Fluorescent aptasensor mediated with multiple ssDNA for sensitive detection of acetamiprid in vegetables based on magnetic Fe3O4/C-assisted separation.

Acetamiprid (ACE) is a highly effective broad-spectrum insecticide, and its widespread use is potentially harmful to human health and environmental safety. In this study, magnetic Fe3O4/carbon (Fe3O4/C), a derivative of metal-organic framework MIL-101 (Fe), was synthesized by a two-step calcination method. And a fluorescent sensing strategy was developed for the efficient and sensitive detection of ACE using Fe3O4/C and multiple complementary single-stranded DNA (ssDNA). By using aptamer with multiple complementary ssDNA, the immunity of interference of the aptasensor was improved, and the aptasensor showed high selectivity and sensitivity. When ACE was present, the aptamer (Apt) combined with ACE. The complementary strand of Apt (Cs1) combined with two short complementary strands of Cs1, fluorophore 6-carboxyfluorescein-labeled complementary strand (Cs2-FAM) and the other strand Cs3. The three strands formed a double-stranded structure, and fluorescence would not be quenched by Fe3O4/C. In the absence of ACE, Cs2-FAM would be in a single-chain state and would be adsorbed by Fe3O4/C, and the fluorescence of FAM would be quenched by Fe3O4/C via photoelectron transfer. This aptasensor sensitively detected ACE over a linear concentration range of 10-1000 nM with a limit of detection of 3.41 nM. The recoveries of ACE spiked in cabbage and celery samples ranged from 89.49% to 110.76% with high accuracy.

Heterologous expression and fibrillary characterization of the microtubule-binding domain of tau associated with tauopathies.

BACKGROUND: Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer's disease. The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity. METHODS AND RESULTS: Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tauMBD) were performed. The tauMBD was heterologously expressed in E. coli. Ni-chelating affinity chromatography was then performed to purify the target protein. Thereafter, tauMBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tauMBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments. CONCLUSIONS: The final yield of the recombinant tauMBD was ~ 20 mg L-1. It is shown that TauMBD, in the absence of an inducer, self-assembled into the typical fibrils at a faster rate than wild-type tau. Finally, the in vitro cytotoxicity of tauMBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.

Genetic Contributions to Attachment Stability Over Time: the Roles of CRHR1 Polymorphisms.

Corticotropin-releasing hormone receptor 1 (CRHR1), a hormone receptor essential to the activation of HPA axis and the subsequent release of cortisol, plays critical roles in emotional and behavioral responses relevant to attachment. However, the specific roles of CRHR1 polymorphisms in attachment remain unclear. To further clarify these genetic effects, this research conducted a three-wave study to investigate whether the CRHR1 polymorphisms (i.e., rs110402 and rs242924) are associated with the stability and variability of attachment by using a sample of freshmen (N = 604; Mage = 18.57 years, SD = 1.90; 68.8% girls). The results showed that rs110402 and rs242924 were associated with the stability of closeness-dependence. The G alleles of the both polymorphisms were found not to be related to lower attachment stability. However, these polymorphisms were not associated with the variability of attachment. Overall, these findings provide evidence for the contribution of CRHR1 to attachment stability.

Genetic Contributions on Attachment in Emerging Adults: Cumulative Effects of Serotonergic Polymorphisms.

Attachment in emerging adults is closely intertwined with emotion regulation, stress coping, and social bonding during the transition from childhood to early adulthood. Due to the critical roles of serotonin in these mental functions, this research explored whether the cumulative genetic effects of serotonergic polymorphisms are associated with individual differences and contextual variations in attachment dimensions over time in emerging adults. Study 1 utilized a cross-sectional design in college students (N = 1088, mean age = 22.71 ± 2.86 years). The results showed significant correlations between a higher cumulative genetic score and elevated levels of attachment anxiety and avoidance. Study 2 employed a three-wave longitudinal design in a cohort of freshmen (N = 523, mean age = 19.54 ± 1.86 years at wave 1). The results demonstrated that a higher genetic score was associated with both higher levels and greater variability in attachment dimensions compared to a lower genetic score. These findings suggest that the cumulative genetic effects of serotonergic polymorphisms contribute to individual differences and dynamic processes in attachment dimensions in emerging adults.

