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LPL is a secreted enzyme that hydrolyzes triglycerides from circulating lipoproteins. Individuals lacking LPL suffer from severe hypertriglyceridemia, a risk factor for acute pancreatitis. One potential treatment is to administer recombinant LPL as a protein therapeutic. However, use of LPL as a protein therapeutic is limited because it is an unstable enzyme that is difficult to produce in large quantities. Furthermore, these considerations also limit structural and biochemical studies that are needed for large-scale drug discovery efforts. We demonstrate that the yield of purified LPL can be dramatically enhanced by coexpressing its maturation factor, LMF1, and by introducing novel mutations into the LPL sequence to render it resistant to proteolytic cleavage by furin. One of these mutations introduces a motif for addition of an N-linked glycan to the furin-recognition site. Furin-resistant LPL has previously been reported, but is not commonly used. We show that our modifications do not adversely alter LPL's enzymatic activity, stability, or in vivo function. Together, these data show that furin-resistant LPL is a useful reagent for both biochemical and biomedical studies.

Recent genetic studies have established that hypertriglyceridemia (HTG) is causally related to cardiovascular disease, making it an active area for drug development. We describe a strategy for lowering triglycerides (TG) with an apolipoprotein C-II (apoC-II) mimetic peptide called D6PV that activates lipoprotein lipase (LPL), the main plasma TG-hydrolyzing enzyme, and antagonizes the TG-raising effect of apoC-III. The design of D6PV was motivated by a combination of all-atom molecular dynamics simulation of apoC-II on the Anton 2 supercomputer, structural prediction programs, and biophysical techniques. Efficacy of D6PV was assessed ex vivo in human HTG plasma and was found to be more potent than full-length apoC-II in activating LPL. D6PV markedly lowered TG by more than 80% within a few hours in both apoC-II-deficient mice and hAPOC3-transgenic (Tg) mice. In hAPOC3-Tg mice, D6PV treatment reduced plasma apoC-III by 80% and apoB by 65%. Furthermore, low-density lipoprotein (LDL) cholesterol did not accumulate but rather was decreased by 10% when hAPOC3-Tg mice lacking the LDL-receptor (hAPOC3-Tg x Ldlr(−/−)) were treated with the peptide. D6PV lowered TG by 50% in whole-body inducible Lpl knockout (iLpl(−/−)) mice, confirming that it can also act independently of LPL. D6PV displayed good subcutaneous bioavailability of approximately 80% in nonhuman primates. Because it binds to high-density lipoproteins, which serve as a long-term reservoir, it also has an extended terminal half-life (42–50 h) in nonhuman primates. In summary, D6PV decreases plasma TG by acting as a dual apoC-II mimetic and apoC-III antagonist, thereby demonstrating its potential as a treatment for HTG.

Lipoprotein lipase (LPL) is a secreted lipase that clears triglycerides from the blood. Proper LPL folding and exit from the endoplasmic reticulum (ER) require lipase maturation factor 1 (LMF1), an ER‐resident transmembrane protein, but the mechanism involved is unknown. We used proteomics to identify LMF1‐binding partners necessary for LPL secretion in HEK293 cells and found these to include oxidoreductases and lectin chaperones, suggesting that LMF1 facilitates the formation of LPL's five disulfide bonds. In accordance with this role, we found that LPL aggregates in LMF1‐deficient cells due to the formation of incorrect intermolecular disulfide bonds. Cells lacking LMF1 were hypersensitive to depletion of glutathione, but not DTT treatment, suggesting that LMF1 helps reduce the ER. Accordingly, we found that loss of LMF1 results in a more oxidized ER. Our data show that LMF1 has a broader role than simply folding lipases, and we identified fibronectin and the low‐density lipoprotein receptor (LDLR) as novel LMF1 clients that contain multiple, non‐sequential disulfide bonds. We conclude that LMF1 is needed for secretion of some ER client proteins that require reduction of non‐native disulfides during their folding.

Tailoring coupled plasmonic structures is an alternative way to obtain new optical properties of plasmonic materials. Recently, film-coupled nanoparticle systems with high stability and controllability have been used to probe the ultimate limits of field enhancement/confinement and near-field interaction with other quantum emitters. When the gap between particles and films is below a few nanometers, induced high order (HO) gap modes become significant. In this report, we investigate these HO modes associated with the system of a gold nanosphere positioned above a gold film, separated by a nanometer-scale spacer layer. The shift in far-field scattering profile under different excitation conditions and collection wavelengths indicates the influence of HO modes, and the results are compared to the corresponding simulations. In addition, the far-field scattering spectra/patterns by multipole expansion and the near-field distributions by FEM, both calculated with the dielectric function of the low damping fact...

