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A network of protein interactions that centre around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells is identified. Rich1 and Amot maintain tight junctions (TJ) integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

Abstract Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.

In III–V IC processing, lift-off patterning is universally used. Historically used negative resist has its limitations; hence the image reverse process using positive tone resist has been utilized as the main work horse in lift-off applications. Metal lift-off processes using a specially designed chemically amplified (CAMP) negative tone resist have been evaluated for thin and thick metal layers to have a varying degree of success. Lift-off using CAMP photoresists has made it possible to reduce cycle time (~57% reduction in $1\times$ theoretical cycle time per layer) and reduce cost (due to elimination of steps) as compared to the image reverse process. However, achieving consistent lift-off and critical dimension (CD) control presents challenges, which are discussed in this paper. [ABSTRACT FROM AUTHOR]

The objective of this paper is to find a sequence of jobs for the permutation flow shop to minimise the makespan. The shop consists of 10 machines. A feed-forward back-propagation artificial neural network (ANN) is used to solve the problem. The network is trained with the optimal sequences for five-, six- and seven-job problems. This trained network is then used to solve a problem with a greater number of jobs. The sequence obtained using the neural network is used to generate the initial population for the genetic algorithm (GA) using the random insertion perturbation scheme (RIPS). The makespan of the sequence obtained by this approach (ANN-GA-RIPS) is compared with that obtained using GA starting with a random population (ANN-GA). It was found that the ANN-GA-RIPS approach performs better than ANN-GA starting with a random population. The results obtained are compared with those obtained using the Nawaz, Enscore and Ham (NEH) heuristic and upper bounds of Taillard's benchmark problems. ANN-GA-RIPS performs better than the NEH heuristic and the results are found to be within 5% of the upper bounds. [ABSTRACT FROM AUTHOR]

An asymmetric synthesis of anti-aldol segments via a nonaldol route is described, where the strategy has involved a highly diastereoselective synthesis of functionalized tetrahydrofuran derivatives from optically active 4-phenylbutyrolactone. The utility of the method is shown by preparing statine derivatives and pancreatic lipase inhibitor, (-)-tetrahydrolipstatin.

In this paper, the evolution of various lithographic processes aimed at fabricating robust tantalum nitride (TaN) resistors in GaAs-based BiFET and BiHEMT technologies are discussed in detail. Such approaches include the common image reverse photograph process (via amine poisoning) using a positive tone resist, the use of a chemically amplified negative tone resist, and also straight patterning of a positive tone resist. The aforementioned approaches were found to have their own unique challenges as it applied to printing robust TaN-based resistors approaching nearly a micron in width which is needed for high density mixed signal designs. We will explore many of these challenges in detail for the various lithography schemes. [ABSTRACT FROM AUTHOR]

Abstract: A practical and metal free method for methylthiolation of imidazo[1,2-a]pyridines and other imidazo-fused heterocycles using DMSO–POCl3 complex is reported. The reactions are carried out at room temperature that give the corresponding methylthiolated products in good to excellent yield. Further, its application to indole has been demonstrated. [Copyright &y& Elsevier]

Background: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics. [ABSTRACT FROM AUTHOR]

The polo-like kinase Plk4 (also called Sak) is required for late mitotic progression, cell survival and postgastrulation embryonic development. Here we identified a phenotype resulting from Plk4 haploinsufficiency in Plk4 heterozygous cells and mice. Plk4+/− embryonic fibroblasts had increased centrosomal amplification, multipolar spindle formation and aneuploidy compared with wild-type cells. The incidence of spontaneous liver and lung cancers was ∼15 times high in elderly Plk4+/− mice than in Plk4+/+ littermates. Using the in vivo model of partial hepatectomy to induce synchronous cell cycle entry, we determined that the precise regulation of cyclins D1, E and B1 and of Cdk1 was impaired in Plk4+/− regenerating liver, and p53 activation and p21 and BubR1 expression were suppressed. These defects were associated with progressive cell cycle delays, increased spindle irregularities and accelerated hepatocellular carcinogenesis in Plk4+/− mice. Loss of heterozygosity occurs frequently (∼60%) at polymorphic markers adjacent to the PLK4 locus in human hepatoma. Reduced Plk4 gene dosage increases the probability of mitotic errors and cancer development. [ABSTRACT FROM AUTHOR]

The human transcription factor CA150 modulates human immunodeficiency virus type 1 gene transcription and contains numerous signaling elements, including six FF domains. Repeated FF domains are present in several transcription and splicing factors and can recognize phosphoserine motifs in the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Using mass spectrometry, we identify a number of nuclear binding partners for the CA150 FF domains and demonstrate a direct interaction between CA150 and Tat-SF1, a protein involved in the coupling of splicing and transcription. CA150 FF domains recognize multiple sites within the Tat-SF1 protein conforming to the consensus motif (D/E)2/5-F/W/Y-(D/E)2/5. Individual FF domains are capable of interacting with Tat-SF1 peptide ligands in an equivalent and noncooperative manner, with affinities ranging from 150 to 500 μM. Repeated FF domains therefore appear to bind their targets through multiple weak interactions with motifs comprised of negatively charged residues flanking aromatic amino acids. The RNAPII CTD represents a consensus FF domain-binding site, contingent on generation of the requisite negative charges by phosphorylation of serines 2 and 5. We propose that CA150, through the dual recognition of acidic motifs in proteins such as Tat-SF1 and the phosphorylated CTD, could mediate the recruitment of transcription and splicing factors to actively transcribing RNAPII. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions. [ABSTRACT FROM AUTHOR]