Target-Induced Electrochemical Sensor Based on Foldable Aptamer and MoS2@MWCNTs-PEI for Enhanced Detection of AFB1 in Peanuts.

Herein, a sensitive and selective electrochemical sensor based on aptamer folding was constructed to detect aflatoxin B1 (AFB1) in peanuts. Specifically, polyethylenimine-functionalized multiwalled carbon nanotubes modified with molybdenum disulfide (MoS2@MWCNTs-PEI) were used as the electrode matrix to enable a large specific surface area, which were characterized by the Randles-Sevcik equation. Additionally, AuNPs were used to immobilize the aptamer via the Au-S covalent bond and provide a favorable microenvironment for signal enhancement. Methylene blue (MB) was modified at the proximal 3' termini of the aptamer as the capture probe, while the signal transduction of the sensor was obtained through changes in conformation and position of MB induced by the binding between AFB1 and the probe. Changes in spatial conformation could be recorded by electrochemical methods more readily. This electrochemical aptasensor demonstrated remarkable sensitivity to AFB1 with an extensive detection range (1 pg/mL to 100 ng/mL) and a lower limit detection (1.0 × 10-3 ng/mL). Moreover, using the constructed aptasensor, AFB1 was identified successfully in peanut samples, with recoveries ranging from 95.83 to 107.53%, illustrating its potential use in determining AFB1 in food.

An "on-off-on" electrochemiluminescence aptasensor based on a self-enhanced luminophore for ochratoxin A detection.

A highly selective and sensitive "on-off-on" electrochemiluminescence (ECL) aptasensor based on a self-enhanced luminophore was developed for the detection of ochratoxin A (OTA). Specifically, polyethyleneimine functionalized multi-walled carbon nanotubes decorated with gold nanoparticles (AuNPs-PEI-MWCNTs) were used as the electrode matrix to accelerate electron transfer and provide a favorable microenvironment for self-enhanced luminophore loading and ECL signal enhancement. In addition, black phosphorus quantum dots (BPQDs) were used as co-reactants of the ECL reagent tris (2,2'-bipyridyl) ruthenium(II) (Ru(bpy)32+) in ECL experiments, and the reaction mechanism was investigated. The self-enhanced luminophore Ru@SiO2-BPQDs was obtained by encapsulating Ru(bpy)32+ in silica (SiO2) nanoparticles and then combining it with BPQDs through electrostatic interaction. In conventional ECL systems, the emitter and its co-reactants reacted via the inter-nanoparticle pathway, leading to long distance electron transfer. However, the electron transfer distance in the self-enhanced luminophore was significantly shortened due to the intra-nanoparticle electron transfer pathway because BPQDs and oxidized Ru(bpy)32+ were bound within one nanoparticle, thereby improving ECL efficiency to achieve the first "switch-on" state. Then, the luminophore was quenched using ferrocenes (Fc) modified on an aptamer to achieve the "switch-off" state. Finally, OTA was specifically identified by the adapter, causing Fc to be released from the sensor interface, restoring the ECL intensity to achieve the second "switch-on" state. Under optimal conditions, the aptasensor exhibited good sensitivity, stability, and reproducibility, with a linear detection range from 0.1 to 320 ng/mL and a detection limit of 0.03 ng/mL. The novel ECL aptasensor provided a common analytical tool for the detection of mycotoxins and other small molecules.

Effect of dexmedetomidine administration on analgesic, respiration and inflammatory responses in patients undergoing percutaneous endoscopic lumbar discectomy: a prospective observational study.