Age-related macular degeneration (AMD) is the foremost cause of irreversible blindness in people over the age of 65 especially in developing countries. Therefore, an exploration of effective and alternative therapeutic interventions is an unmet medical need. It has been established that oxidative stress plays a key role in the pathogenesis of AMD, and hence, neutralizing oxidative stress is an effective therapeutic strategy for treatment of this serious disorder. Owing to autoregenerative properties, nanoceria has been widely used as a nonenzymatic antioxidant in the treatment of oxidative stress related disorders. Yet, its potential clinical implementation has been greatly hampered by its poor water solubility and lack of reliable tracking methodologies/processes and hence poor absorption, distribution, and targeted delivery. The water solubility and surface engineering of a drug with biocompatible motifs are fundamental to pharmaceutical products and precision medicine. Here, we report an engineered water-soluble, biocompatible, trackable nanoceria with enriched antioxidant activity to scavenge intracellular reactive oxygen species (ROS). Experimental studies with in vitro and in vivo models demonstrated that this antioxidant is autoregenerative and more active in inhibiting laser-induced choroidal neovascularization by decreasing ROS-induced pro-angiogenic vascular endothelial growth factor (VEGF) expression, cumulative oxidative damage, and recruitment of endothelial precursor cells without exhibiting any toxicity. This advanced formulation may offer a superior therapeutic effect to deal with oxidative stress induced pathogeneses, such as AMD.

A protocol for the rapid detection and semi-quantification of human enteric adenovirus based on the quantification of viral mRNA during cell culture infectivity assay was developed. Infectivity assays for adenovirus incorporated cell culture and reverse transcription real-time PCR, where viral mRNA detection was used to monitor the progress of adenovirus infection (CC/mRNA qPCR). The cell line used was G293. This specific infectivity assay was calibrated against different initial concentrations of human adenovirus 41. In addition, the expression of the host's housekeeping (HK) gene, GAPDH, served as internal control for the mRNA assays for quality assurance of the mRNA extraction and reverse transcription steps. The concentrations of infectious human adenoviruses in different sewage samples were estimated semi-quantitatively using the CC/mRNA qPCR assay and calibration obtained for adenovirus 41. A linear relationship between concentrations of viral mRNA (hexon gene) and infectious units was observed between 10 7 to 10 1 infectious units per assay ( R 2 = 0.97) in samples analyzed 3–5 days post infection. The expressions of host cell GAPDH gene were not significantly affected by infections with different concentrations of human adenovirus 41, and between virus positive and negative cell cultures ( p > 0.1). The estimated concentrations of human adenoviruses in sewage samples ranged between 10 2 to 10 3 mRNA-IU/L. Most of the viruses detected in sewage samples were from human adenovirus species F. The CC/mRNA qPCR assay can be used for quantifying infectious human adenovirus 41, estimating the levels of human adenoviruses in sewage samples, and applied to other sample settings. The CC/mRNA qPCR protocol described here represents an improvement in the detection of human enteric adenoviruses by reducing incubation time (5 days); whereas the conventional cell culture method requires longer incubation periods (10–20 days). More importantly, this protocol can be used to more rapidly and semi-quantitatively estimate the levels of infectious human adenoviruses in environmental samples.

Auxin is a multi-functional hormone essential for plant development and pattern formation. A nuclear auxin signaling system controlling auxin-induced gene expression is well established, but cytoplasmic auxin signaling as in its coordination of cell polarization is unexplored. We found a cytoplasmic auxin signaling mechanism that modulates the interdigitated growth of Arabidopsis leaf epidermal pavement cells (PCs), which develop interdigitated lobes and indentations to form a puzzle-piece shape in a two-dimensional plane. PC interdigitation is compromised in leaves deficient in either auxin biosynthesis or its export mediated by PINFORMED 1 localized at the lobe tip. Auxin coordinately activates two Rho GTPases, ROP2 and ROP6, which promote the formation of complementary lobes and indentations, respectively. Activation of these ROPs by auxin occurs within 30 seconds and depends on AUXIN-BINDING PROTEIN 1. These findings reveal Rho GTPase-based novel auxin signaling mechanisms, which modulate the spatial coordination of cell expansion across a field of cells.

