Joint instability causes catabolic enzyme production in chondrocytes prior to synovial cells in novel non-invasive ACL ruptured mouse model.

OBJECTIVE: The Anterior Cruciate Ligament (ACL)-deficient model helps to clarify the mechanism of knee osteoarthritis (OA); however, the conventional ACL injury model could have included concurrent onset factors such as direct compression stress to cartilage and subchondral bone. In this study, we established a novel Non-invasive ACL-Ruptured mouse model without concurrent injuries and elucidated the relationship between OA progression and joint instability. DESIGN: We induced the ACL-Rupture non-invasively in twelve-week-old C57BL/6 male mice and evaluated histological, macroscopical, and morphological analysis at 0 days. Next, we created the ACL-R, controlled abnormal tibial translation (CATT), and Sham groups. Then, the joint stability and OA pathophysiology were analyzed at 2, 4, and 8 weeks. RESULTS: No intra-articular injuries, except for ACL rupture, were observed in the ACL-R model. ACL-R mice increased anterior tibial displacement compared to the Sham group (P < 0.001, 95% CI [-1.509 to -0.966]) and CATT group (P < 0.001, 95% CI [-0.841 to -0.298]) at 8 weeks. All mice in the ACL-R group caused cartilage degeneration. The degree of cartilage degeneration in the ACL-R group was higher than in the CATT group (P = 0.006) at 8 weeks. The MMP-3-positive cell rate of chondrocytes increased in the ACL-R group than CATT group from 4 weeks (P = 0.043; 95% CI [-28.32 to -0.364]) while that of synovial cells increased at 8 weeks (P = 0.031; 95% CI [-23.398 to -1.021]). CONCLUSION: We successfully established a Non-invasive ACL-R model without intra-articular damage. Our model revealed that chondrocytes might react to abnormal mechanical stress prior to synovial cells while the knee OA onset.

The difference in joint instability affects the onset of cartilage degeneration or subchondral bone changes.

OBJECTIVE: It has been debated whether the onset of knee osteoarthritis is initiated in cartilage or subchondral bone. The purpose of this study was to clarify the effects of increasing or decreasing joint instability on cartilage degeneration and subchondral bone changes in knee OA by comparing different models of joint instability. DESIGN: We used the anterior cruciate ligament transection (ACL-T) model and the destabilization of the medial meniscus (DMM) model. In addition, we created a controlled abnormal tibial translation (CATT) model and a controlled abnormal tibial rotation (CATR) model. We performed joint instability analysis, micro-computed tomography analysis, histological and immunohistological analysis in 4 and 6 weeks. RESULTS: The CATT group suppressed joint instability in the ACL-T group (6 weeks; P = 0.032), and the CATR group suppressed joint instability in the DMM group (6 weeks; P = 0.032). Chondrocyte hypertrophy in the ACL-T and DMM groups was increased compared to the Sham group (6 weeks; [ACL-T vs Sham], P = 0.002, 95%CI [5.983-33.025]; [DMM vs Sham], P = 0.022, 95%CI [1.691-28.733]). In the subchondral bone, the BV/TV in the DMM and CATR groups was increased compared to the ACL-T and CATT groups (6 weeks; [DMM vs ACL-T], P = 0.002, 95%CI [7.404-37.582]; [DMM vs CATT], P = 0.014, 95%CI [2.881-33.059]; [CATR vs ACL-T], P = 0.006, 95%CI [4.615-34.793]; [CATR vs CATT], P = 0.048, 95%CI [0.092-30.270]). CONCLUSIONS: This study showed that joint instability promotes chondrocyte hypertrophy, but subchondral bone changes were influenced by differences in ACL and meniscus function.

Prognostic impact of interhospital variation in adjuvant chemotherapy for patients with Stage II/III colorectal cancer: a nationwide study.

