BACKGROUND

Long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are the primary vector control interventions used to prevent malaria in Africa. Although both interventions are effective in some settings, high-quality evidence is rarely available to evaluate their effectiveness following deployment by a national malaria control program. In Uganda, we measured changes in key malaria indicators following universal LLIN distribution in three sites, with the addition of IRS at one of these sites.

RESULTS

Comprehensive malaria surveillance was conducted from October 1, 2011, to March 31, 2016, in three sub-counties with relatively low (Walukuba), moderate (Kihihi), and high transmission (Nagongera). Between 2013 and 2014, universal LLIN distribution campaigns were conducted in all sites, and in December 2014, IRS with the carbamate bendiocarb was initiated in Nagongera. High-quality surveillance evaluated malaria metrics and mosquito exposure before and after interventions through (a) enhanced health-facility-based surveillance to estimate malaria test positivity rate (TPR), expressed as the number testing positive for malaria/number tested for malaria (number of children tested for malaria: Walukuba = 42,833, Kihihi = 28,790, and Nagongera = 38,690); (b) cohort studies to estimate the incidence of malaria, expressed as the number of episodes per person-year [PPY] at risk (number of children observed: Walukuba = 340, Kihihi = 380, and Nagongera = 361); and (c) entomology surveys to estimate household-level human biting rate (HBR), expressed as the number of female Anopheles mosquitoes collected per house-night of collection (number of households observed: Walukuba = 117, Kihihi = 107, and Nagongera = 107). The LLIN distribution campaign substantially increased LLIN coverage levels at the three sites to between 65.0% and 95.5% of households with at least one LLIN. In Walukuba, over the 28-mo post-intervention period, universal LLIN distribution was associated with no change in the incidence of malaria (0.39 episodes PPY pre-intervention versus 0.20 post-intervention; adjusted rate ratio [aRR] = 1.02, 95% CI 0.36-2.91, p = 0.97) and non-significant reductions in the TPR (26.5% pre-intervention versus 26.2% post-intervention; aRR = 0.70, 95% CI 0.46-1.06, p = 0.09) and HBR (1.07 mosquitoes per house-night pre-intervention versus 0.71 post-intervention; aRR = 0.41, 95% CI 0.14-1.18, p = 0.10). In Kihihi, over the 21-mo post-intervention period, universal LLIN distribution was associated with a reduction in the incidence of malaria (1.77 pre-intervention versus 1.89 post-intervention; aRR = 0.65, 95% CI 0.43-0.98, p = 0.04) but no significant change in the TPR (49.3% pre-intervention versus 45.9% post-intervention; aRR = 0.83, 95% 0.58-1.18, p = 0.30) or HBR (4.06 pre-intervention versus 2.44 post-intervention; aRR = 0.71, 95% CI 0.30-1.64, p = 0.40). In Nagongera, over the 12-mo post-intervention period, universal LLIN distribution was associated with a reduction in the TPR (45.3% pre-intervention versus 36.5% post-intervention; aRR = 0.82, 95% CI 0.76-0.88, p < 0.001) but no significant change in the incidence of malaria (2.82 pre-intervention versus 3.28 post-intervention; aRR = 1.10, 95% 0.76-1.59, p = 0.60) or HBR (41.04 pre-intervention versus 20.15 post-intervention; aRR = 0.87, 95% CI 0.31-2.47, p = 0.80). The addition of three rounds of IRS at ~6-mo intervals in Nagongera was followed by clear decreases in all outcomes: incidence of malaria (3.25 pre-intervention versus 0.63 post-intervention; aRR = 0.13, 95% CI 0.07-0.27, p < 0.001), TPR (37.8% pre-intervention versus 15.0% post-intervention; aRR = 0.54, 95% CI 0.49-0.60, p < 0.001), and HBR (18.71 pre-intervention versus 3.23 post-intervention; aRR = 0.29, 95% CI 0.17-0.50, p < 0.001). High levels of pyrethroid resistance were documented at all three study sites. Limitations of the study included the observational study design, the lack of contemporaneous control groups, and that the interventions were implemented under programmatic conditions.