A metal film coupled with a metal nanoparticle is a simple and stable nanoantenna structure with plasmonic characteristics. This film-coupled nanoparticle system also has potential for the signal enhancement due to the highly confined field between the film and the nanoparticle. Recently, this structure has been used to probe the limit of the enhanced field and the interaction with quantum emitters. The well-known mode in this nanoantenna structures is the gap dipole mode. However, the high order modes become significant when the gap between the nanoparticle and the film is reduced. In this work, the high order modes are investigated. The size of the whole nanoantenna structure is around λ/10. In experiments, the far field scattering spectra/images under different excitation and collection conditions indicate the influence and the existence of the high order modes. The calculated far-field scattering spectra and spatial intensity profiles have good agreement with the experimental results. In addition, among these high order modes, the simulated near-field distributions reveal distinguishable features which include the different symmetry of field distributions and the various size of confined field. The investigation of these high order modes may provide the information for designing the interaction between this nanoantenna structure and other plasmonic devices.

The DnaQ-H family exonuclease Snipper (Snp) is a 33-kDaDrosophila melanogasterhomolog of 3′hExo and ERI-1, exoribonucleases implicated in the degradation of histone mRNA in mammals and in the negative regulation of RNA interference (RNAi) inCaenorhabditis elegans, respectively. In metazoans, Snp, Exod1, 3′hExo, ERI-1, and the prpip nucleases define a new subclass of structure-specific 3′-5′ exonucleases that bind and degrade double-stranded RNA and/or DNA substrates with 3′ overhangs of 2–5 nucleotides (nt) in the presence of Mg2+with no apparent sequence specificity. These nucleases are also capable of degrading linear substrates. Snp efficiently degrades structured RNA and DNA substrates as long as there exists a minimum 3′ overhang of 2 nt to initiate degradation. We identified aSnpmutant and used it to test whether Snp plays a role in regulating histone mRNA degradation or RNAi in vivo.Snpmutant flies are viable, and display no obvious developmental abnormalities. The expression pattern and level of histone H3 mRNA inSnpmutant embryos and third instar imaginal eye discs was indistinguishable from wild type, suggesting that Snp does not play a significant role in the turnover of histone mRNA at the end of the S phase. The loss ofSnpwas also unable to enhance the silencing capability of two different RNAi transgenes targeting thewhiteandyellowgenes, suggesting that Snp does not negatively modulate RNAi. Therefore, Snp is a nonessential exonuclease that is not a functional ortholog of either 3′hExo or ERI-1.

The Arabidopsis (Arabidopsis thaliana) genome contains 15 genes encoding protein homologs of the barley mildew resistance locus o (MLO) protein biochemically shown to have a seven-transmembrane domain topology and localize to the plasma membrane. Towards elucidating the functions of MLOs, the largest family of seven-transmembrane domain proteins specific to plants, we comprehensively determined AtMLO gene expression patterns by a combination of experimental and in silico studies. Experimentation comprised analyses of transgenic Arabidopsis lines bearing promoter::Beta-glucuronidase (GUS) transcriptional fusions as well as semi-quantitative determination of transcripts by reverse transcription coupled to polymerase chain reaction (RT-PCR). These results were combined with information extracted from public gene profiling databases, and compared to the expression patterns of genes encoding the heterotrimeric G-protein subunits. We found that each AtMLO gene has a unique expression pattern and is regulated differently by a variety of biotic and/or abiotic stimuli, suggesting that AtMLO proteins function in diverse developmental and response processes. The expression of several phylogenetically closely-related AtMLO genes showed similar or overlapping tissue specificity and analogous responsiveness to external stimuli, suggesting functional redundancy, co-function, or antagonistic function(s).

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by defined sets of transcription factors. We previously described reprogramming of monolayer-cultured adult mouse ciliary body epithelial (CE) cells by Oct4 and Klf4, but not with Oct4 alone. In this study, we report that Oct4 alone is sufficient to reprogram CE cells to iPS cells through sphere formation. Furthermore, we demonstrate that sphere formation induces a partial reprogramming state characterized by expression of retinal progenitor markers, upregulation of reprogramming transcription factors, such as Sall4 and Nanog, demethylation in the promoter regions of pluripotency associated genes, and mesenchymal to epithelial transition. The Oct4-iPS cells maintained normal karyotypes, expressed markers for pluripotent stem cells, and were capable of differentiating into derivatives of all three embryonic germ layers in vivo and in vitro. These findings suggest that sphere formation may render somatic cells more susceptible to reprogramming.