BACKGROUND: Local anesthesia has been recommended for percutaneous endoscopic lumbar discectomy (PELD) in recent years; however, the efficacy, including oxidative stress, inflammatory reactions and ventilation effects, when intravenous dexmedetomidine (DEX) is administered during PELD has not been described. METHODS: Sixty adult patients undergoing PELD were randomly allocated to either an intravenous DEX sedation group (Group A) or a normal saline group (Group B). Respiratory data, including minute ventilation (MV), tidal volume (TV), and respiratory rate (RR), were recorded using a respiratory volume monitor (RVM), and peripheral oxygen saturation (SpO2) was monitored by pulse oximetry. The visual analog score (VAS) was used to assess the level of pain. The serum levels of inflammatory biomarkers including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were to assess inflammatory reactions. The serum levels of oxidative stress biomarkers including malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) were also recorded to evaluate oxidative stress. RESULTS: There were no significant differences in RR, MV, TV and SpO2 between the two groups at any time point (P > 0.05). Group B exhibited lower serum levels of GSH-PX (P < 0.0001) and higher serum levels of MDA (p < 0.0001) than Group A at the end of surgery. Twenty-four hours after surgery, Group B exhibited higher serum levels of IL-6 (P = 0.0033), TNF-α (P = 0.0002), and MDA (P < 0.0001) and lower serum levels of GSH-PX (P < 0.0001) than Group A. In addition, Group A exhibited lower VAS (P < 0.0001) than Group B during surgery. CONCLUSIONS: DEX administration using RVM not only provides analgesia without ventilatory depression but also alleviates oxidative stress and inflammatory reactions in patients undergoing PELD.

Expression and purification of amyloid ß-protein, tau, and α-synuclein in Escherichia coli: a review.

Misfolding and accumulation of amyloidogenic proteins into various forms of aggregated intermediates and insoluble amyloid fibrils is associated with more than 50 human diseases. Large amounts of high-quality amyloid proteins are required for better probing of their aggregation and neurotoxicity. Due to their intrinsic hydrophobicity, it is a challenge to obtain amyloid proteins with high yield and purity, and they have attracted the attention of researchers from all over the world. The rapid development of bioengineering technology provides technical support for obtaining large amounts of recombinant amyloidogenic proteins. This review discusses the available expression and purification methods for three amyloid proteins including amyloid ß-protein, tau, and α-synuclein in microbial expression systems, especially Escherichia coli, and discusses the advantages and disadvantages of these methods. Importantly, these protocols can also be referred to for the expression and purification of other hydrophobic proteins.

Design and realization of antireflection coatings for the visible and the infrared based on mesoporous SiO2 and SiO2-TiO2 hybrid materials.

Five-layer antireflective coating for the visible and the infrared has been successfully fabricated by a solgel route on quartz substrate. The quarter-half-quarter multilayer structure with two buffer layers is optimized by the optical admittance approach. Mesoporous SiO2 film templated by an ethylene oxide-propylene oxide-ethylene oxide triblock copolymer is used as the top layer, and SiO2-TiO2 hybrid films are employed as the other layers. The grazing incidence small-angle x-ray scattering results indicate the ordered mesopores in the low-index SiO2 film, as well as the homogeneity of the SiO2-TiO2 hybrid films. Transmittance spectra are fitted to obtain film thicknesses and refractive indices, providing the main technological parameters for theory designs. A short heat-treatment step followed by a shock cooling process is introduced to suppress crack formation in film stacks. The transmittance of the obtained five-layer antireflective coating exceeds 99% at 500-700 nm, and the maximum value in the infrared region is 97.84% at 1100 nm. This work is a beneficial attempt to fabricate antireflective coatings with multilayer structure through the soft solgel route.

The Use of Three-Dimensional Printing Model in the Training of Choledochoscopy Techniques.

AIM: To evaluate the application value of a three-dimensional (3D) printing model in the training of choledochoscopy techniques. MATERIALS AND METHODS: Imaging data from two patients with biliary dilatation were used to produce two 3D reconstruction models which were subsequently constructed into 3D printing models (No. 1 and No. 2). Four hepatobiliary surgeons evaluated the anatomical accuracy and academic teaching value of the printed models. Twenty resident trainees with no prior experience in any kind of endoscopic techniques were randomly and symmetrically divided into two groups. The training group (A) used the 3D model No. 1 in the learning of biliary tract anatomy and practice techniques of choledochoscopy. The control group (B) got the virtual 3D image of the same model on computer for learning. After 4 weeks, the model No. 2 was used to reassess the trainees' subjective and objective progress in anatomy familiarity and choledochoscopy manipulations. RESULTS: All consulted surgeons agreed that the 3D models realistically reproduced the anatomy of the biliary system. All trainees in group A agreed or strongly agreed that the 3D models provided good anatomical realism, enhanced their experience in the training of choledochoscopy techniques, and aided in their learning of biliary anatomy. With the practice went on, they increased the accuracy and showed a reduction in operation time on the model No. 1. During final examination with model No. 2, the rate of correct anatomical structure identification in training group was significantly higher than group B (p < 0.05). CONCLUSION: The 3D printed biliary tract model is an excellent teaching tool in the training of choledochoscopy techniques. The 3D model is anatomically realistic and can improve the trainee's anatomical knowledge and endoscopic skills.