CONCLUSIONS

Universal distribution of LLINs at three sites with varying transmission intensity was associated with modest declines in the burden of malaria for some indicators, but the addition of IRS at the highest transmission site was associated with a marked decline in the burden of malaria for all indicators. In highly endemic areas of Africa with widespread pyrethroid resistance, IRS using alternative insecticide formulations may be needed to achieve substantial gains in malaria control.

OBJECTIVE

Peptide YY (PYY) and pancreatic polypeptide (PPY) are members of the neuropeptide Y peptide family. The neuropeptide Y receptor signaling pathway has been implicated in a number of physiologic processes, including the regulation of energy balance and bone mass. To investigate the contribution of endogenous PYY and PPY to these processes, we generated both Pyy- and Ppy-deficient mice.

METHODS

Pyy(-/-) and Ppy(-/-) mice and their respective wild-type littermates were studied from 8 weeks to 9 months of age. Food intake, metabolic parameters, and locomotor activity were monitored using indirect calorimetry. Body composition and bone parameters were analyzed using dual energy x-ray absorptiometry, histomorphometry, and vertebral compression testing.

RESULTS

Studies in these mice showed an osteopenic phenotype specific to the Pyy-deficient line, which included a reduction in trabecular bone mass and a functional deficit in bone strength. Furthermore, female Pyy(-/-) mice showed a greater sensitivity to ovariectomy-induced bone loss compared with wild-type littermates. No food intake or metabolic phenotype was apparent in male or female Pyy(-/-) mice on standard chow. However, female Pyy(-/-) mice on a high-fat diet showed a greater propensity to gain body weight and adiposity. No metabolic or osteopenic phenotype was observed in Ppy-deficient mice.

CONCLUSIONS

These results indicate that endogenous PYY plays a critical role in regulating bone mass. In comparison, its role in regulating body weight is minor and is confined to situations of high-fat feeding.

We report on the spectroscopic, electrochemical, and electroluminescent properties of [Ir(ppy)(2)(dtb-bpy)](+)(PF(6))(-) (ppy: 2-phenylpyridine, dtb-bpy: 4,4'-di-tert-butyl-2,2'-dipyridyl). Single-layer devices were fabricated and found to emit yellow light with a brightness that exceeds 300 cd/m(2) and a luminous power efficiency that exceeds 10 Lm/W at just 3 V. The PF(6)(-) space charge was found to dominate the device characteristics.

The photoredox-catalyzed coupling of N-aryltetrahydroisoquinoline and Michael acceptors was achieved using Ru(bpy)(3)Cl(2) or [Ir(ppy)(2)(dtb-bpy)]PF(6) in combination with irradiation at 455 nm generated by a blue LED, demonstrating the trapping of visible light generated α-amino radicals. While intermolecular reactions lead to products formed by a conjugate addition, in intramolecular variants further dehydrogenation occurs, leading directly to 5,6-dihydroindolo[2,1-a]tetrahydroisoquinolines, which are relevant as potential immunosuppressive agents.

A novel electrically conductive biodegradable composite material made of polypyrrole (PPy) nanoparticles and poly(d,l-lactide) (PDLLA) was prepared by emulsion polymerization of pyrrole in a PDLLA solution, followed by precipitation. The composite was characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. The electrical stability of the composite containing 5 wt% PPy was investigated in a cell culture environment for 1000 h with 100 mV DC applied voltage. Fibroblasts were cultured on the composite membranes and were stimulated with various DC currents. The PPy particles formed aggregations and constituted microdomains and networks embedded in the PDLLA. With the 1-17% increase in the PPy content, the conductivity of the composite increased by six orders of magnitude. The surface resistivity of the PPy/PDLLA membrane with 3% PPy was as low as 1x10(3) Omega/square. The electrical stability was significantly better in the PPy/PDLLA composite than in the PPy-coated polyester fabrics. For the composite with 5% PPy, the test membrane retained 80% and 42% of the initial conductivity in 100 and 400 h, respectively, following the addition of the MEM solution, compared to 5% and 0.1% for the PPy-coated polyester fabrics. Under 100 mV, a composite membrane 3.0x2.5x0.03cm3 in size and containing 5% PPy sustained a biologically meaningful electrical conductivity in a typical cell culture environment for 1000 h. The growth of fibroblasts was up regulated under the stimulation of medium range intensity of DC current.

