Preferential Loading Research Articles

Case Study on Failure of Ball Bearing of an Aeroengine

Heavy damage was reported on the outer race and rolling elements of a ball bearing belonging to jet engine of a military aircraft. Flaking type of damage was noticed only at one particular location on the outer race of the bearing and on almost all the balls of the bearing. The inner race and the cage of the bearing were free from any damage. The chemistry, hardness, and microstructure of the bearing components met the specification requirements. In the actual engine assembly, the bearing was mounted on shaft of bevel gear. Careful examination of the inner diameter (ID) of the inner ring of the bearing revealed nonuniform contact marks. The contact marks were observed in the form of discoloration of the ID surface. The nonuniform contact was attributed to eccentricity caused by improper mounting of the bearing onto the shaft of the bevel gear. This resulted in preferential loading, causing flaking at one particular location on the raceway of the outer ring and damage to the balls of the bearing. Hence, it was concluded that the ball bearing had failed because of assembly error after the last repair of the engine.

Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-target Silencing

Off-target gene silencing is a major concern when using RNA interference. Imperfect pairing of the antisense strand with unintended mRNA targets is one of the main causes of small interfering RNA (siRNA) off-target silencing. To overcome this, we have developed "bulge-siRNA," a modified siRNA backbone structure with a single nucleotide (nt) bulge placed in the antisense strand. We found that siRNAs with a bulge at position 2 of the antisense strand were able to discriminate better between perfectly matched and mismatched targets, with no loss in silencing of the intended target. Genome-wide analysis also revealed that the bulge-siRNAs significantly reduced off-target silencing of transcripts with complementarity to the seed region of the siRNA antisense strand. When compared to 2'-methoxy ribosyl (2'-OMe) modified siRNAs previously developed to alleviate antisense off-target silencing; the bulge modification could better discriminate between on- versus off-targets. Our results suggest that the bulge-siRNA structure is a simple, yet superior alternative to chemical modifications for minimizing off-target silencing triggered by conventional siRNA structures.

A Preliminary Research on Operation Mode of Condensing Heat Recovery Hot Water Supply System for Water-Cooled Chillers

The objective of the present paper is to investigate the condenser heat recovery in water-cooled chiller system. According to the technology characteristics of vapor compression refrigerating cycle, the viewpoint of condensing heat reclaim was proposed, and heat recovery system can be used in buildings. Air-cooled and water-cooled chillers with heat reclaim capability were discussed. Three optimized water system designs were introduced for more heat recovery or higher heating water temperature. The results show that it is very useful for designing and developing the new heat recovery unit with high efficiency, energy conservation, and environmental protection by analyzing the feature feasibility of condenser heat recovery system. The control of entering-condenser water temperature is recommended for higher chiller efficiency and operating steadily, the water system designs of preferential loading and side stream are for more heat recovery.

Target-Mediated Protection of Endogenous MicroRNAs in C. elegans

MicroRNAs (miRNAs) are tightly regulated through transcriptional and posttranscriptional mechanisms, including degradation by nucleases. Here, we report that in C. elegans, target mRNAs can protect their cognate miRNAs from degradation invivo. We show that the let-7(n2853) mutation destabilizes the mature let-7 miRNA by impairing this protection. Moreover, presence of a cognate target or depletion of the xrn-1 (XRN1) or xrn-2 (XRN2/Rat1p) exoribonucleases enforces accumulation of certain miRNA passenger (miR(∗)) strands. Thus, following biased miRNA strand loading into Argonaute, elimination of nonfunctional RNAs can further refine miRNA strand selection. Conversely, by aligning the levels of miRNAs with those of their targets, the opposing activities of mature miRNA degradation and target-mediated miRNA protection (TMMP) may enable dynamic expression of either mature strand ofa pre-miRNA, and evolution of miRNAs. Thus, it seems that mRNAs are more than inert targets and function with miRNAs in a network of mutual regulation.