Lone-Pair-Electron-Driven Ionic Displacements in a Ferroelectric Metal-Organic Hybrid.

A displacive-type mechanism, which accounts for the occurrence of ferroelectricity in most inorganic ferroelectrics, is rarely found in molecule-based ferroelectrics. Its role is often covered by the predominant order-disorder one. Herein, we report a lone-pair-electron-driven displacive-type ferroelectric organic-inorganic hybrid compound, [H2dmdap][SbCl5] (1; dmdap = N,N-dimethyl-1,3-diaminopropane). The structure of 1 features a typical zigzag chain of [SbCl5]∞ containing cis-connected anionic octahedra. The compound undergoes a second-order paraelectric-ferroelectric phase transition at 143 K (P21/c ↔ Pc) with a saturation polarization of 1.36 µC·cm-2 and a coercive field of 3.5 kV·cm-1 at 119 K. Theoretical study discloses the ferroelectricity mainly originating from the relative displacements of the Sb and Cl ions in the crystal lattice, which are driven by the 5s2 lone-pair electrons of the SbIII center. Furthermore, on the basis of analysis, possible routes are suggested to enhance ferroelectric polarization in this class of compounds.

An electrochemical immunosensor based on interdigitated array microelectrode for the detection of chlorpyrifos.

An electrochemical immunosensor based on interdigitated array microelectrodes (IDAMs) was developed for sensitive, specific and rapid detection of chlorpyrifos. Anti-chlorpyrifos monoclonal antibodies were orientedly immobilized onto the gold microelectrode surface through protein A. Chlorpyrifos were then captured by the immobilized antibody, resulting in an impedance change in the IDAMs surface. Electrochemical impedance spectroscopy was used in conjunction with the fabricated sensor to detect chlorpyrifos. Under optimum conditions, the impedance value change of chlorpyrifos was proportional to its concentrations in the range of 10(0)-10(5) ng/mL. The detection limit was found to be 0.014 ng/mL for chlorpyrifos. The proposed chlorpyrifos immunosensor could be used as a screening method in pesticide determination for the analysis of environmental, agricultural and pharmaceutical samples due to its rapidity, sensitivity and low cost.

Predictive value of diaphragm ultrasound for mechanical ventilation outcome in patients with acute exacerbation of chronic obstructive pulmonary disease.

BACKGROUND: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is often combined with respiratory failure, which increases the patient's morbidity and mortality. Diaphragm ultrasound (DUS) has developed rapidly in the field of critical care in recent years. Studies with DUS monitoring diaphragm-related rapid shallow breathing index have demonstrated important results in guiding intensive care unit patients out of the ventilator. Early prediction of the indications for withdrawal of non-invasive ventilator and early evaluation of patients to avoid or reduce disease progression are very important. AIM: To explore the predictive value of DUS indexes for non-invasive ventilation outcome in patients with AECOPD. METHODS: Ninety-four patients with AECOPD who received mechanical ventilation in our hospital from January 2022 to December 2023 were retrospectively analyzed, and they were divided into a successful ventilation group (68 cases) and a failed ventilation group (26 cases) according to the outcome of ventilation. The clinical data of patients with successful and failed noninvasive ventilation were compared, and the independent predictors of noninvasive ventilation outcomes in AECOPD patients were identified by multivariate logistic regression analysis. RESULTS: There were no significant differences in gender, age, body mass index, complications, systolic pressure, heart rate, mean arterial pressure, respiratory rate, oxygen saturation, partial pressure of oxygen, oxygenation index, or time of inspiration between patients with successful and failed mechanical ventilation (P > 0.05). The patients with successful noninvasive ventilation had shorter hospital stays and lower partial pressure of carbon dioxide (PaCO2) than those with failed treatment, while potential of hydrogen (pH), diaphragm thickening fraction (DTF), diaphragm activity, and diaphragm movement time were significantly higher than those with failed treatment (P < 0.05). pH [odds ratio (OR) = 0.005, P < 0.05], PaCO2 (OR = 0.430, P < 0.05), and DTF (OR = 0.570, P < 0.05) were identified to be independent factors influencing the outcome of mechanical ventilation in AECOPD patients. CONCLUSION: The DUS index DTF can better predict the outcome of non-invasive ventilation in AECOPD patients.