AIM: Clinical guidelines recommend adjuvant chemotherapy for high-risk patients with Stage II-III colorectal cancer. However, chemotherapeutic administration rates differ significantly between hospitals. We assessed the prognostic benefit of adjuvant chemotherapy in patients with Stage IIb/c colorectal cancer, and the prognostic impact of interhospital variations in the administration of adjuvant chemotherapy for Stage II-III colorectal cancer. METHOD: We conducted a multicentre, retrospective study of 17 757 patients with Stage II-III colorectal cancer treated between 1997 and 2008 in 23 hospitals in Japan. Hospitals were classified as high-rate (rate > 42.8%) or low-rate (rate ≤ 42.8%), chemotherapy prescribing clinics. RESULTS: The 5-year overall survival (OS) of patients with Stage II-III colorectal cancer receiving adjuvant chemotherapy was significantly higher than for those not receiving adjuvant chemotherapy (85.7% vs 79.2%, P < 0.01 and 79.9% vs 72.5%, P < 0.01, respectively). For patients with Stage II disease, adjuvant chemotherapy was an independent factor for longer OS (P < 0.01, hazard ratio = 0.71). Both adjuvant chemotherapy and high-rate hospital independently improved OS for patients with Stage III colorectal cancer (both P < 0.01; hazard ratio = 0.68 and 0.87, respectively). CONCLUSION: Significant prognostic benefit was found for patients with Stage IIb/c colorectal cancer who received adjuvant chemotherapy, with patients who were treated in hospitals with high adjuvant chemotherapy rates demonstrating better prognoses.

Detection of one-dimensional migration of single self-interstitial atoms in tungsten using high-voltage electron microscopy.

The dynamic behaviour of atomic-size disarrangements of atoms-point defects (self-interstitial atoms (SIAs) and vacancies)-often governs the macroscopic properties of crystalline materials. However, the dynamics of SIAs have not been fully uncovered because of their rapid migration. Using a combination of high-voltage transmission electron microscopy and exhaustive kinetic Monte Carlo simulations, we determine the dynamics of the rapidly migrating SIAs from the formation process of the nanoscale SIA clusters in tungsten as a typical body-centred cubic (BCC) structure metal under the constant-rate production of both types of point defects with high-energy electron irradiation, which must reflect the dynamics of individual SIAs. We reveal that the migration dimension of SIAs is not three-dimensional (3D) but one-dimensional (1D). This result overturns the long-standing and well-accepted view of SIAs in BCC metals and supports recent results obtained by ab-initio simulations. The SIA dynamics clarified here will be one of the key factors to accurately predict the lifetimes of nuclear fission and fusion materials.

Terrestrial gamma radiation dose rate in Ryukyu Islands, subtropical region of Japan.

In order to explain the distribution of natural radiation level in the Asia, in situ measurements of dose rate in air due to terrestrial gamma radiation have been conducted in a total of 21 islands that belong to Ryukyu Islands (Ryukyu Archipelago), subtropical rejoin of southwest Japan. Car-borne surveys have also been carried out in Okinawa-jima, the biggest island of the archipelago. Based on the results for these measurements, arithmetic mean, the maximum and the minimum of the dose rates at 1 m in height from the unpaved soil ground in the archipelago were estimated to be 47, 165 and 8 nGy h(-1), respectively. A comparative study of car-borne data obtained prior to and subsequent to the 2011 Fukushima nuclear accident, as for Okinawa-jima, indicated that the nuclear accident has no impact on the environmental radiation at the present time.

Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk.

Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious peptides to enteric lactobacilli including L. gasseri after peptic digestion in the gastrointestinal tract. This is the first report showing peptide requirement of L. gasseri and efficacy of pepsinolysis on the growth of L. gasseri and its relatives in milk. This study would contribute to increasing usability of L. gasseri and its relatives as probiotics in dairy foods.

Prognostic significance of L-type amino-acid transporter 1 expression in surgically resected pancreatic cancer.

BACKGROUND: The expression of L-type amino-acid transporter 1 (LAT1) is tumour-specific and has been shown to have essential roles in cell growth and survival. However, little is known regarding the clinical significance of LAT1 expression in pancreatic cancer. This study was conducted to determine the prognostic significance of LAT1 expression. METHODS: A total of 97 consecutive patients with surgically resected pathological stage I-IV pancreatic ductal adenocarcinoma were retrospectively reviewed. Tumour sections were stained by immunohistochemistry for LAT1, CD98, Ki-67 and vascular endothelial growth factor (VEGF), and microvessel density was determined by CD34 and p53. RESULTS: L-type amino-acid transporter 1 and CD98 were highly expressed in 52.6% (51/97) and 56.7% (55/97) of cases, respectively (P=0.568). The expression of LAT1 within pancreatic cancer cells was significantly associated with disease stage, tumour size, Ki-67, VEGF, CD34, p53 and CD98. L-type amino-acid transporter 1 expression was confirmed to be a significant prognostic factor for predicting poor outcome by multivariate analysis. CONCLUSION: L-type amino-acid transporter 1 expression is a promising pathological marker for the prediction of outcome in patients with pancreatic cancer.

