Modulation of intramolecular interactions in superhelical DNA by curved sequences: a Monte Carlo simulation study.

A Monte Carlo model for the generation of superhelical DNA structures at thermodynamic equilibrium (Klenin et al., 1991; Vologodskii et al., 1992) was modified to account for the presence of local curvature. Equilibrium ensembles of a 2700-bp DNA chain at linking number difference delta Lk = -15 were generated, with one or two permanent bends up to 120 degrees inserted at different positions. The computed structures were then analyzed with respect to the number and positions of the end loops of the interwound superhelix, and the intramolecular interaction probability of different segments of the DNA. We find that the superhelix structure is strongly organized by permanent bends. A DNA segment with a 30 degrees bend already has a significantly higher probability of being at the apex of a superhelix than the control, and for a 120 degrees bend the majority of DNAs have one end loop at the position of the bend. The entropy change due to the localization of a 120 permanent bend in the end loop is estimated to be -17 kJ mol-1 K-1. When two bends are inserted, the conformation of the superhelix is found to be strongly dependent on their relative positions: the straight interwound form dominates when the two bends are separated by 50% of the total DNA length, whereas the majority of the superhelices are in a branched conformation when the bends are separated by 33%. DNA segments in the vicinity of the permanent bend are strongly oriented with respect to each other.     

Steering with the head. the visual strategy of a racing driver

We studied the eye movements of a racing driver during high-speed practice to see whether he took in visual information in a different way from a normal driver on a winding road [1, 2]. We found that, when cornering, he spent most of the time looking close to, but not exactly at, the tangent points on the inside edges of the bends. Each bend was treated slightly differently, and there was a highly repeatable pattern to the way the track edge was viewed throughout each bend. We also found a very close relationship between the driver's head direction and the rate of rotation of the car 1 s later. We interpret these observations as indicating that the driver's gaze is not driven directly by tangent point location, as it is in ordinary driving. Instead, we propose that his head direction is driven by the same information that he uses to control steering and speed, namely his knowledge of the track and his racing line round it. If he directs his head at an angle proportional to his estimate of car rotation speed, this will automatically bring his head roughly into line with the tangent points of the bends. From this standardized position, he can use the expected movements of the tangent points in his field of view to verify, and if necessary modify, his racing line during the following second.     

A novel motility pattern in quail spermatozoa with implications for the mechanism of flagellar beating

Background information. The spermatozoon of the quail (Coturnix coturnix L., var japonica) has a ‘9+2’ flagellum that is unusually long. When it moves in a viscous medium, near to the coverslip, it develops a meander waveform. Because of the high viscosity, the meander bends are static in relation to the field of view; bend propagation is therefore manifest as the forward movement of the flagellum through the meander shape. At the same time, the origin of the oscillation typically shifts proximally in a stepwise fashion. These movements have been analysed in the hope of contributing to the resolution of problems in flagellar mechanics. Results. (1) Meander waves originate from spontaneous sigmoid bend complexes. (2) On a given flagellum, fully developed meander bends are uniform in their large angle, curvature and propagation speed; interbends can vary in length and shape. (3) No intra‐axonemal sliding is transmitted through formed bends; sliding related to new bends is accommodated proximally. (4) Sliding reversal is initiated at a threshold shear angle of approx. 1 rad. (5) The arc wavespeed is the product of the arc wavelength and the beat frequency. (6) Physical obstruction to bend development causes a pause in the oscillation. (7) New bend initiation can thus be dissociated from bend propagation on the distal flagellum. (8) The steps in the forward advance of the oscillation site occur during the early phase of bend growth. Conclusions. (1) The main conclusion is that, in meander waves, the mechanical basis of the oscillation appears to be that the propulsive thrust arising from bend propagation acts as a bending stress to trigger sliding reversal, thus perpetuating the rhythmic beating. (2) Oscillations can originate at any position, provided the position is distal to a location where doublet sliding is restrained. (3) Meander waves are an example of new bend development without ‘paradoxical’ classes of sliding.     