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis. [ABSTRACT FROM AUTHOR]

The abstract in is incorrect. The word “photograph” was used in error rather than the more correct, “photo.” The abstract should read: In this paper, the evolution of various lithographic processes aimed at fabricating robust tantalum nitride (TaN) resistors in GaAs-based BiFET and BiHEMT technologies are discussed in detail. Such approaches include the common image reverse photo process (via amine poisoning) using a positive tone resist, the use of a chemically amplified negative tone resist, and also straight patterning of a positive tone resist. The aforementioned approaches were found to have their own unique challenges as it applied to printing robust TaN-based resistors approaching nearly a micron in width which is needed for high density mixed signal designs. We will explore many of these challenges in detail for the various lithography schemes. [ABSTRACT FROM AUTHOR]

The article presents chemical equations related to synthesis of thioethers where from methylthiolation of Imidazo-fused heterocycles such as Imidazo [1,2-a] pyridines a reagent is obtained, published in the volume 38 of 2013 issue of the journal "Tetrahedron."

Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3. [Copyright &y& Elsevier]

Presents information pertaining to the cell and growth cone migrations in animals. How the extracellular polarity information is transduced; Suggestion of results; What are these migrations guided by.

Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate β-hydroxyl or β-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite β-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.

This data article presents a description of a benchmark dataset for the multiple depot vehicle scheduling problem (MDVSP). The MDVSP is to assign vehicles from different depots to timetabled trips to minimize the total cost of empty travel and waiting. The dataset has been developed to evaluate the heuristics of the MDVSP that are presented in "A new formulation and a column generation-based heuristic for the multiple depot vehicle scheduling problem" (Kulkarni et al., 2018). The dataset contains 60 problem instances of varying size. Researchers can use the dataset to evaluate the future algorithms for the MDVSP and compare the performance with the existing algorithms. The dataset includes a program that can be used to generate new problem instances of the MDVSP.

Sulfated molecules with diverse functions are common in biology, but sulfonation as a method to activate a metabolite for chemical catalysis is rare. Catalytic activity was characterized and crystal structures were determined for two such "activating" sulfotransferases (STs) that sulfonate β-hydroxyacyl thioester substrates. The CurM polyketide synthase (PKS) ST domain from the curacin A biosynthetic pathway of Moorea producens and the olefin synthase (OLS) ST from a hydrocarbon-producing system of Synechococcus PCC 7002 both occur as a unique acyl carrier protein (ACP), ST, and thioesterase (TE) tridomain within a larger polypeptide. During pathway termination, these cyanobacterial systems introduce a terminal double bond into the β-hydroxyacyl-ACP-linked substrate by the combined action of the ST and TE. Under in vitro conditions, CurM PKS ST and OLS ST acted on β-hydroxy fatty acyl-ACP substrates; however, OLS ST was not reactive toward analogues of the natural PKS ST substrate bearing a C5-methoxy substituent. The crystal structures of CurM ST and OLS ST revealed that they are members of a distinct protein family relative to other prokaryotic and eukaryotic sulfotransferases. A common binding site for the sulfonate donor 3'-phosphoadenosine-5'-phosphosulfate was visualized in complexes with the product 3'-phosphoadenosine-5'-phosphate. Critical functions for several conserved amino acids in the active site were confirmed by site-directed mutagenesis, including a proposed glutamate catalytic base. A dynamic active-site flap unique to the "activating" ST family affects substrate selectivity and product formation, based on the activities of chimeras of the PKS and OLS STs with exchanged active-site flaps.