Influence of tissue particles on Fas expression in idiopathic interstitial pneumonia.

Idiopathic interstitial pneumonia (IIP) is a progressive fibrosing interstitial pneumonia of unknown etiology with a poor prognosis. The aim of this study is to prove the occurrence of particle deposition and particle-induced tissue damage in IIP by examining proapoptotic Fas expression with in-air microparticle induced X-ray emission (in-air micro-PIXE) analysis. A total of 21 patients were enrolled. Lung tissues from 12 IIP patients and nontumorous lung tissues from 9 lung cancer patients (as a control) were subjected to in-air micro-PIXE analysis. The distribution of particles in lung tissue was compared with the localization of Fas expression by immunohistochemistry. Silicon (Si) was identified in 58.3% of IIP samples and 44.4% of control samples. Iron (Fe) was identified 25% in IIP samples and 11.1% in control samples. The mean lung tissue content of Si and Fe relative to S did not differ between IIP and control patients. Only two IIP patients showed the co-localization of Si and Fe deposition with Fas expression. Adaptation of this method would contribute to assess the influence of particles on IIP.

Oxytocin and vasopressin in the medial amygdala differentially modulate approach and avoidance behavior toward illness-related social odor.

Animals are known to recognize a specific odorant informing conspecific health condition, which plays a significant role in regulating their social communication. Here, we assess neural mechanisms regulating innate approach/avoidance response toward such conspecific odor cues in rats. Odor scent from healthy conspecifics induced approach behavior, while those from sick conspecifics produced avoidance response in odor-recipient male rats. Analysis of mRNA expression in several brain sites of odor recipient rats illustrated that induction of c-fos mRNA expression was found in the olfactory bulb (OB), the medial amygdala (MeA), the bed nucleus of stria terminalis (BnST), and the paraventricular nucleus of the hypothalamus (PVN), when exposed to conspecific odor. Moreover, in the MeA, expression of oxytocin (OT) receptor mRNA was increased when rats were exposed to healthy conspecific odor, while induction of arginine vasopressin (AVP) receptor 1a and 1b mRNA were found only when exposed to sick conspecific odor. Bilateral infusion of OT receptor (OTR) antagonist, (d(CH2)5(1),Tyr(Me)(2),Thr(4),Orn(8),des-Gly-NH2(9))-Vasotocin, into the MeA blocked approach behavior to healthy odor, while those of AVP receptor antagonists, V1a selective: (Phenylac(1),D-Tyr(Me)(2),Arg(6.8),Lys-NH2(9))-Vasopressin, and type 1 receptor antagonist: (Deamino-Pen(1), Try(Me)(2), Arg(8))-Vasopressin, into the MeA inhibited avoidance response to sick odor. These findings provide evidence for an essential role of OT and AVP receptors, especially type 1a, in the MeA in regulating approach/avoidance behaviors, respectively, in social odorant communication.

Two-dimensional metallic tungsten nanowire network fabricated by electron-beam-induced deposition.

One-dimensional tungsten nanowires and two-dimensional tungsten nanowire networks were fabricated on a tungsten substrate by using electron-beam-induced deposition (EBID) without precursor injection. The as-prepared tungsten nanostructures were studied using a combination of energy-dispersive x-ray spectroscopy and selected area electron diffraction. It was revealed that the tungsten nanostructures were composed of pure metallic tungsten. The WO(3) oxide formed in the chemical preparation of tungsten foils was probably the source of tungsten for the fabrication of tungsten nanostructures by EBID.

Analysis on the co-localization of asbestos bodies and Fas or CD163 expression in asbestos lung tissue by in-air micro-pixe.

To prevent and control disease caused by exposure to various agents, it is necessary to determine the harmful level of intervention and to establish a method for measuring that level. In-air microparticle-induced X-ray emission (in-air micro-PIXE) analysis is based on irradiation of specimens with a proton ion microbeam, and has been modified for biological application. Two-dimensional analysis and quantitative analysis using the system confirmed that asbestos induced apoptosis by upregulating Fas expression and also revealed the accumulation of CD163-expressing macrophages in the lungs of patients with asbestosis. By quantitative comparison of the area of Fas or CD163 expression and the Fas- or CD163-negative area in asbestos lung tissue, the harmful levels which caused the expression of Fas or CD163 could be estimated on Silica, Ferrous iron, and Magnesium (the components of asbestos) deposition. These results indicate that the system could be useful for investigating the pathogenesis of inhaled particle-induced immune reactions and for determining harmful levels of exogenous agents.