Beta-turns in proteins

The X-ray atomic co-ordinates from 29 proteins of known sequence and structure were utilized to elucidate 459 β-turns in regions of chain reversals. Tetrapeptides whose αC_iαC_{(i + 3)} distances were below 7 Å and not in a helical region were characterized as β-turns. In addition, β-turns were considered to have hydrogen bonding if their computed O_(i)N_{(i + 3)} distances were ≤3.5 Å. The torsion angles of 26 proteins containing 421 β-turns were examined and classified into 11 bend types based on the (φ, ψ) dihedral angles of the i + 1 and i + 2 bend residues. The average frequency of β-turns is 32% as compared to the 38% helices and 20% β-sheets in the 29 proteins. The most frequently occurring bend residues are Asn, Cys, Asp in the first position, Pro, Ser, Lys in the second position, Asn, Asp, Gly in the third position, and Trp, Gly, Tyr in the fourth position. Residues with the highest β-turn potential in all four positions are Pro, Gly, Asn, Asp, and Ser with the most hydrophobic residues (i.e. Val, IIe, and Leu) showing the lowest bend potential. However, in the region just beyond the β-turns, hydrophobic residues occur with greater frequency than do hydrophilic residues. An environmental analysis of β-turn neighboring residues shows that reverse chain folding is stabilized by anti-parallel β-sheets as well as helix-helix and α-β interactions. The β-turn potential at the 12 positions adjacent to and including the bend were plotted for the 20 amino acids and showed dramatic positional preferences, which may be classified according to the nature of the side-chains. An examination of the 27 β-turns in elastase showed that 21 were found in identical positions as those in α-chymotrypsin. However, only 37 of the 84 bend residues were conserved, indicating that structural similarity may persist despite differences in sequence homology. A survey of residues occupying bend types I′, II′ and III′ showed that Gly appeared most frequently in the third position in bend types I′ and III′ as well as in the second position in bend types II′ and III′. Fourteen hydrogenbonded type II bends were found without a Gly at the third position, contrary to the energy calculations. Eight type VI bends with a cis Pro at the third position were also elucidated.     

Negative staining of myosin molecules

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.     

A Unidirectional Cell Switching Gate by Engineering Grating Length and Bending Angle

On a microgrooved substrate, cells migrate along the pattern, and at random positions, reverse their directions. Here, we demonstrate that these reversals can be controlled by introducing discontinuities to the pattern. On “V-shaped grating patterns”, mouse osteogenic progenitor MC3T3-E1 cells reversed predominately at the bends and the ends. The patterns were engineered in a way that the combined effects of angle- and length-dependence could be examined in addition to their individual effects. Results show that when the bend was placed closer to one end, migration behaviour of cells depends on their direction of approach. At an obtuse bend (135°), more cells reversed when approaching from the long segment than from the short segment. But at an acute bend (45°), this relationship was reversed. Based on this anisotropic behaviour, the designed patterns effectively allowed cells to move in one direction but blocked migrations in the opposing direction. This study demonstrates that by the strategic placement of bends and ends on grating patterns, we can engineer effective unidirectional switching gates that can control the movement of adherent cells. The knowledge developed in this study could be utilised in future cell sorting or filtering platforms without the need for chemotaxis or microfluidic control.     

Cycling around a Curve: The Effect of Cycling Speed on Steering and Gaze Behavior

Although it is generally accepted that visual information guides steering, it is still unclear whether a curvature matching strategy or a ‘look where you are going’ strategy is used while steering through a curved road. The current experiment investigated to what extent the existing models for curve driving also apply to cycling around a curve, and tested the influence of cycling speed on steering and gaze behavior. Twenty-five participants were asked to cycle through a semicircular lane three consecutive times at three different speeds while staying in the center of the lane. The observed steering behavior suggests that an anticipatory steering strategy was used at curve entrance and a compensatory strategy was used to steer through the actual bend of the curve. A shift of gaze from the center to the inside edge of the lane indicates that at low cycling speed, the ‘look where you are going’ strategy was preferred, while at higher cycling speeds participants seemed to prefer the curvature matching strategy. Authors suggest that visual information from both steering strategies contributes to the steering system and can be used in a flexible way. Based on a familiarization effect, it can be assumed that steering is not only guided by vision but that a short-term learning component should also be taken into account.     