Discriminative analysis of aroma profiles in diverse cigar products varieties through integrated sensory evaluation, GC-IMS and E-nose.

Cigars, treasured for their rich aromatic profiles, occupy a notable segment in the global consumer market. The objective of this study was to characterize the volatile aroma compounds that shape the flavor profiles of six distinct varieties of Great Wall cigars, contributing to the understanding of cigar aroma analysis. Utilizing HS-GC-IMS and sensory evaluation, the study discerned the aroma profiles of GJ No. 6 (GJ), Animal from the Chinese zodiac (SX), Range Rover No. 3 Classic (JD), Miracle 132 (QJ), Sheng Shi No. 5 (SS), and Red 132 (HS) cigars. The analysis uncovered a spectrum of characteristic aromas, including tobacco, creaminess, cocoa, leather, baking, herbaceous, leathery, woodsy, and fruity notes. A total of 88 compounds were identified, categorized into 11 chemical classes, with their quantities varying among the cigars in a descending order of QJ, JD, GJ, SS, HS, and SX. 24 compounds, such as 2-heptanone, n-butanol, 2,6-dimethylpyrazine and 2-furfuryl methyl sulfide were considered as key differential components. The volatile components were effectively differentiated using principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and cluster analysis, revealing correlations between sensory attributes, key components, and electronic nose (E-nose). This research introduces a novel method for analyzing volatile aroma components in cigars, offering insights to enhance cigar quality and to foster the development of new products with unique aroma profiles.

Epigenetic profiling reveals key genes and cis-regulatory networks specific to human parathyroids.

In all terrestrial vertebrates, the parathyroid glands are critical regulators of calcium homeostasis and the sole source of parathyroid hormone (PTH). Hyperparathyroidism and hypoparathyroidism are clinically important disorders affecting multiple organs. However, our knowledge regarding regulatory mechanisms governing the parathyroids has remained limited. Here, we present the comprehensive maps of the chromatin landscape of the human parathyroid glands, identifying active regulatory elements and chromatin interactions. These data allow us to define regulatory circuits and previously unidentified genes that play crucial roles in parathyroid biology. We experimentally validate candidate parathyroid-specific enhancers and demonstrate their integration with GWAS SNPs for parathyroid-related diseases and traits. For instance, we observe reduced activity of a parathyroid-specific enhancer of the Calcium Sensing Receptor gene, which contains a risk allele associated with higher PTH levels compared to the wildtype allele. Our datasets provide a valuable resource for unraveling the mechanisms governing parathyroid gland regulation in health and disease.

Strip biosensors based on broad-spectrum aptamers and cationic polymers for the on-site rapid detection of tetracycline antibiotics residues in milk.

A lateral flow chromatography strip (LFS) is a chromatography-based biosensor with advantages of convenient portability, simple operation and rapid detection. In this study, a novel rapid detection technique of aptamer-based chromatography strip was developed and used for the first time for the residue detection of tetracycline antibiotics (TCs) in various milk samples. In this method, gold nanoparticles (AuNPs) modified by TCs specific aptamers were used as probes, and cationic polymers as capture molecules in a test line (T-line). Meanwhile, the analysis of the gray value of the T-line enabled a linear detection range of 1-300 nM and a limit of detection (LOD) of 0.33 nM for quantitative analysis. The biosensor demonstrated high specificity, good stability, and successfully detected tetracyclines in milk samples with recovery rate of 93.60%-106.20%. This method sets a basis for multi-residue antibiotics detection in diverse samples, showing potential for on-site applications.

