Applications and continued evolution of glycan imaging mass spectrometry.

Glycosylation is an important posttranslational modifier of proteins and lipid conjugates critical for the stability and function of these macromolecules. Particularly important are N-linked glycans attached to asparagine residues in proteins. N-glycans have well-defined roles in protein folding, cellular trafficking and signal transduction, and alterations to them are implicated in a variety of diseases. However, the non-template driven biosynthesis of these N-glycans leads to significant structural diversity, making it challenging to identify the most biologically and clinically relevant species using conventional analyses. Advances in mass spectrometry instrumentation and data acquisition, as well as in enzymatic and chemical sample preparation strategies, have positioned mass spectrometry approaches as powerful analytical tools for the characterization of glycosylation in health and disease. Imaging mass spectrometry expands upon these strategies by capturing the spatial component of a glycan's distribution in-situ, lending additional insight into the organization and function of these molecules. Herein we review the ongoing evolution of glycan imaging mass spectrometry beginning with widely adopted tissue imaging approaches and expanding to other matrices and sample types with potential research and clinical implications. Adaptations of these techniques, along with their applications to various states of disease, are discussed. Collectively, glycan imaging mass spectrometry analyses broaden our understanding of the biological and clinical relevance of N-glycosylation to human disease.

Quaternary pulse width modulation based ultra-high frame rate scene projector used for hard-ware-in-the-loop testing.

The scene projector (SP) can provide simulated scene images with same optical characteristics as the real scenes to evaluate imaging systems in hard-ware-in-the-loop (HWIL) simulation testing. The single scene generation device (SGD) based SP typically projects 8-bit images at 220 fps, which is insufficient to fulfill the requirements of ultra-high frame rate imaging systems, such as star trackers and space debris detectors. In this paper, an innovative quaternary pulse width modulation (PWM) based SP is developed and implemented to realize the ultra-high frame rate projection. By optically overlapping modulation layers of two digital micro-mirror devices (DMDs) in parallel, and illuminating them with light intensities, a quaternary SGD is built up to modulate quaternary digit-planes (QDs) with four grayscale levels. And the quaternary digit-plane de-composition (QDD) is adopted to decompose an 8-bit image into 4 QDs. In addition, the exposure time of each QD is controlled by quaternary PWM, and the base time is optimized to 8 µs. The experimental results prove that the total exposure time of all QDs sequentially modulated by quaternary PWM is approximately 760 µs, namely projecting 8-bit images at 1300 fps. The quaternary PWM using two DMDs in parallel dramatically improves the grayscale modulation efficiency compared to the existing projection technologies, which provides a new approach for the SP design with ultra-high frame rate.

Simulation and On-Site Detection of the Failure Characteristics of Overlying Strata under the Mining Disturbance of Coal Seams with Thin Bedrock and Thick Alluvium.

When mining deep coal seams with thin bedrock and thick alluvium, the collapse and fracture of thin bedrock layers may cause geological disasters, such as water inrush and sand inrush of the mining face. Comprehensively obtaining the response data of coal mining and reasonably analyzing the failure characteristics of overlying strata are helpful in guiding safe production. In this study, the caving zone heights of overlying strata are obtained by field detection during layered mining. Then, the caving zone heights during the once-full-height mining are evaluated by theoretical analysis. Further, the force and failure characteristics of coal-rock structures under different mining conditions are compared by the simulation detection and analysis. Finally, the results of on-site observation, theoretical analysis, and simulation detection are compared and discussed, and an optimized mining technology is proposed to ensure safe mining. The research shows the caving zone heights of on-site and simulation detections are, respectively, 14.65 m and 13.5 m during bottom-layer mining, which is larger than the caving zone heights of the top-layer coal mining. During once-full-height mining, the maximum caving zone height of simulation detection is 21 m, which is in between two standard results. For the mechanical responses of an aquiclude clay layer under thick loose alluvium, the maximum disturbance displacement of clay aquiclude is 5.8 m during layered mining, which is slightly larger than the disturbance displacement of once full-height mining; however, the maximum stress of the clay layer is 25 MPa during once-full-height mining, which is larger than the maximum stress of clay layer during layered mining. For the clay aquiclude failure, the clay layer during layered mining is in the deflection deformation area, and there is no obvious fracture structure to inrush the water and sand of thick loose alluvium; however, the clay layer during once-full-height mining is prone to produce obvious fracture structure. Therefore, the layered mining technology can effectively reduce and prevent the water/sand inrush disaster of mining working face.