Saturation mutagenesis of a DNA region of bend. Base steps other than ApA influence the bend

A 60 base-pair region of a simian virus 40 DNA fragment was mutagenized to determine base-pairs that are critical for the fragment to bend. The site-directed mutagenesis saturated this region with all possible single base-pair substitutions. The mobility of each mutated fragment was measured by polyacrylamide electrophoresis at 4 degrees C and at 65 degrees C to assess the degree of bend. Four conclusions can be drawn. First, interruptions within the A tracts and changes in the phasing of the A tracts alter the degree of bend. Second, G tracts phased at a half-helical turn from an A tract are additive to the bend. Third, guanine residues in a nearest-neighbor contact with the A tracts modify the bend. Fourth, some mutations that do not obviously relate to the A tracts also alter the DNA bend and suggest clearly that base steps other than ApA are involved in sequence-directed DNA bends.     

Assessing the rider's seat and horse's behavior: difficulties and perspectives

A correct seat and position are the basis for a good performance in horseback riding. This study aimed to measure deviations from the correct seat, test a seat improvement program (dismounted exercises), and investigate whether horse behavior was affected by the rider's seat. Five experienced trainers defined 16 seat deviations and scored the occurrence in 20 riders in a dressage test. Half the riders then carried out an individual training program; after 9 weeks, riders were again scored. The study took no video or heart-rate recordings of horses and riders. Panel members did not agree on the deviations in the rider's seat; the study detected no differences-with the exception of improvement of backward-tilted pelvis-between the groups. Horse behavior, classified as "evasive," increased; horse heart rate decreased in the experimental group. Heart rates of riders in both groups decreased. Seven of 9 riders in the experimental group had the impression that the exercises improved their riding performance. There is a clear need to develop a robust system that allows trainers to objectively evaluate the rider's seat.     

Cre induces an asymmetric DNA bend in its target loxP site

Cre initiates recombination by preferentially exchanging the bottom strands of the loxP site to form a Holliday intermediate, which is then resolved on the top strands. We previously found that the scissile AT and GC base pairs immediately 5' to the scissile phosphodiester bonds are critical in determining this order of strand exchange. We report here that the scissile base pairs also influence the Cre-induced DNA bends, the position of which correlates with the initial site of strand exchange. The binding of one Cre molecule to a loxP site induces a approximately 35 degrees asymmetric bend adjacent to the scissile GC base pair. The binding of two Cre molecules to a loxP site induces a approximately 55 degrees asymmetric bend near the center of the spacer region with a slight bias toward the scissile A. Lys-86, which contacts the scissile nucleotides, is important for establishing the bend near the scissile GC base pair when one Cre molecule is bound but has little role in positioning the bend when two Cre molecules are bound to a loxP site. We present a model relating the position of the Cre-induced bends to the order of strand exchange in the Cre-catalyzed recombination reaction.     

Factors Associated with Injuries among Commercial Motorcyclists: Evidence from a Matched Case Control Study in Kampala City,Uganda

Introduction

Road traffic injuries are the eighth leading cause of death globally and the most affected are young people aged 15–29. By 2030 road traffic deaths will become the fifth leading cause of death unless urgent action is taken. Motorcyclists are among the most vulnerable road users and in Uganda they contribute 41% of all road traffic injuries. This paper establishes factors associated with the injuries of commercial motorcycle riders also known as boda-boda riders in Kampala, Uganda’s capital city.

Methods

The study was matched case-control with a case being a boda-boda rider that was seen at one of the 5 major city hospitals with a road traffic injury while a control was a boda-boda rider that was at the parking stage where the case operated from before the injury. The sample size was 289 riders per arm and data collection took 7 months. A structured questionnaire was used to collect data on background and exposing factors. Being matched case-control data conditional logistic regression was used in the analysis.