Cycas micronesica (Cycadales) plants devoid of endophytic cyanobacteria increase in β-methylamino- l-alanine

Cycads are among the most ancient of extant Spermatophytes, and are known for their pharmacologically active compounds. β-methylamino- l-alanine (BMAA) is one metabolite that been implicated as causal of human neurodegenerative diseases in Guam. We grew Cycas micronesica seedlings without endophytic cyanobacteria symbiosis, and quantified initial and ending BMAA in various plant tissues. BMAA increased 79% during nine months of seedling growth, and root tissue contained 75% of the ultimate BMAA pool. Endophytic cyanobacteria symbionts were not the source of BMAA increase in these seedlings, which contradicts previously reported claims that biosynthesis of this toxin by cyanobacteria initiates its accumulation in the Guam environment. The preferential loading of root tissue with BMAA does not support earlier reports that this toxin serves a defensive role against herbivory of leaf or seed tissues. The long history of conflicting results in Guam’s cycad toxin research continues, and recent developments underscore the sense of urgency in continued research as this endangered cycad population approaches extirpation from the island.

The Clamp Loader Assembles the β Clamp onto Either a 3′ or 5′ Primer Terminus: THE UNDERLYING BASIS FAVORING 3′ LOADING

Clamp loaders assemble sliding clamps onto 3' primed sites for DNA polymerases. Clamp loaders are thought to be specific for a 3' primed site, and unable to bind a 5' site. We demonstrate here that the Escherichia coli gamma complex clamp loader can load the beta clamp onto a 5' primed site, although with at least 20-fold reduced efficiency relative to loading at a 3' primed site. Preferential clamp loading at a 3' site does not appear to be due to DNA binding, as the clamp loader forms an avid complex with beta at a 5' site. Preferential loading at a 3' versus a 5' site occurs at the ATP hydrolysis step, needed to close the ring around DNA. We also address DNA structural features that are recognized for preferential loading at a 3' site. Although the single-stranded template strand extends in opposite directions from 3' and 5' primed sites, thus making it a favorite candidate for distinguishing between 3' and 5' sites, the single-strand polarity at a primed template junction does not determine 3' site selection for clamp loading. Instead, we find that clamp loader recognition of a 3' site lies in the duplex portion of the primed site, not the single-strand portion. We present evidence that the beta clamp facilitates its own loading specificity for a 3' primed site. Implications to eukaryotic clamp loader complexes are proposed.

Regulated and Multiple miRNA and siRNA Delivery Into Primary Cells by a Lentiviral Platform

RNA interference (RNAi) has tremendous potential for investigating gene function and developing new therapies. However, the design and validation of proficient vehicles for stable and safe microRNA (miR) and small interfering RNA (siRNA) delivery into relevant target cells remains an active area of investigation. Here, we developed a lentiviral platform to efficiently coexpress one or more natural/artificial miR together with a gene of interest from constitutive or regulated polymerase-II (Pol-II) promoters. By swapping the stem-loop (sl) sequence of a selected primary transcript (pri-miR) with that of other miR or replacing the stem with an siRNA of choice, we consistently obtained robust expression of the chimeric/artificial miR in several cell types. We validated our platform transducing a panel of engineered cells stably expressing sensitive reporters for miR activity and on a natural target. This approach allowed us to quantitatively assess at steady state the target suppression activity and expression level of each delivered miR and to compare it to those of endogenous miR. Exogenous/artificial miR reached the concentration and activity typical of highly expressed natural miR without perturbing endogenous miR maturation or regulation. Finally, we demonstrate the robust performance of the platform reversing the anergic/suppressive phenotype of human primary regulatory T cells (Treg) by knocking-down their master gene Forkhead Transcription Factor P3 (FOXP3).

Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference activity and strand selection

Short interfering RNAs (siRNAs) are valuable reagents for sequence-specific inhibition of gene expression via the RNA interference (RNAi) pathway. Although it has been proposed that the relative thermodynamic stability at the 5′-ends of siRNAs plays a crucial role in siRNA strand selection, we demonstrate here that a character of the 2-nt 3′-overhang of siRNAs is the predominant determinant of which strand participates in the RNAi pathway. We show that siRNAs with a unilateral 2-nt 3′-overhang on the antisense strand are more effective than siRNAs with 3′-overhangs at both ends, due to preferential loading of the antisense strand into the RNA-induced silencing complex (RISC). Regardless of the relative thermodynamic stabilities at the ends of siRNAs, overhang-containing strands are predominantly selected as the guide strand; whereas, relative stability markedly influences opposite strand selection. Moreover, we show that sense strand modifications, such as deletions or DNA substitutions, of siRNAs with unilateral overhang on the antisense strand have no negative effect on the antisense strand selection, but may improve RNAi potency. Our findings provide useful guidelines for the design of potent siRNAs and contribute to understanding the crucial factors in determining strand selection in mammalian cells.

Identification of ETS-like transcription factor 4 as a novel androgen receptor target in prostate cancer cells

Transcriptional control by androgens via androgen receptor (AR) is strongly involved in prostate cancer development, but the critical target genes have remained elusive. We have characterized E twenty-six-like transcription factor 4 (ELK4) (also known as serum response factor accessory protein 1) as a novel AR target in human prostate cancer cells. In-silico screening identified three putative AR response elements (AREs) within -10 kb from the transcription start site of ELK4. Both ARE1 at -167/-153 and ARE2 at -481/-467 bound AR in vitro and mediated androgen induction as isolated elements in transcription assays in non-prostate cells. However, merely the ARE2 that cooperates with a proximal forkhead box A1-binding site was critical for the AR-dependent activation of ELK4 promoter in prostate cancer cells. Preferential loading of holo-AR onto the ARE2 and concomitant recruitment of RNA polymerase II onto the ELK4 promoter was confirmed in prostate cancer cells by chromatin immunoprecipitation. Database searches indicated that the expression of ELK4 is markedly increased in prostate cancers relative to normal prostates. Moreover, prostate cancer tissue immunostainings showed that nuclear ELK4 levels are significantly increased in androgen-refractory prostate cancers compared to untreated tumours. Reduction of the amount of ELK4 in LNCaP cells by RNAi retarded cell growth. In conclusion, ELK4 is a direct AR target in prostate cancer cells. Androgens may thus contribute to the growth of prostate cancer via influencing ELK4 levels.

Extracellular-loop peptide antibodies reveal a predominant hemichannel organization of connexins in polarized intestinal cells

Shigella, the causative agent of bacillary dysentery, invades colonic epithelial cells to elicit an intense inflammatory reaction leading to destruction of the mucosa. ATP-dependent paracrine signalling induced by connexin (Cx) hemichannel opening was previously shown to favor Shigella flexneri invasion and dissemination in transfectants of HeLa cells [G. Tran Van Nhieu, C. Clair, R. Bruzzone, M. Mesnil, P. Sansonetti and L. Combettes. (2003). Connexin-dependent intercellular communication increases invasion and dissemination of Shigella in epithelial cells. Nat. Cell Biol. 5, 720–726.]. However, although Cxs have been described in polarized epithelial cells, little is known about their structural organization and the role of hemichannels during S. flexneri invasion. We show here that polarized Caco-2/TC7 cells express significant amounts of Cx26, Cx32 and Cx43, but that unexpectedly, cell–cell coupling assessed by dye-transfer experiments is inefficient. Consistent with a predominant Cx organization in hemichannels, dye loading induced by low calcium was readily observed, with preferential loading at the basolateral side. Antibodies (Abs) against connexin extracellular loop peptides (CELAbs) demonstrated the importance of hemichannel signalling since they inhibited dye uptake at low calcium and at physiological calcium concentrations during S. flexneri invasion. Importantly, CELAbs allowed the visualization of hemichannels at the surface of epithelial cells, as structures distinct from gap intercellular junctions.