BACKGROUND

Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99.

RESULTS

Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate.

CONCLUSIONS

We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting β-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-β-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.

Two endonucleases, AvaI and AvaII, were isolated from Anabaena variabilis on the basis of their ability to make a limited number of breaks at specific points in bacteriophage lambda DNA. Neither enzyme has cofactor requirements beyond Mg2+. Endonuclease AvaI makes eight breaks in the phage lambda chromosome at which the 5'-terminal sequence is pPy-C-G-N. AvaII endonuclease cuts phage lambda DNA more extensively, yielding fragments with the 5'-terminal sequence G-T-C-N or G-A-C-N. Neither enzyme generates cohesive ends.

To facilitate the positional cloning of the breast-ovarian cancer gene BRCA1, we constructed a high-density genetic map of the 8.3-cM interval between D17S250 and GIP on chromosome 17q12-q21. Markers were mapped by linkage in the CEPH and in extended kindreds in our breast cancer series. The map comprises 33 ordered polymorphisms, including 12 genes and 21 anonymous markers, yielding an average of one polymorphism every 250 kb. Twenty-five of the markers are PCR-based systems. The order of polymorphic genes and markers is cen-D17S250-D17S518-HER2-THRA1-RARA-D17S80 -KRT10-[D17S800-D17S857]-GAS- D17S856-EDH17B-D17S855-D17S859-D17S858-[++ +PPY-D17S78]-D17S183-EPB3-D17S579- D17S509-[D17S508-D17S190 = D17S810]-D17S791-[D17S181 = D17S806]-D17S797- HOX2B-GP3A-[D17S507 = GIP]-qter. BRCA1 lies in the middle of the interval, between THRA1 and D17S183. Markers from this map can be used to determine whether cancer is linked to BRCA1 in families, to evaluate whether tumors have lost heterozygosity at loci in the region, and to identify probes for characterizing chromosomal rearrangements from patients and from tumors.

Magnetically guided ultrasound-powered nanowire motors, functionalized with bioreceptors and a drug-loaded polymeric segment, are described for "capture and transport" and drug-delivery processes. These high-performance fuel-free motors display advanced capabilities and functionalities, including magnetic guidance, coordinated aligned movement, cargo towing, capture and isolation of biological targets, drug delivery, and operation in real-life biological and environmental media. The template-prepared three-segment Au-Ni-Au nanowire motors are propelled acoustically by mechanical waves produced by a piezoelectric transducer. An embedded nickel segment facilitates a magnetically guided motion as well as transport of large "cargo" along predetermined trajectories. Substantial improvement in the speed and power is realized by the controlled concavity formation at the end of the motor nanowire using a sphere lithography protocol. Functionalization of the Au segments with lectin and antiprotein A antibody bioreceptors allows capture and transport of E. coli and S. aureus bacteria, respectively. Potential therapeutic applications are illustrated in connection to the addition of a pH-sensitive drug-loaded polymeric (PPy-PSS) segment. The attractive capabilities of these fuel-free acoustically driven functionalized Au-Ni-Au nanowires, along with the simple preparation procedure and minimal adverse effects of ultrasonic waves, make them highly attractive for diverse in vivo biomedical applications.