Results

Factors independently associated with injury among motorcyclists were younger age group, being a current alcohol drinker (OR = 2.30, 95%CI: 1.19–4.45), lower engine capacity (<100cc)(OR = 5.03, 95%CI: 2.91–8.70), riding experience of less than 3 years, not changing a motorcycle in past 1 year (OR = 2.04, 95%CI: 1.19–3.52), riding for a longer time in a day (OR = 6.05, 95%CI: 2.58–14.18) and sharing a motorcycle (OR = 8.25, 95%CI:2.62–25.9). Other factors associated with injury were low level of knowledge of traffic rules, being stopped by police for checks on condition of motorcycle/license/insurance, working till late.

Recommendations

More road safety sensitization is required among riders to raise awareness against sharing motorcycles, working for a longer time and alcohol consumption. Police enforcement of drink-driving laws should include riders of commercial motorcycles. Investigate the validity of motorcycle riding licenses and test the riding competency of all who got licenses in last 3 years.     

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.     

Transient Pinning and Pulling: A Mechanism for Bending Microtubules

Microtubules have a persistence length of the order of millimeters in vitro, but inside cells they bend over length scales of microns. It has been proposed that polymerization forces bend microtubules in the vicinity of the cell boundary or other obstacles, yet bends develop even when microtubules are polymerizing freely, unaffected by obstacles and cell boundaries. How these bends are formed remains unclear. By tracking the motions of microtubules marked by photobleaching, we found that in LLC-PK1 epithelial cells local bends develop primarily by plus-end directed transport of portions of the microtubule contour towards stationary locations (termed pinning points) along the length of the microtubule. The pinning points were transient in nature, and their eventual release allowed the bends to relax. The directionality of the transport as well as the overall incidence of local bends decreased when dynein was inhibited, while myosin inhibition had no observable effect. This suggests that dynein generates a tangential force that bends microtubules against stationary pinning points. Simulations of microtubule motion and polymerization accounting for filament mechanics and dynein forces predict the development of bends of size and shape similar to those observed in cells. Furthermore, simulations show that dynein-generated bends at a pinning point near the plus end can cause a persistent rotation of the tip consistent with the observation that bend formation near the tip can change the direction of microtubule growth. Collectively, these results suggest a simple physical mechanism for the bending of growing microtubules by dynein forces accumulating at pinning points.     

Hyperactivation is the mode conversion from constant-curvature beating to constant-frequency beating under a constant rate of microtubule sliding

Flagellar beating of hyperactivated golden hamster spermatozoa was analyzed in detail using digital image analysis and was compared to that of nonhyperactivated (activated) spermatozoa in order to understand the change in flagellar beating during hyperactivation and the active microtubule sliding that brought about the change in flagellar beating. Hyperactivated flagellar beating, which was characterized by a sharp bend in the proximal midpiece and low beat frequency, was able to alter the waveform with little change in beat frequency (constant-frequency beating), whereas activated flagellar beating, which was characterized by a slight bend in the proximal midpiece and high beat frequency, was able to alter beat frequency with little change in the waveform (constant-curvature beating). These results demonstrate that flagellar beating of hyperactivated and activated spermatozoa were essentially different modes and that hyperactivation was the mode conversion from constant-curvature beating to constant-frequency beating. Detailed analysis of flagellar bends revealed that the increase in curvature in the proximal midpiece during hyperactivation was due to the increase in total length of microtubule sliding in a nearly straight region between bends, while the rate of microtubule sliding remained almost constant.     

Skip residues correlate with bends in the myosin tail

Sharp bends have previously been observed in the tail of the skeletal myosin molecule at well-defined positions 44, 75 and 135 nm from the head-tail junction, and in vertebrate smooth myosin at two positions about 45 and 96 nm from this junction. The amino acid sequence of the heavy chain does not straightforwardly account for such bending on the original model of the tail in which an invariant proline residue is present at the head-tail junction and the repeating seven amino acid pattern of hydrophobic residues lies entirely in the tail. Recently, a revised model has been proposed by Rimm et al. in which the first seven to eight heptads lie in the heads. It is shown here that with this model the observed bends in the tail of skeletal myosin coincide with three of the four additional (skip) residues that interrupt the heptad repeat. It is concluded that the skip residues, by causing localized instability of the coiled-coil, are responsible for the bends. Smooth myosin lacks the second of these skip residues explaining the absence of a bend at 75 nm.     