HPLC purification and re-evaluation of chemical identity of two circular bacteriocins, gassericin A and reutericin 6.

AIM: The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. METHODS AND RESULTS: Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H](+) and both had the same specific activity. D/L-amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and O-phthalaldehyde/N-acetyl-L-cystein revealed neither gassericin A nor reutericin 6 contained D-alanine residues contrary to our previous results. CONCLUSION: Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no D-alanine residues. SIGNIFICANCE AND IMPACT OF THE STUDY: The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.

Effects of gassericins A and T, bacteriocins produced by Lactobacillus gasseri, with glycine on custard cream preservation.

Lactobacillus gasseri LA39 and LA158 isolated from human-infant feces produce bacteriocins named gassericins A and T, respectively. Both gassericins have high heat stability (121 degrees C, 10 min), good pH tolerance (pH 2-11), and strong bactericidality against many gram-positive bacteria, especially lactic acid bacteria, and thus are expected to be effective food preservatives. A microwell plate assay against 12 strains of custard cream spoilage bacteria showed that the gassericins had broader antibacterial spectra than nisin A. Although the gassericins allowed gram-negative isolates to grow, they successfully inhibited the growth of all tested bacterial strains in microwells with the addition of glycine. Glycine was bacteriostatic against many strains except lactic acid bacteria. For practical use, gassericin A was efficiently produced by cultivation in a food-grade medium improved using cheese whey, nourishing proteose peptone, and surfactant yolk lecithin. The practical preservative effect of gassericin A and glycine was verified from the viability of 4 isolated strains, Bacillus cereus, Lactococcus lactis ssp. lactis, Achromobacter denitrificans, and Pseudomonas fluorescens, in custard creams. Custard cream containing 123 arbitrary units of gassericin A per milliliter entirely growth-inhibited the 2 gram-positive strains. In custard cream containing an insufficient amount of gassericin A (49 arbitrary units/mL), the gram-positive strains gradually grew but were completely inhibited by the addition of 0.5% (wt/wt) glycine. The 2 gram-negative strains did not multiply even in the additive-free custard cream, probably because of the unsuitable growth environment. This is the first report showing the combined effect of bacteriocin and glycine and their application for food preservation, which may be helpful for future use in the food industry.

In-air micro-particle induced X-ray emission analysis of asbestos and metals in lung tissue.

Inhalation of asbestos increases the risk of lung cancer and pulmonary fibrosis. It is difficult to directly assess the distribution and content of inhaled particles in lung tissue sections. The purpose of this study is to employ an in-air micro particle induced X-ray emission (in-air micro-PIXE) system for assessment of the spatial distribution and content of asbestos and other metals in lung tissue. A proton ion-microbeam from this system was applied to irradiate lung tissue of patients with or without asbestosis, tumor tissue from both groups, and asbestos fibers (in vitro). The content of each element composing asbestos and those of other metals were calculated and their distribution was assessed from the characteristic X-ray pattern for each element obtained after irradiation. This in-air micro-PIXE system could identify the location of asbestos bodies composed of Si, Mg, and Fe in lung tissue sections. Macrophage and lymphocytes accumulated in that area. This new system also revealed deposits of titanium, nickel, and cobalt in the lung tissues, in addition to asbestos bodies. The Si and Fe content were higher in lungs with asbestosis than in lungs without asbestosis or in tumor tissue. Analysis of asbestos fibers composed of chrysotile, crocidolite, and amosite showed that the ratios of Si, Fe, and Mg corresponded with those for the chemical structures. In-air micro-PIXE analysis is useful for assessing the distribution and quantities of asbestos bodies and also other metals in lung tissue comparing to immune-related cell localizations, and is also useful for analysis of standard asbestos fibers.

Microbial community analysis of food-spoilage bacteria in commercial custard creams using culture-dependent and independent methods.

