Strand Formation Research Articles

Effect of Molecular Crowding and Ionic Strength on the Isothermal Hybridization of Oligonucleotides

The isothermal hybridization of complementary oligonucleotides, 15-mer, 25-mer, 35-mer, and a molecular beacon, was investigated under varying conditions of molecular crowding and ionic strength, using hypochromicity to follow strand pairing and polyethylene glycol as a crowding agent. Thermodynamic analysis of the results revealed the addition of counterions to the oligonucleotide backbones, DeltaPsi, to be dependent on the strand GC content and the molecular crowding. A decrease in DeltaPsi was observed, with both increasing GC% and solution PEG content. In contrast, the number of bound water molecules depended on the activity of Na(+), where two regimes were observed. At a(Na(+)) < 0.05 and increasing molecular crowding, water molecules were released into the DNA solutions, and oligonucleotide pairing was favored with both increasing hydrophobic forces, whereas at a(Na(+)) >or= 0.05, water molecules were bound to the strands, and the extent of double strand formation decreased with increasing PEG wt %.

4-(4-Methoxyphenyl)-2-methylbut-3-yn-2-ol

The mol­ecular structure of the title compound, C12H14O2, features a nearly coplanar arrangement including the aromatic ring, the C C—C group and the ether O atom. The maximum deviation from the least-squares plane of these ten atoms is 0.0787 (8) Å for the ether O atom. In the crystal, mol­ecules are connected via O—H⋯O hydrogen bonds (involving the hy­droxy O atom both as hydrogen-bond donor and acceptor) and weaker (ar­yl)C—H⋯π(ar­yl) contacts, leading to the formation of strands running parallel to the b axis. Further stabilization results from weaker (meth­yl)C—H⋯π(acetyl­ene) inter­actions between different strands.

ROTEM and multiplate – a suitable tool for POC?

The early recognition of perioperative coagulation disorders is essential to identify non-surgical reasons of bleeding, to initiate appropriate haemostatic treatment and finally to reduce perioperative blood loss. Conventional laboratory coagulation tests include prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen levels and platelet counts. Although these parameters are frequently assessed, their value has been challenged at least in the setting of acute perioperative bleeding [1]. PT and aPTT are performed in an artificial ‘in vitro’ system, i.e. in plasma separated from whole blood, which is then warmed to 37 C and buffered to a pH of 7AE4. These standardized conditions often do not reflect the patient’s situation [2]. Plasmatic ‘in vitro’ tests merely reflect the time elapsing until the activation of thrombin, but provide only marginal information about the functional state of the coagulation system (e.g. platelet function, interaction of plasmatic coagulation factors with platelets and red blood cells, clot firmness, fibrinolysis). Routine coagulation tests are mostly performed in the central laboratory, i.e. remote from the operating theatre. As a consequence, test results are often not available in time or with a considerable delay [3]. Current devices designed for point-of-care (POC) monitoring of coagulation have been designed for the assessment of coagulation directly at the bedside, so that results could be available earlier. Moreover, POC tests are usually performed in whole blood, thereby comprising interactions between cellular components (i.e. platelets and red blood cells) and plasmatic coagulation factors. Commonly used POC devices for assessment of plasmatic coagulation and platelet function are listed in Table 1. This review focuses on rotation thrombelastography (ROTEM ) as a global coagulation test and multiple electrode aggregometry (MEA, Multiplate ) for assessment of platelet function. Rotation thrombelastometry (ROTEM )

Physical properties of acid milk gels prepared at 37°C up to gelation but at different incubation temperatures for the remainder of fermentation

