Single-Stranded DNA within Nanopores: Conformational Dynamics and Implications for Sequencing; a Molecular Dynamics Simulation Study

Engineered protein nanopores, such as those based on α-hemolysin from Staphylococcus aureus have shown great promise as components of next-generation DNA sequencing devices. However, before such protein nanopores can be used to their full potential, the conformational dynamics and translocation pathway of the DNA within them must be characterized at the individual molecule level. Here, we employ atomistic molecular dynamics simulations of single-stranded DNA movement through a model α-hemolysin pore under an applied electric field. The simulations enable characterization of the conformations adopted by single-stranded DNA, and allow exploration of how the conformations may impact on translocation within the wild-type model pore and a number of mutants. Our results show that specific interactions between the protein nanopore and the DNA can have a significant impact on the DNA conformation often leading to localized coiling, which in turn, can alter the order in which the DNA bases exit the nanopore. Thus, our simulations show that strategies to control the conformation of DNA within a protein nanopore would be a distinct advantage for the purposes of DNA sequencing.     

Molecular dynamics simulations of a bacterial autotransporter: NalP from Neisseria meningitidis

NalP is an autotransporter secretory protein found in the outer membrane of Neisseria meningitidis. The crystal structure of the NalP translocator domain revealed a transmembrane beta-barrel containing a central alpha-helix. The role of this alpha-helix, and of the conformational dynamics of the beta-barrel pore have been studied via atomistic molecular dynamics simulations. Three simulations, each of 10 ns duration, of NalP embedded within a solvated DMPC bilayer were performed. The helix was removed from the barrel interior in one simulation. The conformational stability of the protein is similar to that of other outer membrane proteins, e.g., OmpA, in comparable simulations. The transmembrane beta-barrel is stable even in the absence of the alpha-helix. Removal of the helix results in an influx of water into the pore region, suggesting the helix acts as a 'plug'. Water molecules entering the resultant pore form hydrogen bonds with the barrel lining that compensate for the loss of helix-barrel hydrogen bonds. The dimensions of the pore fluctuate over the course of the simulation revealing it to be flexible, but only wide enough to allow transport of the passenger domain in an unfolded or extended conformation. The simulations help us to understand the role of the central helix in plugging the pore and in maintaining the width of the barrel, and show that the NalP monomer is sufficient for the transport of the passenger domain in an unfolded or extended conformation.     

Optimization of an improved,efficient and rapid in vitro micropropagation protocol for Petunia hybrida Vilm. Cv. “Bravo”

An efficient protocol for in-vitro propagation of an important ornamental crop, Petunia hybrida Vilm. Cv. “Bravo” was developed. The explants that were used to carry out the experiment were Leaf segments, nodal segments and shoot tips. Nodal segments recorded highest per cent asepsis followed by shoot tips and leaf segments. Asepsis was found to be highest when the explants were sterilized with Fungicide (Carbendazim) 0.02% for the duration of 30 min followed by 0.1% HgCl₂ for duration of 10 min and then ethanol 70% for 10 s. Longer duration of the sterilant treatment showed more necrotic effects on the explants, thus mercuric chloride treatment when given for 5 min proved to be more effective in terms of survival of the explants. Maximum establishment per cent was recorded in Murashige and Skoog (MS) media fortified with BAP (1.5 mg L⁻¹) and IBA (0.5 mg L⁻¹) in shoot tips and nodal segments, i.e. 97.90 and 95.74% respectively. Callus was efficiently induced and developed when PGR amalgamation of BAP (0.1 mg L⁻¹) and 2,4-D (1.5mg L⁻¹) was used. Kinetin at the concentration of 2.0 mg L⁻¹ along with IBA at 0.5mg L⁻¹ recorded highest callus regeneration in both leaf and internodal segment derived callus. Maximum proliferation percent of shoots (97.90%), highest number of shoots (20.50 explant⁻¹) and maximum length of shoot (2.70 cm) was recorded in PGR combination of IBA and BAP both at 0.5 mg L⁻1 concentration level. Rhizogenesis was recorded to be highest in the MS media containing IBA 1.00 mg L⁻¹. Best hardening media which recorded maximum survival per cent 92.50% was noticed on the media formulation comprised of equal ratio of perlite and vermiculite mix, under poly house conditions.     

Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (K_D) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (K_D = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.     

Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.     

The effect of estradiol on COX-2, EP2, and EP4 mRNA expression and the extracellular matrix in the cervix of the hypogonadotrophic, ovariectomized ewe

There is a degree of cervical relaxation in the ewe at estrus that is regulated by changes in prostaglandin synthesis, prostaglandin receptor expression, and changes in the cervical extracellular matrix. It is likely that these are regulated by changes in periovulatory hormones, particularly estradiol. This study determined the effect of estradiol benzoate on the mRNA expression of cyclooxygenase-2 (COX-2) and the prostaglandin E receptors EP₂ and EP₄, the concentration of cervical hyaluronan, and the proportion of smooth muscle and collagen in the cervix of the hypogonadotrophic ovariectomized ewe (Ovis aries). Ovariectomized hypogonadotrophic ewes were given 100 μg estradiol benzoate, and their cervices were collected 0, 24, and 48 h thereafter to determine the expression of cervical COX-2, EP₂, and EP₄ mRNA by in situ hybridization, the concentration of hyaluronan by ELISA, and the proportion of smooth muscle and collagen by Masson's trichrome staining. Estradiol benzoate increased the mRNA expression of COX-2 and EP₄ within 24 h after treatment (P < 0.05), whereas EP₂ mRNA, hyaluronan, and the ratio of smooth muscle to collagen did not change within 48 h after treatment. The COX-2, EP₂, and EP₄ mRNA expression were greatest in the smooth muscle layers (P < 0.05) and least in the luminal epithelium (P < 0.05). In conclusion, we inferred that estradiol regulates cervical COX-2 and EP₄ mRNA expression and may regulate cervical relaxation via the synthesis of prostaglandin E₂ and activation of the PGE₂ receptors EP₂ and EP₄.     

Prawn lipocalin: characteristics and expressional pattern in subepidermal adipose tissue during reproductive molting cycle

In crustaceans, the fascinating processes of maturation, reproductive molting and carapace coloration are regulated by hydrophobic molecules. Interestingly, most of the molecules are ligands of lipocalin. To understand the role of lipocalin in the aforementioned processes at molecular level, we isolated a cDNA that belongs to the lipocalin family, from a central nervous system cDNA library of Macrobrachium rosenbergii. We monitored the spatial and temporal distributions of the mRNA by using Northern Blotting analysis. Our results demonstrated that this gene expresses abundantly in the subepidermal adipose tissue, while faintly in the hepatopancreas and central nervous system. However, no signal was detected in other tissues including muscle, gill and ovary. Its expression levels in subepidermal adipose tissue during various stages of maturation as well as through the whole molting cycle showed that prawn lipocalin is involved in sexual maturation, as the maximal level was observed just after molt.     

Tissue distribution of a menthyl-conjugated oligodeoxyribonucleotide antisense to PAI-1 mRNA

The inhibitory effect of numerous analogues of PO-16, an hexadecadeoxyribonucleotide antisense to sequences -22 to -17 of PAI-1 mRNA coding for a fragment of the signal peptide, on the expression of PAI-1 in endothelial cells, and physiological consequences of the subsequently reduced PAI-1 activity tested in vitro and in vivo, were described in our previous studies. Of particular interest was PO-16 5'-O-conjugated with menthyl phosphorothioate (MPO-16R). In this work, tissue localisation of MPO-16R labelled with [(35)S] phosphorothioate at the 3'-end, was determined. [(35)S]MPO-16R and control [(35)S]MPO-16R-SENSE oligonucleotides were administered intravenously into 22 rats and organ distribution of the labelled bioconjugates was assessed after 24 and 48 h. For this purpose, tissue sections were subjected to autoradiography, and quantitated by liquid scintillation after solubilisation. Overall clearance of radioactivity was already seen after 24 h, with the radioactivity recovered mainly in the kidney and liver. A smaller fraction of radioactivity was also retained in the spleen and heart. The kidney concentration of the labelled probe was higher than that of liver by 50%. The distribution of PAI-1 mRNA in untreated rat kidney, liver, spleen and heart established by two independent techniques: Ribonuclease Protection Assay and Real-Time PCR, shows the same pattern as that observed for [(35)S]MPO-16R antisense.