Development of an Antibody-Based Platform for the Analysis of Immune Cell-Specific N-linked Glycosylation.

N-linked glycosylation plays an important role in both the innate and adaptive immune response through the modulation of cell surface receptors as well as general cell-to-cell interactions. The study of immune cell N-glycosylation is gaining interest but is hindered by the complexity of cell-type-specific N-glycan analysis. Analytical techniques such as chromatography, LC-MS/MS, and the use of lectins are all currently used to analyze cellular glycosylation. Issues with these analytical techniques include poor throughput, which is often limited to a single sample at a time, lack of structural information, the need for a large amount of starting materials, and the requirement for cell purification, thereby reducing their feasibility for N-glycan study. Here, we report the development of a rapid antibody array-based approach for the capture of specific nonadherent immune cells coupled with MALDI-IMS to analyze cellular N-glycosylation. This workflow is adaptable to multiple N-glycan imaging approaches such as the removal or stabilization and derivatization of terminal sialic acid residues providing unique avenues of analysis that have otherwise not been explored in immune cell populations. The reproducibility, sensitivity, and versatility of this assay provide an invaluable tool for researchers and clinical applications, significantly expanding the field of glycoimmunology.

Bioorthogonal Chemical Labeling Probes Targeting Sialic Acid Isomers for N-Glycan MALDI Imaging Mass Spectrometry of Tissues, Cells, and Biofluids.

Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3- and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.

High Thermoelectric Performance of Non-Stoichiometric and Oriented GeTe Thin Films.

The increasing demand for micro-thermoelectric coolers and generators promotes the research on thermoelectric (TE) thin films. As a promising medium-temperature TE material, GeTe has attracted wide attention recently. However, the thermoelectric performance of thin-film GeTe remains inferior. Herein, oriented GeTe films with excessive Ge are obtained by magnetron co-sputtering technique, which can not only reduce the carrier concentration but also increase the carrier mobility, maintaining the high electrical conductivity of GeTe. Furthermore, higher structural symmetry and grain boundary scattering enhance the Seebeck coefficient of oriented GeTe films. As a result, the power factor (PF) value can reach as high as 2848 µW m-1 K-2 at room temperature and increase to 5263 µW m-1 K-2 at 600 K. Furthermore, a TE device with the Ge-rich GeTe thin film is fabricated and the maximum output power density (power per unit area) reaches 0.3 W cm-2 at ΔT = 250 K. This work demonstrates that the stoichiometry and orientation modulations are effective strategies to improve the thermoelectric performance of GeTe thin films.

Identification of nonfunctional PABPC1L causing oocyte maturation abnormalities and early embryonic arrest in female primary infertility.

Oocyte maturation arrest, fertilization failure, and early embryonic arrest are important causes of female infertility, whereas the genetic events that contribute to these processes are largely unknown. Loss-of-function of PABPC1L in mice has been suggested to cause female infertility involved in the absence of mature oocytes or embryos in vivo or in vitro. However, the role of PABPC1L in human female reproduction remains largely elusive. In this study, we identified a homozygous missense mutation (c.536G>A, p.R179Q) and a compound heterozygous mutation (c.793C>T, p.R265W; c.1201C>T, p.Q401*) in PABPC1L in two unrelated infertile females characterized by recurrent oocyte maturation abnormalities and early embryonic arrest. These variants resulted in nonfunctional PABPC1L protein and were associated with impaired chromatin configuration and transcriptional silencing in GV oocytes. Moreover, the binding capacity of mutant PABPC1L to mRNAs related to oocyte maturation and early embryonic development was decreased significantly. Our findings revealed novel PABPC1L mutations causing oocyte maturation abnormalities and early embryonic arrest, confirming the essential role of PABPC1L in human female fertility.

Local OAM manipulation of a terahertz wave from the air filament by chirping the few-cycle vortex pump laser.

We propose a scheme to manipulate the local orbital angular momentum (OAM) of the ultra-broadband (0.1-30 THz) terahertz (THz) waves from the laser-induced short air filament via chirping the few-cycle vortex laser pump. The simulation results show that either the THz vortex pulses with linear azimuth-dependent phases or the THz angular accelerating vortex beams (AAVBs) with nonlinear azimuth-dependent phases can be produced by tuning the chirp parameter of the pump. Thus, the dominant physical mechanism for THz generation can be determined. The THz temporal and transverse spatial distributions can be also controlled by the chirp parameter. Furthermore, their local OAM density distributions present very complex structures because most of the modulated azimuthal intensity and the corresponding local angular helicity distributions are not able to cancel out completely. Via analyzing the simulated THz results at the different pump intensities, we classify the initial pump intensity into three cases. For the low intensity case, the Kerr effect comes into prominence, so the generated THz radiation shall be vortex pulses. While for the high intensity case, the leading plasma effect dominates. In contrast, when the pump intensity is at the medium level, the Kerr nonlinearity and the plasma effect may be comparable and competitive. Basically, THz AAVBs are generated for both high and medium intensity cases. Our study will provide the possibility for studying the optically induced rotation technology more intuitively from the perspective of angular momentum transfer.