We investigated the effect of altering temperature immediately after gels were formed at 37°C. We defined instrumentally measurable gelation (IMG) as the point at which gels had a storage modulus (G′) ≥5Pa. Gels were made at constant incubation temperature (IT) of 37°C up to IMG, and then cooled to 30 or 33.5, or heated to 40.5 or 44°C, at a rate of 1°C/min and maintained at those temperatures until pH 4.6. Control gel was made at 37°C (i.e., no temperature change during gelation/gel development). Gel formation was monitored using small strain dynamic oscillatory rheology, and the resulting structure and physical properties at pH 4.6 were studied by fluorescence microscopy, large deformation rheology, whey separation (WS), and permeability (B). A single strain of Streptococcus thermophilus was used to avoid variations in the ratios of strains that could have resulted from changes in temperature during fermentation. Total time required to reach pH 4.6 was similar for samples made at constant IT of 37°C or by cooling after IMG from 37 to either 30 or 33.5°C, but gels heated to 40 or 44°C needed less time to reach pH 4.6. Cooling gels after IMG resulted in an increase in G′ values at pH 4.6, a decrease in LTmax, WS, and B, and an increase in the area of protein aggregates of micrographs compared with the control gel made at constant IT of 37°C. Heating gels after IMG resulted in a decrease in G′ values at pH 4.6 and an increase in LTmax values and WS. The physical properties of acid milk gels were dominated by the temperature profile during the gel-strengthening phase that occurs after IMG. This study indicates that the final properties of yogurt greatly depend on the environmental conditions (e.g., temperature, time/rate of pH change) experienced by the casein particles/clusters during the critical early gel development phase when bonding between and within particles is still labile. Cooling of gels may encourage inter-cluster strand formation, whereas heating of gels may promote intra-cluster fusion and the breakage of strands between clusters.

CAMP via Epac/Rap1 enhanced microvascular endothelial barrier functions by Rac1‐mediated reorganization of adherens and tight junctions

cAMP stabilizes the endothelial barrier, in part by activation of Rac1. However, the effects on cell junctions on the ultrastructural level are largely unknown. Here, in human dermal microvascular endothelial cells, we used forskolin/rolipram (F/R) to increase cAMP as well as O‐Me‐cAMP to stimulate Epac/Rap 1 signaling. Under both conditions, endothelial barrier properties were enhanced as detected by increased transendothelial electrical resistance (TER) which was paralleled by activation of Rac 1. Compared to controls, electron microscopy revealed a significantly higher frequency of complex intercellular junctions containing both adherens and tight junctions which may reflect the increase in VE‐cadherin and claudin 5 immunostaining intensity at cell borders. In contrast, cleft length and intercellular overlap were not significantly reduced on the ultrastructural level but adherens and tight junction immunostaining appeared to be lineralized indicating recruitment of adhesion molecules to and formation of junction strands within the intercellular cleft. Barrier stabilization and junction reorganization in response to O‐Me‐cAMP were completely abolished in the presence of NSC‐23766 which specifically interferes with Rac1 activation. These results indicate that Rac1‐mediated junction reorganization was required for Epac/Rap1‐mediated cAMP‐induced barrier stabilization. DFG SFB688, A4

Modeling and Simulation of Rodlet Assembly of Hydrophobin EAS in an All-Atom Free-Energy Forcefield

In this investigation we simulate the rodlet assembly of an Class-I hydrophobin mutant EAS D15D8 in all-atom free-energy Monte-Carlo simulations. Results show that rodlet assembly is possible without additional beta strand formation in the flexible loop regions.Hydrophobins are small amphipathic proteins characterized by their unique bond pattern of eight Cysteine amino acids and a resulting similar tertiary structure, although only marginal sequence conservation exists within the family. In this work we elucidate the structure of the assembled rodlets in Class-I hydrophobins.The initial model of the EAS mutant is created using homology modeling of the solution structure of EAS allowing for a rigid structure omitting most of the flexible loop regions. A large population of possible docking structures is further generated using a generic protein-protein docking approach and filtered for candidates with distinct amphipathicity to account for the presence of an air-water interface.The resulting structures are further relaxed in the all-atom-free energy forcefield PFF02[1] using the POEM (Protein Optimization using Energy Methods) program package in parallel relaxation runs. Relaxation of the whole population was possible using the distributed volunteer computing platform POEM@HOME (http://boinc.fzk.de/).[1] A.Verma, W.Wenzel, A Free-Energy Approach for All-Atom Protein Simulation, Biophysical Journal, Volume 96, Issue 9, 2009, P. 3483