A simple and cost-effective, all-electrochemical method to fabricate and assemble single conducting polymer nanowire based biosensors was developed. Polypyrrole (Ppy) nanowires were synthesized by electrochemical polymerization using an alumina template. The single-nanowire chemoresistive sensor device was assembled using ac dielectrophoretic alignment followed by maskless anchoring on a pair of gold electrodes separated by 3 microm. To establish an efficient covalent surface biofunctionalization route, glutaraldehyde (GA) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) chemistries were compared. EDC was established to be the most effective chemistry and was used to surface-functionalize a single Ppy nanowire with cancer antigen (CA 125) antibody to fabricate a nanoimmunosensor for CA 125 biomarker detection and quantification. The immunosensor had excellent sensitivity with a lower detection limit of 1 U/mL CA 125 and dynamic range up to 1000 U/mL in 10 mM phosphate buffer. Furthermore, there was no loss of performance upon exposure to CA 125 in spiked human blood plasma. This demonstrates the clinical importance of these sensors for cancer marker detection with cost benefits and great portability for diagnosis of patients at the point of care.

Posttranslational methylation of release factors on the glutamine residue of a conserved GGQ motif is required for efficient termination of protein synthesis. This methylation is performed by an N(5)-glutamine methyltransferase called PrmC/HemK, whose crystal structure we report here at 2.2 A resolution. The electron density at the active site appears to contain a mixture of the substrates, S-adenosyl-L-methionine (AdoMet) and glutamine, and the products, S-adenosyl-L-homocysteine (AdoHcy) and N(5)-methylglutamine. The C-terminal domain of PrmC adopts the canonical AdoMet-dependent methyltransferase fold and shares structural similarity with the nucleotide N-methyltransferases in the active site, including use of a conserved (D/N)PPY motif to select and position the glutamine substrate. Residues of the PrmC (197)NPPY(200) motif form hydrogen bonds that position the planar Gln side chain such that the lone-pair electrons on the nitrogen nucleophile are oriented toward the methyl group of AdoMet. In the product complex, the methyl group remains pointing toward the sulfur, consistent with either an sp(3)-hybridized, positively charged Gln nitrogen, or a neutral sp(2)-hybridized nitrogen in a strained conformation. Due to steric overlap within the active site, proton loss and formation of the neutral planar methylamide product are likely to occur during or after product release. These structures, therefore, represent intermediates along the catalytic pathway of PrmC and show how the (D/N)PPY motif can be used to select a wide variety substrates.

The significant drawbacks and lack of success associated with current methods to treat critically sized nerve defects have led to increased interest in neural tissue engineering. Conducting polymers show great promise due to their electrical properties, and in the case of polypyrrole (PPY), its cell compatibility as well. Thus, the goal of this study is to synthesize a conducting composite nerve conduit with PPY and poly(d, l-lactic acid) (PDLLA), assess its ability to support the differentiation of rat pheochromocytoma 12 (PC12) cells in vitro, and determine its ability to promote nerve regeneration in vivo. Different amounts of PPY (5%, 10%, and 15%) are used to synthesize the conduits resulting in different conductivities (5.65, 10.40, and 15.56 ms/cm, respectively). When PC12 cells are seeded on these conduits and stimulated with 100 mV for 2 h, there is a marked increase in both the percentage of neurite-bearing cells and the median neurite length as the content of PPY increased. More importantly, when the PPY/PDLLA nerve conduit was used to repair a rat sciatic nerve defect it performed similarly to the gold standard autologous graft. These promising results illustrate the potential that this PPY/PDLLA conducting composite conduit has for neural tissue engineering.

BACKGROUND

Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA assembly that uses cell extracts from the Escherichia coli PPY strain, which expresses the components of the λ prophage Red/ET recombination system. This method facilitates restriction endonuclease cleavage site-free DNA cloning by performing recombination between short stretches of homologous DNA (≥ 15 base pairs).