Custard cream is made from highly nutritive raw materials such as milk and sugar and is easily spoiled by the multiplication of specific microbial contaminants or residents. However, this spoilage microbial community has not been studied. We determined the spoilage microbiota in commercial custard creams using culture-dependent and independent methods. Using the culture-dependent analysis with various agar media, 185 bacterial colonies and 43 eukaryal colonies were isolated from 7 commercial custard cream products. All bacterial isolates were morphologically, physiologically, and genetically identified as bacilli, staphylococci, lactic acid bacteria, and psychrotrophic gram-negative rods. Using culture-independent molecular analysis, the PCR-denaturing gradient gel electrophoresis technique, spoilage of the commercial custard creams was found to be caused by bacilli, staphylococci, lactic acid bacteria, psychrotrophic gram-negative rods, Anoxybacillus sp., Caurobacter sp., and Streptococcus sp. bacteria. The detected spoilage bacteria were the same species as previously detected in spoiled milk products and shown in other reports, suggesting that spoilage bacteria in a raw material easily grow in processed foods made from milk. We determined the spoilage microbial communities in commercial custard creams, and these are the first data concerning spoilage microbiota in nonfermented processed foods using a culture-independent analysis. Our study will be useful for the manufacture and safe preservation of dairy products because the first step toward safe food preservation by food manufacturers is to understand the spoilage microbiota in a target food to select optimal preservatives and to reduce the use of food additives.

Intracellular changes of metal elements by fucoidan extracted from brown seaweed (Cladosiphon okamuranus).

This study was undertaken to elucidate the intracellular changes of metal elements after the administration of fucoidan extracted from Cladosiphon okamuranus. TRL1215 cells (normal rat liver cell line) were treated with 0, 0.1, or 1.0 mg/ml fucoidan and incubated in 5% CO2 at 37 degrees C. The cellular levels of Mg, Al, Fe, and Zn were significantly increased in the 1.0 mg/ml fucoidan-treated cells compared to those of the 0.1 mg/ml fucoidan-treated cells and the control. Next, TRL1215 cells were cultured on Mylar film overnight. At 24 h after 5-bromo-2'-deoxyuridine dosing, 0, 0.1, or 1.0 mg/ml fucoidan was treated for 9 h. The cellular distribution of elements was analyzed using in-air micro-micro-particle induced X-ray emission. The X-ray spectra showed that yields of Al, Mg, and Zn were high in order of the 1.0 mg/ml fucoidan-treated sample, the 0.1 mg/ml fucoidan-treated sample, and the control. Fe yield was mildly increased by fucoidan administration. In fucoidan-treated cells, the focal accumulation of Br was correlated spatially with phosphorous-rich region, suggesting that Br was localized within the nucleus. Al distribution provided a spatial association with Br map. These data suggest that fucoidan increases the accumulations of Al, Mg, Fe, and Zn in normal rat hepatocytes, and fucoidan-binding Al is postulated to be transferred into the nucleus.

Changes of mental stress biomarkers in ultramarathon.

We investigated the possible influence of an exhaustive physical exercise on mental stress biomarkers (serotonin, tryptophan, and beta-endorphin) along with dopamine, noradrenaline and free fatty acids in an ultramarathon race in which 45 km was run on the first day and 90 km on the second. We obtained serum samples at 6 different time points during and after the race from 18 Japanese male runners who completed the marathon. Overall changes of serum serotonin and tryptophan concentrations were statistically significant according to ANOVA for repeated measurements (p < 0.05). Serum serotonin levels elevated rapidly on the first day with the post hoc Tukey's test. Tryptophan concentrations inversely decreased during the race, possibly because of utilization for synthesis of serotonin. Levels of beta-endorphin appeared to increase on the first and second days, but were not statistically significant. In conclusion, serum serotonin, tryptophan and beta-endorphin appeared to be used for mental stress markers in physical exercise.

Long- and short-term freezing induce different types of injury in Arabidopsis thaliana leaf cells.

In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in "fracture-jump lesions" in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.

Observation of the one-dimensional diffusion of nanometer-sized dislocation loops.

Dislocations are ubiquitous linear defects and are responsible for many of the properties of crystalline materials. Studies on the glide process of dislocations in bulk materials have mostly focused on the response of dislocations with macroscopic lengths to external loading or unloading. Using in situ transmission electron microscopy, we show that nanometer-sized loops with a Burgers vector of (1/2)111 in alpha-Fe can undergo one-dimensional diffusion even in the absence of stresses that are effective in driving the loops. The loop size dependence of the loop diffusivity obtained is explained by the stochastic thermal fluctuation in the numbers of double kinks.