Bends in human mitotic metaphase chromosomes, including a bend marking the X-inactivation center.

Bends in mitotic metaphase chromosomes are not distributed randomly throughout the karyotype. The frequency of bends at centromeres is positively correlated with the relative length of the chromosomes and negatively correlated with the centromere index (more bends in metacentrics, fewer in acrocentrics). The frequency of bends in the noncentromeric regions (except at Xq13-Xq21) is positively correlated with the relative length of chromosome arms. A bend at Xq13.3 to Xq21.1 was more frequent than a bend in any other region of the karyotype, centromeric or noncentromeric. It was observed in one member of the X-chromosome pair in 63% of 46,XX cells. In contrast, it was observed in only 2% of 46,XY cells. RBG-staining showed that this specific bend is confined to the lyonized X chromosome. These observations in cells from normal subjects were confirmed using G-banding and RBG-staining on cells from nine subjects with different X-chromosome abnormalities and on metaphases from amniotic fluid cell and lymphocyte cultures. The "center for Barr body condensation" has been localized to the region between Xq11.2 and Xq21.1. The functional and structural relationship is unclear, but we believe this highly specific bend may represent a visible manifestation of the condensation process; it could represent the first folded (and last unfolded) position, upon or around which the rest of the chromosome condenses. The late replication of this region may also be a factor. The smallest region of overlap (SRO) for the X-chromosome inactivation center and the specific chromosome bend is Xq13.3 to Xq21.1.     

Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa

Caudal epididymal spermatozoa of golden hamsters were incubated in capacitation medium. Their movement patterns changed as they became hyperactivated and underwent the acrosome reaction. To understand the basic mechanism by which changes in movement pattern are brought about, digital image analysis was carried out on the flagellar movements recorded with a video system. The degree of flagellar bending increased with incubation time, especially in the proximal midpiece. The hyperactivated spermatozoa had remarkably asymmetrical flagellar waves of large amplitude because either the bends in the same direction as the hook of the head (referred as the "pro-hook bend") or the bends in the opposite direction to the hook of the head (referred as the "anti-hook bend") extremely increased their curvature; whereas, the acrosome-reacted spermatozoa had relatively symmetrical flagellar waves of large amplitude because both the pro- and anti-hook bends remarkably increased their curvature. Beat frequency significantly decreased while wavelength of flagellar waves increased after hyperactivation and further after the acrosome reaction. These results suggest that both extreme pro- and anti-hook bends are essential in the acrosome-reacted spermatozoa even though beat frequency decreased markedly.     

Intrinsically bent DNA in the promoter regions of the yeast GAAL1-10 and GAL80 genes

Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1-10 and GAL80 genes. These bends lie in functionally suggestive locations. On the promoter of the GAL1-10 structural genes, strong bends bracket nucleosome B, which lies between the UAS(G) and the GAL1 TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1-10 expression is induced. The strongest intrinsic bend ( approximately 70 degrees ) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it to be involved in GAL1 activation in several ways, including the recruitment of a yeast HMG protein that is required for the normally robust level of GAL1 expression in the induced state (Paull, T., Carey, M., and Johnson, R. (1996) Genes Dev. 10, 2769-2781). On the regulatory gene GAL80, the single bend lies in the non-nucleosomal hypersensitive region, between a GAL80-specific far upstream promoter element and the more gene-proximal promoter elements. GAL80 promoter region nucleosomes contain no intrinsically bent DNA.     

Form of developing bends in reactivated sperm flagella.

1. Dark-field, multiple-exposure photographs of reactivated tritonated sea urchin sperm flagella swimming under a variety of conditions were analysed. 2. The length, radius and subtended angle of bends increased during bend development. The pattern of development was essentially the same under all conditions observed. 3. The angles of the two bends nearest the base tend to increase at the same rate, cancelling one another, so that the development of new bends causes little if any net microtubular sliding. 4. The direction of microtubular sliding within a bend is initially in the same direction as that within the preceding bend, and reverses as the bend develops.