ADDITION OF THE FORCE MEASUREMENT CAPABILITY TO A COMMERCIALLY AVAILABLE EXTENSIONAL RHEOMETER (CABER)

The Capillary Breakup Extensional Rheometer (CaBER) is the only commercially available elongational rheometer. The CaBER can measure the diameter of a low viscous sample under stretching as a function of time. The analysis of this setup is consequently limited to the time dependent diameter of the capillaries D (t). Some samples do not develop a proper capillary flow, because of, for example, a lack of a substantial surface tension or a cylindrical fluid filament. As a consequence the interpretation of the results based on D (t) is problematic. On the other hand there is a huge fundamental industrial interest in measuring properties like strand formation, stickiness, spreadability, and so on. These properties are related to the extensional properties, specifically the elongational viscosity of a sample. Therefore, additional information beside D (t) is needed. In this publication a newly developed extension of the CaBER is presented and first applications are shown. The viscous sample is placed between two plates and afterward extended quickly by moving the upper plate. The normal force acting on the lower plate is measured as a function of time. To measure these small forces (<0.05 mN) with very short time resolution (e.g., dwell‐times down to 0.2 ms) a piezo transducer is used in combination with modern data oversampling techniques to further increase the sensitivity of the transducer. In this article the CaBER modifications are presented in detail to simultaneously measure the diameter and the force down to 5*10−5 N, with 10−3 s time resolution.

Biochemical Evaluation of Genotoxic Biomarkers for 2-Deoxyribonolactone-Mediated Cross-Link Formation with Histones

Numerous environmental carcinogens involve radical formation interacting with DNA to produce 2-deoxyribonolactone (dL), a major type of oxidized abasic site, implicated in DNA strand breaks, mutagenesis, and formation of covalent DNA–protein cross-links (DPC). Studies showed major dL-specific DPC occurred due to reactions with DNA polymerase β (Polβ) dependent on native conformation, while other DPC formed involved nonenzymatic reactions of DNA binding proteins with dL lesions. Polβ appeared to play a major role in alleviating the cytotoxic effects of neocarzinostatin, which was used as a dL-producing agent. When a duplex DNA containing a dL at a site-specific position was incubated with purified histones, DPC were formed between dL and each histone protein, including H1, H2A, H2B, H3, and H4. Comparative kinetic analysis of DPC formation with histones and Polβ revealed two distinct mechanisms of dL-mediated DPC formation. The rate of DPC formation with Polβ was approximately two orders of magnitude higher than that with various histone proteins. These results indicate that catalytic activity of Polβ mediates rapid DPC formation between dL and this DNA repair enzyme, whereas nonenzymatic reactions of dL with histones form DPC more slowly. The abundance of histones and their constant interaction with DNA may nevertheless yield significant levels of DPC with dL, as biomarkers of dL-induced cytotoxicity. Overall, data suggest that occurrence of dL-mediated DPC with histones may contribute to the genotoxic effects of dL in DNA.

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

Molecular Determinants of the Interaction between Clostridium perfringens Enterotoxin Fragments and Claudin-3

Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116-319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10-13, 15, 16, 18-20, and 22. Substitution peptide mapping identified the central motif (148)NPL(150)VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn(148) and Leu(150) of full-length claudin-3 in the binding. CPE-(116-319) and CPE-(194-319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116-319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116-319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116-319) and CPE-(194-319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.

Differential Effect of Extracellular Acidosis on the Release and Dispersal of Soluble and Membrane Proteins Secreted from the Weibel-Palade Body

Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pH(o)) on the release of von Willebrand factor (VWF), the VWF-propolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pH(o) 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pH(o) range. At pH(o) 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pH(o) 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pH(o) 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pH(o) 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.