Acute and chronic cardiovascular effects of intermittent hypoxia in C57BL/6J mice

We investigated the effects of 1) acute hypoxia and 2) 5 wk of chronic intermittent hypoxia (IH) on the systemic and pulmonary circulations of C57BL/6J mice. Mice were chronically instrumented with either femoral artery or right ventricular catheters. In response to acute hypoxia (4 min of 10% O2; n = 6), systemic arterial blood pressure fell (P < 0.005) from 107.7 +/- 2.5 to 84.7 +/- 6.5 mmHg, whereas right ventricular pressure increased (P < 0.005) from 11.7 +/- 0.8 to 14.9 +/- 1.3 mmHg. Another cohort of mice was then exposed to IH for 5 wk (O2 nadir = 5%, 60-s cycles, 12 h/day) and then implanted with catheters. In response to 5 wk of chronic IH, mice (n = 8) increased systemic blood pressure by 7.5 mmHg, left ventricle + septum weight by 32.2 +/- 7.5 x 10(-2) g/100 g body wt (P < 0.015), and right ventricle weight by 19.3 +/- 3.2 x 10(-2) g/100 g body wt (P < 0.001), resulting in a 14% increase in the right ventricle/left ventricle + septum weight (P < 0.005). We conclude that in C57BL/6J mice 1) acute hypoxia causes opposite effects on the pulmonary and systemic circulations, leading to preferential loading of the right heart; and 2) chronic IH in mice results in mild to moderate systemic and pulmonary hypertension, with resultant left- and right-sided ventricular hypertrophy.

Effect of sample geometry on the apparent biaxial mechanical behaviour of planar connective tissues

Mechanical testing methodologies developed for engineering materials may result in artifactual material properties if applied to soft planar connective tissues. The use of uniaxial tissue samples with high aspect ratios or biaxial samples with slender cruciform arms could lead to preferential loading of only the discrete subset of extracellular fibres that fully extend between the grips. To test this hypothesis, cruciform biaxial connective tissue samples that display distinctly different material properties (bovine pericardium, fish skin), as well as model textile laminates with predefined fibrous orientations, were repeatedly tested with decreasing sample arm lengths. With mechanical properties determined at the sample centre, results demonstrated that the materials appeared to become stiffer and less extensible with less slender sample geometries, suggesting that fibre recruitment increases with decreasing sample arm length. Alterations in the observed shear behaviour and rigid body rotation were also noted. The only truly reliable method to determine material properties is through in vivo testing, but this is not always convenient and is typically experimentally demanding. For the in vitro determination of the biaxial material properties, appropriate sample geometry should be employed in which all of the fibres contribute to the mechanical response.

Voluntary limb-load distribution in horses with acute and chronic laminitis.

To compare limb-load distribution between horses with and without acute or chronic laminitis. 10 horses with carbohydrate-induced acute laminitis, 20 horses with naturally occurring chronic laminitis, and 20 horses without foot abnormalities (controls). Limb-load distribution was determined, using a custom-designed system that allowed simultaneous quantification of the mean percentage of body weight voluntarily placed on each limb (ie, mean limb load) and the SD of the mean load over a 5-minute period (ie, load distribution profile [LDP]). Load distribution profile was used as an index of frequency of load redistribution. Mean loads on fore- and hind limbs in control horses were 58 and 42%, respectively, and loads were equally and normally distributed between left and right limbs. In addition, forelimb LDP was greater, compared with hind limbs, and was affected by head and neck movement. In comparison, limb-load distribution in horses with chronic laminitis was characterized by an increase in the preferential loading of a forelimb, a decrease in total forelimb load, and an increase in LDP that was correlated with severity of lameness. In horses with carbohydrate-induced acute laminitis, mean limb loads after onset of lameness were not different from those prior to lameness; however, LDP was significantly decreased after onset of lameness. Quantification of limb-load distribution may be an applicable screening method for detecting acute laminitis, grading severity of lameness, and monitoring rehabilitation of horses with chronic laminitis.