A 65-kDa subunit, nuclear protein from mung bean was previously identified using anti-idiotypic antibodies directed against IgG recognizing the auxin, indole-3-acetic acid. This protein was proposed to be a putative auxin-binding protein and was designated 65-kDa ABP (Prasad and Jones, Proc. Natl. Acad. Sci. USA 88 (1991) 5479–5483). Despite substantial effort, direct evidence for auxin-binding has not been demonstrated. Therefore, 65-kDa ABP is redesignated as p65 and herein is described its purification and the N-terminal sequence of this protein. Antibodies directed against a synthetic N-terminal peptide of p65 immunoprecipitate a 65-kDa peptide that is recognized by the anti-idiotypic antibodies indicating that the amino terminal sequence is of the originally described 65-kDa ABP. High sequence identity between p65 and the N-terminal sequence of several glutathione-S-transferases (GSTs) was found, although significant GST activity was not detected. We demonstrate that p65 is retained by both glutathione- and IAA-immobilized columns. These and previous observations indicate that p65 has structural homology, but not necessarily functional homology, to both GST and ABP.

The Vimentin gene plays a pivotal role in epithelial-to-mesenchymal transition and is known to be overexpressed in the prognostically poor basal-like breast cancer subtype. Recent studies have reported Vimentin DNA methylation in association with poor clinical outcomes in other solid tumors, but not in breast cancer. We therefore quantified Vimentin DNA methylation using MALDI-TOF mass spectrometry in breast tumors and matched normal pairs in association with gene expression and survival in a hospital-based study of breast cancer patients. Gene expression data via qRT-PCR in cell lines and oligomicroarray data from breast tissues were correlated with percent methylation in the Vimentin promoter. A threshold of 20 percent average methylation compared with matched normal pairs was set for bivariate and multivariate tests of association between methylation and tumor subtype, tumor histopathology, and survival. Vimentin was differentially methylated in luminal breast cancer cell lines, and in luminal A, luminal B, and HER2-enriched breast tumor subtypes, but was rare in basal-like cell lines and tumors. Increased methylation was strongly correlated with decreased mRNA expression in cell lines, and had a moderate inverse correlation in breast tumors. Vimentin methylation predicted poor overall survival independent of race, subtype, stage, nodal status, or metastatic disease and holds promise as a new prognostic biomarker for breast cancer patients.

Induced pluripotent stem (iPS) cells are attractive for cell replacement therapy, because they overcome ethical and immune rejection issues that are associated with embryonic stem cells. iPS cells have been derived from autonomous fibroblasts at low efficiency using multiple ectopic transcription factors. Recent evidence suggests that the epigenome of donor cell sources plays an important role in the reprogramming and differentiation characteristics of iPS cells. Thus, identification of somatic cell types that are easily accessible and are more amenable for cellular reprogramming is critical for regenerative medicine applications. Here, we identify ciliary body epithelial cells (CECs) as a new cell type for iPS cell generation that has higher reprogramming efficiency compared with fibroblasts. The ciliary body is composed of epithelial cells that are located in the anterior portion of the eye at the level of the lens and is readily surgically accessible. CECs also have a reduced reprogramming requirement, as we demonstrate that ectopic Sox2 and c-Myc are dispensable. Enhanced reprogramming efficiency may be due to increased basal levels of Sox2 in CECs. In addition, we are the first to report a cellular reprogramming haploinsufficiency observed when reprogramming with fewer factors (Oct4 and Klf4) in Sox2 hemizygous cells. Taken together, endogenous Sox2 levels are critical for the enhanced efficiency and reduced exogenous requirement that permit facile cellular reprogramming of CECs.