Novel ratiometric electrochemical aptasensor based on broad-spectrum aptamer recognition for simultaneous detection of penicillin antibiotics in milk.

To effectively monitor multi-residues of penicillin antibiotics (PENs) in milk, we developed a novel ratiometric electrochemical aptasensor enabling simultaneous detection of PENs. The aptasensor employed a broad-spectrum aptamer as a recognition element, niobium carbide functionalized with methylene blue (Nb2C-MB) as a reference signal generator, and a ferrocene-labeled aptamer (Fc-Apt) as an output signal. Electrodes were modified with Fe-N-C doped carbon nanotubes (Fe-N-C-CNTs) to amplify detection signals further. During detection, Fc-Apt binding to PENs decreased Fc current intensity (IFc) and increased MB current intensity (IMB). The simultaneous detection of PENs was achieved using IMB/IFc as a quantitative signal. Under optimal conditions, a good linear relationship between IMB/IFc and antibiotic concentration was observed, indicating the aptasensor had a robustness. The limits of detection of aptasensor for four penicillin antibiotics and their mixed targets were 0.093-0.191 nM. This work provides a new approach to multi-residue detection of the same class of antibiotics.

Genetic architecture of well-being: cumulative effect of serotonergic polymorphisms.

Serotonin influences mental health and well-being. To understand the influences of genetic variations in serotonin pathway on well-being, we examined the effects of seven serotonergic polymorphisms on subjective well-being (i.e. affective balance and global life satisfaction) and psychological well-being (i.e. optimal psychological functions in the face of existential challenges) in a larger sample. Results indicated that the cumulative genetic score, but single genetic effects of serotonergic polymorphisms, was related to individual differences in well-being. Specifically, individuals with a greater cumulative genetic score, which is related to a low risk of depression, tended to exhibit high levels of subjective well-being and psychological well-being. These findings suggest that the overall serotoninergic genetic profile, rather than a specific genetic polymorphism, could greatly influence the individual differences in well-being.

Alternating Early Afterdepolarizations Underlying Bradycardia-Dependent Macroscopic T Wave and Discordant Mechanical Alternans.

BACKGROUND: Macroscopic T wave alternans (macro-TWA) often heralds the onset of Torsades de Pointes in patients with QT prolongation. However, the mechanisms underlying macro-TWA remain unclear. We examined the cellular and ionic basis for macro-TWA in rabbits with left ventricular hypertrophy (LVH). METHODS: The renovascular hypertension model was used to induce LVH in rabbits. Action potentials were simultaneously recorded from epicardium and endocardium together with a transmural ECG and isometric contractility in arterially perfused left ventricular wedges. Late sodium current (INa-L) was recorded in single-isolated left ventricular myocytes with the whole cell patch-clamp technique. RESULTS: Macro-TWA and accompanied mechanical alternans occurred spontaneously in 8 of 33 LVH rabbits (P<0.05, versus 0/15 in controls) and were induced by an INa-L enhancer ATX-II at 1 to 3 nM in additional 7. Macro-TWA and mechanical alternans occurred discordantly, that is, that longer QT interval and larger T wave were associated with weaker isometric contvractility. Alternating early afterdepolarizations in the endocardium caused macro-TWA in 12 of 15 LVH rabbits and, therefore, early afterdepolarization-dependent R-from-T extrasystoles and Torsades de Pointes always originated from the beats with longer QT and larger T wave during macro-TWA. INa-L density was significantly larger in LVH myocytes than that of control myocytes. Macro-TWA, mechanical alternans, R-from-T extrasystoles, and Torsades de Pointes were all abolished by INa-L blocker ranolazine or mexiletine. CONCLUSIONS: LVH enhances INa-L density and promotes alternating early afterdepolarizations in the left ventricular endocardium that manifest as macro-TWA with discordant mechanical alternans. INa-L blockade abolishes macro-TWA, mechanical alternans, early afterdepolarization-dependent R-from-T extrasystoles, and Torsades de Pointes.