Effect of physical activity on bone mineral density assessed by limb dominance across the lifespan

Bone mineral density is higher in dominant vs. nondominant limbs, implying that the greater use of dominant limbs in everyday activities results in the deposition of more bone or that the dominant limb is genetically larger. The objective of the present study was to determine whether bone mineral density differences between dominant and nondominant arms were greater in older vs. younger women. To determine whether this was due to a greater lifetime of preferential loading of the dominant arm, differences between dominant and nondominant arms were compared to accumulated amounts of physical activities which emphasized use of the dominant arm. Bone mineral density of dominant and nondominant arms was assessed by dual-energy X-ray absorptiometry in groups of younger (n = 35; age = 20.9) and older (n = 53; age = 57.4) women. The difference between arms was greater in the older vs. the younger group (5.2% vs. 1.9%, respectively, P < 0.01). Within the older group, total lifetime energy expenditure during activities emphasizing loading of the dominant arm correlated with the bone mineral difference between dominant and nondominant arms (r = 0.47, P < 0.01). This implies that a greater lifetime of preferential loading of the dominant arm in the older group resulted in a greater difference between arms. Am. J. Hum. Biol. 12:633–637, 2000. © 2000 Wiley-Liss, Inc.

Effect of physical activity on bone mineral density assessed by limb dominance across the lifespan.

Bone mineral density is higher in dominant vs. nondominant limbs, implying that the greater use of dominant limbs in everyday activities results in the deposition of more bone or that the dominant limb is genetically larger. The objective of the present study was to determine whether bone mineral density differences between dominant and nondominant arms were greater in older vs. younger women. To determine whether this was due to a greater lifetime of preferential loading of the dominant arm, differences between dominant and nondominant arms were compared to accumulated amounts of physical activities which emphasized use of the dominant arm. Bone mineral density of dominant and nondominant arms was assessed by dual-energy X-ray absorptiometry in groups of younger (n = 35; age = 20.9) and older (n = 53; age = 57.4) women. The difference between arms was greater in the older vs. the younger group (5.2% vs. 1.9%, respectively, P < 0.01). Within the older group, total lifetime energy expenditure during activities emphasizing loading of the dominant arm correlated with the bone mineral difference between dominant and nondominant arms (r = 0.47, P < 0.01). This implies that a greater lifetime of preferential loading of the dominant arm in the older group resulted in a greater difference between arms. Am. J. Hum. Biol. 12:633-637, 2000. Copyright 2000 Wiley-Liss, Inc.

Postmortem Analysis of Bone Growth into Porous-Coated Acetabular Components*

Microradiography, backscattered electron microscopy, and histological analysis were used to conduct a quantitative postmortem study of seven consecutively retrieved anatomical porous replacement acetabular components that had been inserted during total hip arthroplasties. Screws had been used for the initial fixation of six components. The microradiographic analysis of all seven components showed that an average (and standard deviation) of 84 +/- 9 per cent (range, 72 to 93 per cent) of the porous coating was in direct apposition to the periprosthetic bone. The backscattered electron images demonstrated that an average of 12 +/- 6 per cent (range, 4 to 21 per cent) of the space available in the porous coating was occupied by ingrown bone. The amount of bone ingrowth was not significantly different among the three zones delineated by DeLee and Charnley. Uniformity of bone growth into the porous coating suggests that the preferential loading that occurs in the superior region did not differentially affect the bone ingrowth. The present study showed that consistent bone growth into anatomical porous replacement acetabular components can be achieved.