RESULTS

To extend the versatility of this system, I examined whether, in addition to bacterial extracts from the PPY strain, other E. coli laboratory strains were suitable for the SLiCE protocol. Indeed, carefully prepared cell extracts from several strains exhibited sufficient cloning activity for seamless gene incorporation into vectors with short homology lengths (approximately 15-20 bp). Furthermore, SLiCE was applied to the polymerase chain reaction (PCR)-based site-directed mutagenesis method, in a process termed "SLiCE-mediated PCR-based site-directed mutagenesis (SLiP site-directed mutagenesis)". SLiP site-directed mutagenesis simplifies the steps of PCR-based site-directed mutagenesis, as it exploits the capability of the SLiCE method to insert multiple fragments.

CONCLUSIONS

SLiCE can be performed in the laboratory with no requirement for a special E. coli strain, and the technique is easily established. This method increases the cloning efficiency, shortens the time for DNA manipulation, and greatly reduces the cost of seamless DNA cloning.

Electrically conducting polymers such as polypyrrole (PPy) are important biomaterials in neural engineering applications, including neural probes, nerve conduits, and scaffolds for tissue and nerve regeneration. Surface modification of these polymers can introduce other valuable characteristics for neural interfacing in addition to electrical conductivity, such as topographical features and chemical bioactivity. Here, the patterning of PPy to create topographical cues for cells is reported. In particular, 1 and 2 µm wide PPy microchannels are fabricated using electron-beam (e-beam) lithography and electropolymerization. A systematic analysis of parameters controlling PPy micropatterning is performed, and finds that microchannel depth, roughness, and morphology are highly dependent on the e-beam writing current, polymerization current, PPy/dopant concentrations, and the polymerization time. Embryonic hippocampal neurons cultured on patterned PPy polarize (i.e., defined an axon) faster on this modified material, with a twofold increase in the number of cells with axons compared to cells cultured on unmodified PPy. These topographical features also have an effect on axon orientation but do not have a significant effect on overall axon length. This is the first investigation that studies controlled PPy patterning with small dimensions (i.e., less than 5 µm) for biological applications, which demonstrates the relevance of expanding microelectronic materials and techniques to the biomedical field.

The dystroglycan gene produces two products from a single mRNA, the extracellular alpha-dystroglycan and the transmembrane beta-dystroglycan. The Duchenne muscular dystrophy protein, dystrophin, associates with the muscle membrane via beta-dystroglycan, the WW domain of dystrophin interacting with a PPxY motif in beta-dystroglycan. A panel of four monoclonal antibodies (MANDAG1-4) was produced using the last 16 amino acids of beta-dystroglycan as immunogen. The mAbs recognized a 43 kDa band on Western blots of all cells and tissues tested and stained the sarcolemma in immunohistochemistry of skeletal muscle over a wide range of animal species. A monoclonal antibody (mAb) against the WW domain of dystrophin, MANHINGE4A, produced using a 16-mer synthetic peptide, recognized dystrophin on Western blots and also stained the sarcolemma. We have identified the precise sequences recognized by the mAbs using a phage-displayed random 15-mer peptide library. A 7-amino-acid consensus sequence SPPPYVP involved in binding all four beta-dystroglycan mAbs was identified by sequencing 17 different peptides selected from the library. PPY were the most important residues for three mAbs, but PxxVP were essential residues for a fourth mAb, MANDAG2. By sequencing five different random peptides from the library, the epitope on dystrophin recognized by mAb MANHINGE4A was identified as PWxRA in the first beta-strand of the WW domain, with the W and R residues invariably present. A recent three-dimensional structure confirms that the two epitopes are adjacent in the dystrophin-dystroglycan complex, highlighting the question of how the two interacting motifs can also be accessible to antibodies during immunolocalization in situ.