FISI: frequency domain integration sequential imaging at 1.26×1013 frames per second and 108 lines per millimeter.

High spatial resolution on the image plane (intrinsic spatial resolution) has always been a problem for ultrafast imaging. Single-shot ultrafast imaging methods can achieve high spatial resolution on the object plane through amplification systems but with low intrinsic spatial resolutions. We present frequency domain integration sequential imaging (FISI), which encodes a transient dynamic by an inversed 4f (IFF) system and decodes it using optical spatial frequencies recognition (OFR), which overcomes the limitation of the spatial frequencies recognition algorithm. In an experiment on the process of an air plasma channel, FISI achieved shadow imaging of the channel with a framing rate of 1.26×1013 fps and an intrinsic spatial resolution of 108 lp/mm (the spatial resolution on the image plane). Owing to its excellent framing time and high intrinsic spatial resolution, FISI can probe both repeatable and unrepeatable ultrafast phenomena, such as laser-induced damage, plasma physics, and shockwave interactions in living cells with high quality.

What is the impact of a novel DEPDC5 variant on an infant with focal epilepsy: a case report.

BACKGROUND: Variants in the DEPDC5 have been proved to be main cause of not only various dominant familial focal epilepsies, but also sporadic focal epilepsies. In the present study, a novel variant in DEPDC5 was detected in the patient with focal epilepsy and his healthy father. We aimed to analyze the pathogenic DEPDC5 variant in the small family of three. CASE PRESENTATION: A 5-month-old male infant presented with focal epilepsy. Whole exome sequencing identified a novel heterozygous variant c.1696delC (p.Gln566fs) in DEPDC5, confirmed by Sanger sequencing. The variant was inherited from healthy father. CONCLUSIONS: Our study expands the spectrum of DEPDC5 variants. Moreover, We discuss the relation between the low penetrance of DEPDC5 and the relatively high morbidity rate of DEPDC5-related sporadic focal epilepsy. Besides, due to interfamilial phenotypic and genetic heterogeneity, we speculate the prevalence of familial focal epilepsy with variable foci might be underestimated in such small families. We emphasize the importance of gene detection in patients with sporadic epilepsy of unknown etiology, as well as their family members. It can identify causative mutations, thus providing help to clinicians in making a definitive diagnosis.

Association between frailty index based on routine laboratory tests and risk of cerebral small vessel disease in elderly patients: a hospital-based observational study.

BACKGROUND: The association between frailty and cerebral small vessel disease (CSVD) remains controversial due to the use of different methods to assess frailty, including physical frailty phenotype and frailty scores containing measures of cognition. A frailty index based on laboratory tests (FI-Lab), which assesses frailty by the combination of routine laboratory measures and several vital signs, is independent of cognition and function status. We aimed to evaluate the association of FI-Lab with CSVD. METHODS: An observational study was carried out in a hospitalized cohort of older patients with minor ischemic stroke or TIA. The FI-Lab was constructed by 20 routine laboratory tests, plus systolic blood pressure, diastolic blood pressure, and pulse pressure. Manifestations of CSVD including white matter hyperintensity (WMH), silent lacunar infarcts, microbleed, enlarged perivascular spaces (EPVS), as well as deep brain atrophy, were measured on magnetic resonance imaging (MRI). An ordinal score system constructed by WMH, EPVS, silent lacunar infarcts, and microbleed was used to reflect the total burden of CSVD. The associations between FI-lab and CSVD were examined by logistic regression analysis and ordinal regression. RESULTS: A total of 398 patients were recruited from January 2016 to December 2018. The mean FI-Lab value was 0.26 ± 0.11. The prevalence of extensive periventricular WMH, extensive deep WMH, extensive basal ganglia EPVS, extensive centrum semiovale EPVS, silent lacunar infarcts, and deep microbleed was 26.1, 66.6, 68.6, 80.7, 32.9, and 6.5%, respectively. A higher FI-Lab value was associated with increased risks of extensive deep WMH (OR = 1.622; 95% CI, 1.253 ~ 2.100), extensive basal ganglia EPVS (OR = 1.535; 95% CI, 1.187 ~ 1.985), extensive centrum semiovale EPVS (OR = 1.584; 95% CI, 1.167 ~ 2.151), silent lacunar infarcts (OR = 1.273; 95% CI, 1.007 ~ 1.608), and higher total burden of CSVD. These associations remained after the adjustment of potential confounding factors. CONCLUSION: This study demonstrated that a higher FI-Lab score might be associated with the presence of WMH, EPVS, silent lacunar infarcts, as well as severe total CSVD burden in older patients with minor stroke or TIA. The FI-Lab provides a basis for the prediction of CSVD.