Natural products with macrocyclic structural features often display intriguing biological properties. Molecular design incorporating macrocycles may lead to molecules with unique protein-ligand interactions. We generated novel human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing a macrocycle and bis-tetrahydrofuranylurethane. Four such compounds exerted potent activity against HIV-1LAI and had 50% effective concentrations (EC50s) of as low as 0.002 microM with minimal cytotoxicity. GRL-216 and GRL-286 blocked the replication of HIV-1NL4-3 variants selected by up to 5 microM saquinavir, ritonavir, nelfinavir, lopinavir, or atazanavir; they had EC50s of 0.020 to 0.046 microM and potent activities against six multi-PI-resistant clinical HIV-1 (HIVmPIr) variants with EC50s of 0.027 to 0.089 microM. GRL-216 and -286 also blocked HIV-1 protease dimerization as efficiently as darunavir. When HIV-1NL4-3 was selected by GRL-216, it replicated progressively more poorly and failed to replicate in the presence of >0.26 microM GRL-216, suggesting that the emergence of GRL-216-resistant HIV-1 variants is substantially delayed. At passage 50 with GRL-216 (the HIV isolate selected with GRL-216 at up to 0.16 microM [HIV216-0.16 microM]), HIV-1NL4-3 containing the L10I, L24I, M46L, V82I, and I84V mutations remained relatively sensitive to PIs, including darunavir, with the EC50s being 3- to 8-fold-greater than the EC50 of each drug for HIV-1NL4-3. Interestingly, HIV216-0.16 microM had 10-fold increased sensitivity to tipranavir. Analysis of the protein-ligand X-ray structures of GRL-216 revealed that the macrocycle occupied a greater volume of the binding cavity of protease and formed greater van der Waals interactions with V82 and I84 than darunavir. The present data warrant the further development of GRL-216 as a potential antiviral agent for treating individuals harboring wild-type and/or HIVmPIr.

We present a new type of non-contact sensor for use in ambulatory cardiac monitoring. The sensor operation is based on a microwave Doppler technique; however, instead of detecting the heart activity from a distance, the sensor is placed on the patient's chest over the clothing. The microwave sensor directly measures heart movement rather than electrical activity, and is thus complementary to ECG. The primary advantages of the microwave sensor includes small size, light weight, low power, low-cost, and the ability to operate through clothing. We present a sample sensor design that incorporates a 2.4 GHz Doppler circuit, integrated microstrip patch antenna, and microntroller with 12-bit ADC data sampling. The prototype sensor also includes a wireless data link for sending data to a remote PC or mobile phone. Sample data is shown for several subjects and compared to data from a commercial portable ECG device. Data collected from the microwave sensor exhibits a significant amount of features, indicating possible use as a tool for monitoring heart mechanics and detection of abnormalities such as fibrillation and akinesia.

The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic macrocyclic HIV protease inhibitors are described. The inhibitors are designed to effectively fill in the hydrophobic pocket in the S1'-S2' subsites and retain all major hydrogen bonding interactions with the protein backbone similar to darunavir (1) or inhibitor 2. The ring size, the effect of methyl substitution, and unsaturation within the macrocyclic ring structure were assessed. In general, cyclic inhibitors were significantly more potent than their acyclic homologues, saturated rings were less active than their unsaturated analogues and a preference for 10- and 13-membered macrocylic rings was revealed. The addition of methyl substituents resulted in a reduction of potency. Both inhibitors 14b and 14c exhibited marked enzyme inhibitory and antiviral activity, and they exerted potent activity against multidrug-resistant HIV-1 variants. Protein-ligand X-ray structures of inhibitors 2 and 14c provided critical molecular insights into the ligand-binding site interactions.

An enantioselective total synthesis of the cytotoxic natural product (+)-largazole (1) is described. It is a potent histone deacetylase inhibitor. Our synthesis is convergent and involves the assembly of thiazole 3-derived carboxylic acid with amino ester 4 followed by cycloamidation of the corresponding amino acid. The synthesis features an efficient cross-metathesis, an enzymatic kinetic resolution of a beta-hydroxy ester, a selective removal of a Boc-protecting group, a HATU/HOAt-promoted cycloamidation reaction, and synthetic manipulations to a sensitive thioester functional group.

Structure-based design and synthesis of a number of potent and selective memapsin 2 inhibitors are described. These inhibitors were designed based upon the X-ray structure of memapsin 2-bound inhibitor 3 that incorporates methylsulfonyl alanine as the P2-ligand and a substituted pyrazole as the P3-ligand. Of particular importance, we examined the ability of the substituted isophthalic acid amide derivative to mimic the key interactions in the S2-S3 regions of the enzyme active sites of 3-bound memapsin 2. We investigated various substituted phenylethyl, alpha-methylbenzyl, and oxazolylmethyl groups as the P3-ligands. A number of inhibitors exhibited very potent inhibitory activity against mempasin 2 and good selectivity against memapsin 1. Inhibitor 5d has shown low nanomolar enzyme inhibitory potency (Ki=1.1 nM) and very good cellular inhibitory activity (IC50=39 nM). Furthermore, in a preliminary study, inhibitor 5d has shown 30% reduction of Abeta40 production in transgenic mice after a single intraperitoneal administration (8 mg/kg). A protein-ligand X-ray crystal structure of 5d-bound memapsin 2 provided vital molecular insight that can serve as an important guide to further design of novel inhibitors.

A novel diastereoselective synthesis of substituted pyrrolidines has been developed. Asymmetric multicomponent reactions of optically active phenyldihydrofuran, N-tosyl imino ester, and silane reagents in a one-pot operation afforded highly substituted pyrrolidine derivatives diastereoselectively. The reaction is quite efficient and constructed up to three stereogenic centers in a single operation.