Electrical stimulation (ES) can dramatically enhance neurite outgrowth through conductive polymers and accelerate peripheral nerve regeneration in animal models of nerve injury. Therefore, conductive tissue engineering graft in combination with ES is a potential treatment for neural injuries. Conductive tissue engineering graft can be obtained by seeding Schwann cells on conductive scaffold. However, when ES is applied through the conductive scaffold, the impact of ES on Schwann cells has never been investigated. In this study, a biodegradable conductive composite made of conductive polypyrrole (PPy, 2.5%) and biodegradable chitosan (97.5%) was prepared in order to electrically stimulate Schwann cells. The tolerance of Schwann cells to ES was examined by a cell apoptosis assay. The growth of the cells was characterized using DAPI staining and a MTT assay. mRNA and protein levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in Schwann cells were assayed by RT-PCR and Western blotting, and the amount of NGF and BDNF secreted was determined by an ELISA assay. The results showed that the PPy/chitosan membranes supported cell adhesion, spreading, and proliferation with or without ES. Interestingly, ES applied through the PPy/chitosan composite dramatically enhanced the expression and secretion of NGF and BDNF when compared with control cells without ES. These findings highlight for the first time the possibility of enhancing nerve regeneration in conductive scaffolds through ES-increased neurotrophin secretion.

Release of neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF) from hair cells in the cochlea is essential for the survival of spiral ganglion neurons (SGNs). Loss of hair cells associated with a sensorineural hearing loss therefore results in degeneration of SGNs, potentially reducing the performance of a cochlear implant. Exogenous replacement of either or both neurotrophins protects SGNs from degeneration after deafness. We previously incorporated NT3 into the conducting polymer polypyrrole (Ppy) synthesized with para-toluene sulfonate (pTS) to investigate whether Ppy/pTS/NT3-coated cochlear implant electrodes could provide both neurotrophic support and electrical stimulation for SGNs. Enhanced and controlled release of NT3 was achieved when Ppy/pTS/NT3-coated electrodes were subjected to electrical stimulation. Here we describe the release dynamics and biological properties of Ppy/pTS with incorporated BDNF. Release studies demonstrated slow passive diffusion of BDNF from Ppy/pTS/BDNF, with electrical stimulation significantly enhancing BDNF release over 7 days. A 3-day SGN explant assay found that neurite outgrowth from explants was 12.3-fold greater when polymers contained BDNF (p < 0.001), although electrical stimulation did not increase neurite outgrowth further. The versatility of Ppy to store and release neurotrophins, conduct electrical charge, and act as a substrate for nerve-electrode interactions is discussed for specialized applications such as cochlear implants.

Previous studies have identified a conserved AG dinucleotide at the 3' splice site (3'SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei alpha-tubulin 3'SS region is required to specify accurate 3'-end formation of the upstream beta-tubulin gene and trans splicing of the downstream alpha-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3'SS identification. Our results indicate that a minimal alpha-tubulin 3'SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by the trans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the alpha-tubulin 3'SS is dependent upon the presence of exon sequences. Furthermore, beta-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace alpha-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the alpha-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar to cis-splicing enhancers described in other systems.

BACKGROUND

Hepatitis C virus (HCV) causes significant morbidity and mortality in injecting drug users (IDU) worldwide. HCV vaccine candidates have shown promise for reducing the infectivity of acute infection and averting chronic infection, yet the impact of varying levels of vaccine efficacy and vaccine delivery strategies on the HCV epidemic in IDU have not been explored.

METHODS

We utilized extensive data on injecting behavior collected in the UFO Study of young IDU in San Francisco to construct a stochastic individual-based model that reflects heterogeneous injecting risk behavior, historical HCV trends, and existing information on viral dynamics and vaccine characteristics.

RESULTS

Our modeled HCV rate closely paralleled observed HCV incidence in San Francisco, with estimated incidence of 59% per person year (ppy) early in the epidemic, and 27% ppy after risk reduction was introduced. Chronic HCV infection, the clinically relevant state of HCV infection that leads to liver disease and hepatocellular cancer, was estimated at 22% ppy (± 3%) early in the epidemic and 14% ppy (± 2%) after risk reduction was introduced. We considered several scenarios, and highlight that a vaccine with 50% to 80% efficacy targeted to high-risk or sero-negative IDU at a high vaccination rate could further reduce chronic HCV incidence in IDU to 2-7% ppy 30 years after its introduction.