Par3 is essential for the establishment of planar cell polarity of inner ear hair cells.

In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3-GEF-Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.

Generation and evolution of different terahertz singular beams from long gas-plasma filaments.

We theoretically and numerically investigate the generation and evolution of different pulsed terahertz (THz) singular beams with an ultrabroad bandwidth (0.1-40 THz) in long gas-plasma filaments induced by a shaped two-color laser field, i.e., a vortex fundamental pulse (ω0) and a Gaussian second harmonic pulse (2ω0). Based on the unidirectional propagation model under group-velocity moving reference frame, the simulating results demonstrate that three different THz singular beams, including the THz necklace beams with a π-stepwise phase profile, the THz angular accelerating vortex beams (AAVBs) with nonlinear phase profile, and the THz vortex beams with linear phase profile, are generated. The THz necklace beams are generated first at millimeter-scale length. Then, with the increase of the filament length, THz AAVBs and THz vortex beams appear in turn almost periodically. Our calculations confirm that all these different THz singular beams result from the coherent superposition of the two collinear THz vortex beams with variable relative amplitudes and conjugated topological charges (TCs), i.e., +2 and -2. These two THz vortex beams could come from the two four-wave mixing (FWM) processes, respectively, i.e., ω0+ω0-2ω0→ωTHz and -(ω0+ω0) + 2ω0→ωTHz. The evolution of the different THz singular beams depends on the combined effect of the pump ω0-2ω0 time delay and the separate, periodical, and helical plasma channels. And the TC sign of the generated THz singular beams can be easily controlled by changing the sign of the ω0-2ω0 time delay. We believe that these results will deepen the understanding of the THz singular beam generation mechanism and orbital angular momentum (OAM) conversion in laser induced gas-filamentation.

Enhance terahertz radiation and its polarization- control with two paralleled filaments pumped by two-color femtosecond laser fields.

We present experimentally an obvious enhancement of the terahertz (THz) radiation with two paralleled filaments pumped by two-color laser fields for a full use of a high laser power, compared with single filament. By mapping the 3-dimensional electric trajectories of generated THz fields with a (111) ZnTe crystal, we observe that the total THz polarization from two filaments can be manipulated by varying the time delay between the two orthogonally polarized pumps, which agrees well with the simulations under the photocurrent model. Notably, the power and spectrum of the THz field almost keep unchanged while manipulating the ellipticity of the THz polarization, which is important for a polarization-controllable THz source.

All-optical high spatial-temporal resolution photography with raster principle at 2 trillion frames per second.

A novel single-shot ultrafast all-optical photography with raster principle (OPR) that can capture real-time imaging of ultrafast phenomena is proposed and demonstrated. It consists of a sequentially timed module (STM), spectral-shaping module (SSM), and raster framing camera (RFC). STM and SSM are used for linearly encoding frequency-time mapping and system calibration, respectively. The function of the RFC is sampling the target by microlens arrays and framing on the basis of frequency-time-spatial positions conversion. We demonstrated the recording of transient scenes with the spatial resolution of ∼90lp/mm, the frame number of 12 and the frame rate of 2 trillion frames per second (Tfps) in single-shot. Thanks to its high spatial-temporal resolution, high frame rate (maximum up to 10 Tfps or more) and sufficient frame number, our OPR can observe the dynamic processes with complex spatial structure at the atomic time scale (10 fs∼1ps), which is promising for application in plasma physics, shock waves in laser-induced damage, and dynamics of condensed matter materials.

Localized modes and dark solitons sustained by nonlinear defects.

Dark solitons and localized defect modes against periodic backgrounds are considered in arrays of waveguides with defocusing Kerr nonlinearity, constituting a nonlinear lattice. Bright defect modes are supported by a local increase in nonlinearity, while dark defect modes are supported by a local decrease in nonlinearity. Dark solitons exist for both types of defects, although in the case of weak nonlinearity, they feature side bright humps, making the total energy propagating through the system larger than the energy transferred by the constant background. All considered defect modes are found stable. Dark solitons are characterized by relatively narrow windows of stability. Interactions of unstable dark solitons with bright and dark modes are described.

