Connector inversion probe technology: a powerful one-primer multiplex DNA amplification system for numerous scientific applications

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. "multiplex multiplexing padlocks" (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.     

Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0–15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.     

Characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis

Human angiogenin (ANG), the first member of the angiogenin family (from the pancreatic ribonuclease A superfamily) to be identified, is an angiogenic factor that induces neovascularization. It has received much attention due to its involvement in the growth of tumors and its elevated expression level in pancreatic and several other cancers. Recently the biological role of ANG has been shown to extend to the nervous system. Mutations in ANG have been linked with familial as well as sporadic forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by selective destruction of motor neurons. Furthermore, mouse angiogenin-1 has been shown to be expressed in the developing nervous system and during the neuronal differentiation of pluripotent stem cells. We have now characterized the seven variants of ANG reported in ALS patients with respect to the known biochemical properties of ANG and further studied the biological properties of three of these variants. Our results show that the ribonucleolytic activity of six of the seven ANG-ALS implicated variants is significantly reduced or lost and some variants also show altered thermal stability. We report a significant reduction in the cell proliferative and angiogenic activities of the three variants that we chose to investigate further. Our studies on the biochemical and structural features of these ANG variants now form the basis for further investigations to determine their role(s) in ALS.     

A predicted amphipathic helix mediates plasma membrane localization of GRK5

G protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied G protein-coupled receptors at the inner surface of the plasma membrane (PM), leading to receptor desensitization. GRKs utilize a variety of mechanisms to bind tightly, and sometimes reversibly, to cellular membranes. Previous studies demonstrated the presence of a membrane binding domain in the C terminus of GRK5. Here we define a mechanism by which this short C-terminal stretch of amino acids of GRK5 mediates PM localization. Secondary structure predictions suggest that a region contained within amino acids 546-565 of GRK5 forms an amphipathic helix, with the key features of the predicted helix being a hydrophobic patch of amino acids on one face of the helix, hydrophilic amino acids on the opposite face, and a number of basic amino acids surrounding the hydrophobic patch. We show that amino acids 546-565 of GRK5 are sufficient to target the cytoplasmic green fluorescent protein (GFP) to the PM, and the hydrophobic amino acids are necessary for PM targeting of GFP-546-565. Moreover, full-length GRK5-GFP is localized to the PM, but mutation of the hydrophobic patch or the surrounding basic amino acids prevents PM localization of GRK5-GFP. Last, we show that mutation of the hydrophobic residues severely diminishes phospholipid-dependent autophosphorylation of GRK5 and phosphorylation of membrane-bound rhodopsin by GRK5. The findings in this report thus suggest the presence of a membrane binding motif in GRK5 and define the importance of a group of hydrophobic amino acids within this motif in mediating its PM localization.     

Comprehensive network map of interferon gamma signaling

Interferon gamma (IFN-γ), is a cytokine, which is an important regulator of host defense system by mediating both innate and adaptive immune responses. IFN-γ signaling is primarily associated with inflammation and cell-mediated immune responses. IFN-γ is also represented as antitumor cytokine which facilitates immunosurveillance in tumor cells. In addition, IFN-γ mediated signaling also elicits pro-tumorigenic transformations and promotes tumor progression. Impact of IFN-γ signaling in mammalian cells has been widely studied which indicate that IFN-γ orchestrates distinct cellular functions including immunomodulation, leukocyte trafficking, apoptosis, anti-microbial, and both anti- and pro-tumorigenic role. However, a detailed network of IFN-γ signaling pathway is currently lacking. Therefore, we systematically curated the literature information pertaining to IFN-γ signaling and develop a comprehensive signaling network to facilitate better understanding of IFN-γ mediated signaling. A total of 124 proteins were catalogued that were experimentally proven to be involved in IFN-γ signaling cascade. These 124 proteins were found to participate in 81 protein-protein interactions, 94 post-translational modifications, 20 translocation events, 54 activation/inhibiton reactions. Further, 236 differential expressed genes were also documented in IFN-γ mediated signaling. IFN-γ signaling pathway is made freely available to scientific audience through NetPath at (http://www.netpath.org/pathways?path_id=NetPath_32). We believe that documentation of reactions pertaining to IFN-γ signaling and development of pathway map will facilitate further research in IFN-γ associated human diseases including cancer.     

Combined effects of temperature and salinity on larval development and attachment of the subtidal barnacle Balanus trigonus Darwin

The combined effects of temperature and salinity on larval development and attachment of Balanus trigonus Darwin (Cirripedia, Balanidae) was examined under controlled laboratory conditions. Whilst larval survivorship was not affected (>70%), the duration of larval development was significantly affected by temperature and salinity. The effect of temperature was comparatively stronger than that of salinity. The majority of nauplius II larvae metamorphosed into cypris stage after 4-5 and 10-11 days at 28 °C (22-34‰) and 18 °C (22-34‰), respectively. Temperature, salinity and the duration of assay had a significant effect on cypris attachment with significant interaction among these main effects. Maximum (>80% in 6 days) and minimum percent attachment (0% in 6 days) on polystyrene surfaces were observed at 24 °C (34‰) and 18 °C (22‰), respectively. At high temperature (28 °C) and low salinity (22-26‰), larvae rapidly (4 days) developed into cyprids, but less than 33% attached. These results suggest that low larval attachment rates may lead to the low recruitment of B. trigonus in Hong Kong waters during summer when the water temperature is high (about 28 °C) and salinity is low (<26‰).     