Bis- and Tris(dimethylgallyl)benzenes: Synthesis, Solid-State Structures, and Redistribution Reactions

Benzene derivatives containing dimethylgallyl substituents in 1,3- (compounds 5 and 6), 1,4- (compound 9), and 1,3,5-position (compound 12) were prepared by reaction of the corresponding chloromercuriobenzenes with an excess of trimethylgallium at higher temperatures. These compounds decompose in solution at room temperature and in the solid state upon mild heating with elimination of trimethylgallium to give oligomeric condensation products of unknown detailed composition. These condensation products can be transformed back into the starting compounds by treatment with an excess of trimethylgallium at higher temperatures. Highly air-sensitive crystals of 5, 6, 9, and 12 suitable for an X-ray analysis are obtained from trimethylgallium as solvent. The X-ray crystallographic studies revealed the presence of higher coordinate gallium atoms, which lead to the formation of strand- or sheet-like polymers. A trigonal-bipyramidal coordination sphere is observed for the gallium atoms in 9. A distorted tetrahedral coordination is found for the gallium atoms in 5, 6, and 12. The latter compounds possess asymmetric aryl-dimethylgallyl bridging units.

Selection for the G4 DNA motif at the 5′ end of human genes

Formation of G4 DNA may occur in the course of replication and transcription, and contribute to genomic instability. We have quantitated abundance of G4 motifs and potential for G4 DNA formation of the nontemplate strand of 5' exons and introns of transcripts of human genes. We find that, for all human genes, G4 motifs are enriched in 5' regions of transcripts relative to downstream regions; and in 5' regulatory regions relative to coding regions. Notably, although tumor suppressor genes are depleted and proto-oncogenes enriched in G4 motifs, abundance of G4 motifs in the 5' regions of transcripts of genes in these categories does not differ. These results support the hypothesis that G4 motifs are under selection in the human genome. They further show that for tumor suppressor genes and proto-oncogenes, independent selection determines potential for G4 DNA formation of 5' regulatory regions of transcripts and downstream coding regions.

Claudin-11 is over-expressed and dislocated from the blood–testis barrier in Sertoli cells associated with testicular intraepithelial neoplasia in men

In mouse testis, claudin-11 is responsible for the formation of specific parallel TJ strands of the blood-testis barrier (BTB). Concerning the human BTB, there is no information about the transmembrane TJ proteins. We recently demonstrated the loss of functional integrity of the BTB in testicular intraepithelial neoplasia (TIN), associated with a dislocation of the peripheral TJ proteins ZO-1 and ZO-2. Here, we determined the expression and distribution of claudin-11 at the human BTB in seminiferous tubules with normal spermatogenesis (NSP) and TIN. Immunostaining of claudin-11 revealed intense signals at the basal BTB region in seminiferous epithelium with NSP. Within TIN tubules, claudin-11 immunostaining became diffuse and cytoplasmic. Double immunogold labeling demonstrated a co-localization of claudin-11 and ZO-1 at the inter-Sertoli cell junctions. Real-time RT-PCR of laser microdissected tubules showed an up-regulation of claudin-11 mRNA in TIN. Additionally, increased claudin-11 protein was observed by Western blot. We conclude that claudin-11 constitutes a TJ protein at the human BTB. In TIN tubules, claudin-11 is up-regulated and dislocated from the BTB. Therefore, the disruption of the BTB is related to a dysfunction of claudin-11 and not to a failure of its expression.

Integration of transport-based models for phyllotaxis and midvein formation.

The plant hormone auxin mediates developmental patterning by a mechanism that is based on active transport. In the shoot apical meristem, auxin gradients are thought to be set up through a feedback loop between auxin and the activity and polar localization of its transporter, the PIN1 protein. Two distinct molecular mechanisms for the subcellular polarization of PIN1 have been proposed. For leaf positioning (phyllotaxis), an "up-the-gradient" PIN1 polarization mechanism has been proposed, whereas the formation of vascular strands is thought to proceed by "with-the-flux" PIN1 polarization. These patterning mechanisms intersect during the initiation of the midvein, which raises the question of how two different PIN1 polarization mechanisms may work together. Our detailed analysis of PIN1 polarization during midvein initiation suggests that both mechanisms for PIN1 polarization operate simultaneously. Computer simulations of the resulting dual polarization model are able to reproduce the dynamics of observed PIN1 localization. In addition, the appearance of high auxin concentration in our simulations throughout the initiation of the midvein is consistent with experimental observation and offers an explanation for a long-standing criticism of the canalization hypothesis; namely, how both high flux and high concentration can occur simultaneously in emerging veins.

