artery, endothelial GR is found to be highly nuclear localized. We report a new shear responsive transcriptional element, GRE. The finding that hemodynamic forces can be as potent as high dose glucocorticoid steroid in activating GR and GRE-regulated expression correlates with the atheroprotective responses of endothelial cells to unidirectional arterial shear stress.

Identification of the human cDNA for new survival/evasion peptide (DSEP): studies in vitro and in vivo of overexpression by neural cells.

We identified the human cDNA encoding a peptide that has been partially purified from the secretions of oxidatively stressed neural cell lines, murine adenocarcinoma cells, and group Abeta-hemolytic steptococci. We then genetically modified mouse and human neural cells to overexpress this peptide and found these modified cells to be remarkably hearty, surviving under conditions of severe oxidative stress, in xenocultures when exposed to activated macrophages, and as xenografts in the brain of rats that were not immunosuppressed. The peptide is called DSEP (dee-sep) for diffusible survival evasion peptide. Part of the survival advantage of DSEP overexpressors may be due to their attenuated response to all-trans-retinoic acid, which regulates differentiation and apoptosis of several cell types including neural and immune cells.