The Translocating RecBCD Enzyme Stimulates Recombination by Directing RecA Protein onto ssDNA in a χ-Regulated Manner

Double-stranded DNA break repair and homologous recombination in E. coli are initiated by the RecBCD enzyme, which unwinds and simultaneously degrades DNA from a double-stranded DNA end. This process is stimulated by cis-acting DNA elements, known as χ sites. Using both in vitro pairing and nuclease protection assays, we demonstrate that the translocating RecBCD enzyme, which has been activated by χ, coordinates the preferential loading of the homologous pairing protein, RecA, onto the resultant single-stranded DNA downstream of χ. This facilitated loading of RecA protein results in a substantial increase in both the efficiency and rate of in vitro recombination reactions and offers an explanation for stimulation of recombination and repair in vivo by χ.

Nonuniform activity distribution in catalyst particles: Benzene hydrogenation on supported nickel in a single pellet diffusion reactor

A single pellet diffusion reactor (SPDR) of new design has been used to investigate the influence of nonuniform distributions of active ingredient on the Ni-catalyzed hydrogenation of benzene. Five different distributions, ranging from preferential shell-loading to uniform to preferential core loading, have been studied under isothermal conditions. Analysis of results has been carried out using a simple one-dimensional diffusion/reaction analysis, which the configuration of the SPDR is set to mimic. The rate of the reaction follows an Eley-Rideal model, effectively positive fractional-order under the experimental conditions, for which kinetics the nonuniform distribution problem has not been studied experimentally before. Preferential shell-loading yields the highest catalytic effectiveness but because of the complex interactions between the reaction kinetics and the activity profiles within the pellets, there is no simple relationship between diffusion lengths and effectiveness even for large values of the Thiele modulus.

ENTROPY-DRIVEN PROTEIN SELF-AGGREGATION AS THE BASIS FOR SELF/NOT-SELF DISCRIMINATION IN THE CROWDED CYTOSOL

Cytotoxic T cells recognize cell surface complexes of MHC class I proteins with peptides derived from proteins synthesized within the recognized cell. A mechanism permitting some intracellular discrimination between self proteins and not-self proteins (encoded by a foreign species) would allow the preferential loading of MHC proteins with peptides derived from not-self proteins. This would decrease competition with peptides derived from self proteins and decrease gaps in the T cell repertoire. A possible mechanism has been derived from studies of the specificity of self-aggregation of erythrocytes and of virus coat protein. It is postulated that genes whose products occupy a common cytosol have co-evolved such that product concentrations are fine-tuned to a maximum consistent with avoiding self-aggregation. Cytosolic proteins collectively generate a pressure tending to drive protein molecules into self-aggregates. Each individual protein species both contributes to, and is influenced by, this pressure. The aggregation involves a liberation of bound water and an increase in entropy. The concentrations of proteins encoded by viral genes (not-self) readily exceed the solubility limits imposed by the crowded host cytosol. This leads to their preferential degradation to peptides which associate with MHC proteins. The intracellular and extracellular self/not-self discrimination systems complement each other to ensure that there is no immunological reaction against normal self tissue components.

Determination of the in situ loads on the human anterior cruciate ligament.

A noncontact, kinematic method was used to determine the lengths and in situ loads borne by portions of the human anterior cruciate ligament (ACL) by the combination of kinematic data from the intact knee and load-length curves of the isolated ACL. Specimens from knees of cadavers of young people were tested in passive flexion and extension as well as with 100 N of anterior tibial drawer at 0, 30, 45, and 90 degrees of flexion. The results showed that the in situ load on the whole ACL (as much as 129 N) can exceed the magnitude of the applied anterior tibial drawer. The load distribution within the ligament changes with flexion of the knee. The anterior and posterior portions share the anterior drawer force equally toward full extension. However, at flexion > 45 degrees, the anterior portion supports 90-95% of the load. This information is important for the determination of the function of the entire ACL and of its subportions in response to external loading of the intact knee. In particular, the preferential loading found for one of the portions of the ACL demonstrates that successful operative reconstruction of this ligament may not be achieved simply by reproduction of its gross anatomy; consideration of the role of the ligament in the overall kinematics of the knee is necessary.