Formation of Strands After Valve Replacement

Fibrin strands are long, thin, mildly echogenic and mobile structures that are usually incidental findings after prosthetic heart valve surgery. They are seen by transesophageal echocardiography in 6% to 45% of patients. The formation of these benign strands may be the result of cardiac manipulation by surgical instrumentation. A case study of strand formation after aortic valve replacement is detailed. In any valvular lesion, differential diagnoses include thrombus and vegetation, and thus the clinical picture is important in diagnosing and managing valvular lesions. Although strands rarely embolize and are not associated with poor prognosis, their management and significance are items of debate.

Numerical study and experimental validation of particle strand formation

Pneumatic conveying of spherical glass particles through a rectangular channel is studied by means of numerical simulation and compared with optical measurements. Thereby, a double-looping is placed in front of the straight channel in order to generate a particle strand at the bottom of the channel. Finally the profiles of particle velocity and volume concentration are measured by Particle Image Velocimetry (PIV). The corresponding numerical simulations are carried out with the Discrete Phase Model using the Fluent software package. Also some additional sub-models have been introduced in order to describe particle-wall collisions, particle-particle collisions and the influence of particle rotation.

Competitive interactions in crystalline 9-pyridyl-9-fluorenols: crossover from O–H⋯O to O–H⋯N hydrogen bonding to construct intra- and intermolecular, helical and linear contact modes

In order to find out the consequences of a phenyl for pyridyl exchange on the crystalline packing behaviour of the parent compound 9-phenyl-9-fluorenol, three analogous 9-pyridyl substituted 9-fluorenols featuring the pyridyl ring in different linkage positions were comparatively studied with regard to their crystal structures. While the molecular structures in the crystal are not noticeably altered, the network structures are, showing the leading influence of the nitrogen atom to cause a variation of contact modes. Corresponding to the substitution site of the pyridyl group, these involve intra- and intermolecular hydrogen bonds, including helical and linear strand formation.

Heparanase 1: a key participant of inner root sheath differentiation program and hair follicle homeostasis

Heparanase is a heparan sulphate endo-glycosidase which was previously detected in the outer root sheath of murine hair follicles. Heparanase overexpression was reported to improve mouse hair (re)growth. In this study, we investigated its involvement in human hair biology. Immunofluorescence detection was used to explore heparanase distribution in both anagen and catagen hair follicles. Heparanase functionality was assessed in in vitro cultured hair follicles, in the presence of a heparanase activity inhibitor. Our results showed that heparanase expression was (i) primarily located in the inner root sheath (IRS) of human hair follicle, and there (ii) restricted to anagen phase. Furthermore, inhibition of heparanase in in vitro cultured hair follicles induced a catagen-like process. Hair shaft retreat upward was accompanied by a decrease in Ki67-positive cells, the formation of an epithelial strand as evidenced by K14 keratin expression, and the loss of IRS as assessed by transglutaminase 1 and desmoglein labelling. IRS distribution of heparanase and the induction of catagen-like involution of hair follicles when a potent heparanase inhibitor is added suggest that heparanase is a key actor of IRS differentiation and hair homeostasis.

Stress singularities and the formation of birefringent strands in stagnation flows of dilute polymer solutions

We consider stagnation point flow away from a wall for creeping flow of dilute polymer solutions. For a simplified flow geometry, we explicitly show that a narrow region of strong polymer extension (a birefringent strand) forms downstream of the stagnation point in the UCM model and extensions, like the FENE-P model. These strands are associated with the existence of an essential singularity in the stresses, which is induced by the fact that the stagnation point makes the convective term in the constitutive equation into a singular point. We argue that the mechanism is quite general, so that all flows that have a separatrix going away from the stagnation point exhibit some singular behaviour. These findings are the counterpart for wall stagnation points of the recently discovered singular behaviour in purely elongational flows: the underlying mechanism is the same while the different nature of the singular stress behaviour reflects the different form of the velocity expansion close to the stagnation point.