Crystal structure of a catalytically active, non-toxic endopeptidase derivative of Clostridium botulinum toxin A

Botulinum neurotoxins (BoNTs) modulate cholinergic nerve terminals to result in neurotransmitter blockade. BoNTs consists of catalytic (LC), translocation (Hn) and cell-binding domains (Hc). The binding function of the Hc domain is essential for BoNTs to bind the neuronal cell membrane, therefore, removal of the Hc domain results in a product that retains the endopeptidase activity of the LC but is non-toxic. Thus, a molecule consisting of LC and Hn domains of BoNTs, termed LHn, is a suitable molecule for engineering novel therapeutics. The structure of LHA at 2.6 Å reported here provides an understanding of the structural implications and challenges of engineering therapeutic molecules that combine functional properties of LHn of BoNTs with specific ligand partners to target different cell types.     

Structures of Complexes of a Metal-independent Glycosyltransferase GT6 from Bacteroides ovatus with UDP-N-Acetylgalactosamine (UDP-GalNAc) and Its Hydrolysis Products

Mammalian members of glycosyltransferase family 6 (GT6) of the CAZy database have a GT-A fold containing a conserved Asp-X-Asp (DXD) sequence that binds an essential metal cofactor. Bacteroides ovatus GT6a represents a GT6 clade found in more than 30 Gram-negative bacteria that is similar in sequence to the catalytic domains of mammalian GT6, but has an Asn⁹⁵-Ala-Asn⁹⁷ (NXN) sequence substituted for the DXD motif and metal-independent catalytic activity. Co-crystals of a low activity mutant of BoGT6a (E192Q) with UDP-GalNAc contained protein complexes with intact UDP-GalNAc and two forms with hydrolysis products (UDP plus GalNAc) representing an initial closed complex and later open form primed for product release. Two cationic residues near the C terminus of BoGT6a, Lys²³¹ and Arg²⁴³, interact with the diphosphate moiety of UDP-GalNAc, but only Lys²³¹ interacts with the UDP product and may function in leaving group stabilization. The amide group of Asn⁹⁵, the first Asn of the NXN motif, interacts with the ribose moiety of the substrate. This metal-independent GT6 resembles its metal-dependent homologs in undergoing conformational changes on binding UDP-GalNAc that arise from structuring the C terminus to cover this substrate. It appears that in the GT6 family, the metal cofactor functions specifically in binding the UDP moiety in the donor substrate and transition state, actions that can be efficiently performed by components of the polypeptide chain.     

Incorporation of alpha-chymotrypsin into the 3D channels of bicontinuous cubic lipid mesophases

The effects of protein entrapment on the structure and phase behavior of periodically curved lipid mesostructures have been examined by synchrotron small-angle X-ray diffraction and FT-IR spectroscopy. The study was directed towards a better understanding of the effect of confinement in a lipid environment on the stability and unfolding behavior of alpha-chymotrypsin, and, vice versa, the effect of the entrapped protein on the lipid's mesophase structure and temperature- and pressure-dependent phase behavior. We compare the interaction of protein molecules of two different sizes (cytochrome c, 12.4 kDa, and alpha-chymotrypsin, 25.8 kDa) with the cubic Ia3d phase of monoolein (MO), which forms spontaneously in water. The cubic structure changes significantly when cyt c is incorporated: above a protein concentration of 0.2 wt.%, the interaction between the positively charged protein and the lipid headgroups leads to an increase in interfacial curvature which promotes the formation of a new micellar cubic phase, presumably of crystallographic space group P4(3)32, which the lipid system does not form on its own. The larger alpha-chymotrypsin leads to a different scenario. On the basis of an examination of the calculated geometric parameters and water volume fractions, it is concluded that the alpha-chymotrypsin molecules cannot be located exclusively in the water channels of the cubic Ia3d or P4(3)32 phases, but rather form new, less ordered (presumably cubic Pn3m) structures. The new structure disappears above the unfolding temperature of chymotrypsin and exhibits a pressure stability, which-- in contrast to cyt c in MO-- decreases with increasing chymotrypsin concentration in the system. While the secondary structure of cyt c remains unaffected in the confining lipid environment, the structure of alpha-chymotrypsin gets destabilized slightly, and the protein tends to aggregate even at relatively low concentrations.     

PathogenMIPer: a tool for the design of molecular inversion probes to detect multiple pathogens

Background  

Here we describe PathogenMIPer, a software program for designing molecular inversion probe (MIP) oligonucleotides for use in pathogen identification and detection. The software designs unique and specific oligonucleotide probes targeting microbial or other genomes. The tool tailors all probe sequence components (including target-specific sequences, barcode sequences, universal primers and restriction sites) and combines these components into ready-to-order probes for use in a MIP assay. The system can harness the genetic variability available in an entire genome in designing specific probes for the detection of multiple co-infections in a single tube using a MIP assay.