The conjugate addition of thioacetic acid (3 equivalents) to (4S)-4-phenyl-N-methacryloyloxazolidin-2-one (3) in the presence of TiCl4 (1.5 equivalents) gave the addition adducts in 87% yield and only 14% de. When this reaction was carried out in the absence of TiCl4, good diastereoselectivity (94% de) was observed. A similar result was obtained by employing thiobenzoic acid as a nucleophile. The reaction of 3 with other thiols, such as thiophenol, 4-methoxythiophenol, 2-methoxythiophenol, methyl 3-mercatopropionate, methyl thioglycolate and methyl thiosalicylate, required TiCl4 (1.5 equivalents) to give the addition adducts in good chemical yields and the diastereoselectivities are ranging from 50 to 82% de. 2-Aminothiophenol was also employed in this study to give the addition adducts in 60% yield and 70% de when the reaction was carried out in the absence of TiCl4. In the presence of TiCl4, this reaction provided the addition adducts as racemic mixture in 28% yield along with 1,5-benzothiazepin-4-one 10 in 56% yield.

The conjugate addition of thioacetic acid (3 equivalents) to (4S)-4-phenyl-N-methacryloyloxazolidin-2-one (3) in the presence of TiCl4 (1.5 equivalents) gave the addition adducts in 87% yield and only 14% de. When this reaction was carried out in the absence of TiCl4, good diastereoselectivity (94% de) was observed. A similar result was obtained by employing thiobenzoic acid as a nucleophile. The reaction of 3 with other thiols, such as thiophenol, 4-methoxythiophenol, 2-methoxythiophenol, methyl 3-mercatopropionate, methyl thioglycolate and methyl thiosalicylate, required TiCl4 (1.5 equivalents) to give the addition adducts in good chemical yields and the diastereoselectivities are ranging from 50 to 82% de. 2-Aminothiophenol was also employed in this study to give the addition adducts in 60% yield and 70% de when the reaction was carried out in the absence of TiCl4. In the presence of TiCl4, this reaction provided the addition adducts as racemic mixture in 28% yield along with 1,5-benzothiazepin-4-one 10 in 56% yield.

Directional root expansion is governed by nutrient gradients, positive gravitropism and hydrotropism, negative phototropism and thigmotropism, as well as endogenous oscillations in the growth trajectory (circumnutation). Null mutations in phylogenetically related Arabidopsis thaliana genes MILDEW RESISTANCE LOCUS O 4 (MLO4) and MLO11, encoding heptahelical, plasma membrane–localized proteins predominantly expressed in the root tip, result in aberrant root thigmomorphogenesis. mlo4 and mlo11 mutant plants show anisotropic, chiral root expansion manifesting as tightly curled root patterns upon contact with solid surfaces. The defect in mlo4 and mlo11 mutants is nonadditive and dependent on light and nutrients. Genetic epistasis experiments demonstrate that the mutant phenotype is independently modulated by the Gβ subunit of the heterotrimeric G-protein complex. Analysis of expressed chimeric MLO4/MLO2 proteins revealed that the C-terminal domain of MLO4 is necessary but not sufficient for MLO4 action in root thigmomorphogenesis. The expression of the auxin efflux carrier fusion, PIN1-green fluorescent protein, the pattern of auxin-induced gene expression, and acropetal as well as basipetal auxin transport are altered at the root tip of mlo4 mutant seedlings. Moreover, addition of auxin transport inhibitors or the loss of EIR1/AGR1/PIN2 function abolishes root curling of mlo4, mlo11, and wild-type seedlings. These results demonstrate that the exaggerated root curling phenotypes of the mlo4 and mlo11 mutants depend on auxin gradients and suggest that MLO4 and MLO11 cofunction as modulators of touch-induced root tropism.

To test the hypothesis that auxin-binding protein 1 (ABP1) is a receptor controlling auxin-mediated plant cell expansion, ABP1 complementary DNAs were expressed in a controllable fashion in tobacco plants and constitutively in maize cell lines. Induction of Arabidopsis ABP1 expression in tobacco leaf strips resulted in an increased capacity for auxin-mediated cell expansion, whereas induction of ABP1 in intact plants resulted in leaves with a normal morphology, but larger cells. Similarly, constitutive expression of maize ABP1 in maize cell lines conferred on them the capacity to respond to auxin by increasing cell size. These results support a role of ABP1 as an auxin receptor controlling plant growth.

This paper presents a method of evaluation of four integrals of Howland type, which involve a Bessel function in the integrands. With the aid of tabulated values, they are evaluated to 10D. Two of the four Howland integrals needed in the evaluation are evaluated anew to 20D in order to provide adequate accuracy.