CONCLUSIONS

Our results underscore the importance of further efforts to develop both HCV vaccines and optimal systems of delivery to IDU populations.

A new and general design strategy is presented for amphiphilic block copolymers whose micellar aggregates can be dissociated by light. A diblock copolymer composed of hydrophilic poly(ethylene oxide) (PEO) and a hydrophobic polymethacrylate bearing pyrene pendant groups (PPy) was synthesized using ATRP. Upon UV light irradiation of polymer micellar solutions, the photosolvolysis of pyrene moieties results in their detachment from the polymer and converts the hydrophobic PPy block into hydrophilic poly(methacrylic acid). This effect leads to complete dissociation of polymer micelles.

A nonemissive cyclometalated iridium(III) solvent complex, without conjugation with a cell-penetrating molecular transporter, [Ir(ppy)(2)(DMSO)(2)](+)PF(6)(-) (LIr1), has been developed as a first reaction-based fluorescence-turn-on agent for the nuclei of living cells. LIr1 can rapidly and selectively light-up the nuclei of living cells over fixed cells, giving rise to a significant luminescence enhancement (200-fold), and shows very low cytotoxicity at the imaging concentration (incubation time <10 min, LIr1 concentration 10 μM). More importantly, in contrast to the reported nuclear stains that are based on luminescence enhancement through interaction with nucleic acids, complex LIr1 as a nuclear stain has a reaction-based mode of action, which relies on its rapid reaction with histidine/histidine-containing proteins. Cellular uptake of LIr1 has been investigated in detail under different conditions, such as at various temperatures, with hypertonic treatment, and in the presence of metabolic and endocytic inhibitors. The results have indicated that LIr1 permeates the outer and nuclear membranes of living cells through an energy-dependent entry pathway within a few minutes. As determined by an inductively coupled plasma atomic emission spectroscopy (ICP-AEC), LIr1 is accumulated in the nuclei of living cells and converted into an intensely emissive adduct. Such novel reaction-based nuclear staining for visualizing exclusively the nuclei of living cells with a significant luminescence enhancement may extend the arsenal of currently available fluorescent stains for specific staining of cellular compartments.

This works introduces hypervalent bis-catecholato silicon compounds as versatile sources of alkyl radicals upon visible-light photocatalysis. Using Ir[(dF(CF3)ppy)2(bpy)](PF6) (dF(CF3)ppy = 2-(2,4-difluorophenyl)-5-trifluoromethylpyridine, bpy = bipyridine) as catalytic photooxidant, a series of alkyl radicals, including highly reactive primary ones can be generated and engaged in various intermolecular homolytic reactions. Based on cyclic voltammetry, Stern-Volmer studies, and supported by calculations, a mechanism involving a single-electron transfer from the silicate to the photoactivated iridium complex has been proposed. This oxidative photocatalyzed process can be efficiently merged with nickel-catalyzed Csp2-Csp3 cross-coupling reactions.

Pt nanoparticles of 2-3 nm and 5-6 nm in diameter were loaded into stable, porous, and phosphorescent metal-organic frameworks (MOFs 1 and 2) built from [Ir(ppy)(2)(bpy)](+)-derived dicarboxylate ligands (L(1) and L(2)) and Zr(6)(μ(3)-O)(4)(μ(3)-OH)(4)(carboxylate)(12) secondary building units, via MOF-mediated photoreduction of K(2)PtCl(4). The resulting Pt@MOF assemblies serve as effective photocatalysts for hydrogen evolution by synergistic photoexcitation of the MOF frameworks and electron injection into the Pt nanoparticles. Pt@2 gave a turnover number of 7000, approximately five times the value afforded by the homogeneous control, and could be readily recycled and reused.