Hypoxia and its possible relationship with endometrial receptivity in adenomyosis: a preliminary study.

BACKGROUND: Adenomyosis (AM) is an important cause of female infertility. However, the underlying mechanism remains unclear. This report describes a preliminary study of hypoxia and its possible association with endometrial receptivity in AM. METHODS: The study was divided into in vitro and in vivo experiments. In vitro, expression levels of the endometrial receptivity markers HOXA10 and HOXA11 in the implantation period were examined using real-time PCR and western blotting. Endometrial expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, and HIF-3α was determined using immunohistochemistry. In vivo, using an AM mouse model established by oral administration of tamoxifen, we inhibited expression of HIF-2α using an HIF-2α antagonist (PT2399; 30 mg/kg body weight, twice daily by oral gavage for 2 days) and then examined expression levels of Hoxa10 and Hoxa11 using real-time PCR and western blotting. RESULTS: Endometrial mRNA and protein expression levels of HOXA10 and HOXA11 were significantly lower in patients with AM than in control patients. Expression of HIF-2α was significantly higher in the AM group than in the control group, whereas that of HIF-1α and HIF-3α was equivalent in both groups. In vivo analysis showed that administration of the HIF-2α antagonist resulted in increased expression of Hoxa10 and Hoxa11 at both the mRNA and protein levels in AM model mice. CONCLUSIONS: HIF-2α overexpression may be one reason for decreased endometrial receptivity in AM. The current findings provide insight into HIF-2α-mediated AM-related infertility and suggest that PT2399 has potential as a treatment for AM. TRIAL REGISTRATION: This trial was retrospectively registered.

LFZ-4-46, a tetrahydroisoquinoline derivative, induces apoptosis and cell cycle arrest via induction of DNA damage and activation of MAPKs pathway in cancer cells.

LFZ-4-46, that is [2-hydroxy-1-phenyl-1,5,6,10b-tetrahydropyrazolo(5,1-a) isoquinolin-3(2H)-yl](phenyl) methanone, a tetrahydroisoquinoline derivative with a pyrazolidine moiety, was synthetically prepared. The anti-cancer mechanism of the compound has not been clarified yet. In this study, the anticancer effects and potential mechanisms of LFZ-4-46 on human breast and prostate cancer cells were explored. (a) 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide assay was first performed to detect the effects of LFZ-4-46 on the viability of human cancer cells. (b) Comet assay was utilized to evaluate DNA damage. (c) Cell cycle, apoptosis and mitochondrial membrane potential were detected by flow cytometry. (d) The expression of relative proteins was detected by western blotting assay. LFZ-4-46 significantly inhibited the viability of cancer cells in a time- and dose-dependent manner and had no obviously inhibitory effect on the viability of mammary epithelial MCF-10A cells. Mechanistic studies demonstrated that LFZ-4-46-induced cell apoptosis and cycle arrest were mediated by DNA damage. It caused DNA damage through activating γ-H2AX and breaking DNA strands. Further studies showed that mitogen-activated protein kinasess pathway was involved in these activated several key molecular events. Finally, LFZ-4-46 showed a potent antitumor effect in vivo. These results suggest that LFZ-4-46 may be a potential lead compound for the treatment of breast and prostate cancer.

A tectorin-based matrix and planar cell polarity genes are required for normal collagen-fibril orientation in the developing tectorial membrane.

The tectorial membrane is an extracellular structure of the cochlea. It develops on the surface of the auditory epithelium and contains collagen fibrils embedded in a tectorin-based matrix. The collagen fibrils are oriented radially with an apically directed slant - a feature considered crucial for hearing. To determine how this pattern is generated, collagen-fibril formation was examined in mice lacking a tectorin-based matrix, epithelial cilia or the planar cell polarity genes Vangl2 and Ptk7 In wild-type mice, collagen-fibril bundles appear within a tectorin-based matrix at E15.5 and, as fibril number rapidly increases, become co-aligned and correctly oriented. Epithelial width measurements and data from Kif3acKO mice suggest, respectively, that radial stretch and cilia play little, if any, role in determining normal collagen-fibril orientation; however, evidence from tectorin-knockout mice indicates that confinement is important. PRICKLE2 distribution reveals the planar cell polarity axis in the underlying epithelium is organised along the length of the cochlea and, in mice in which this polarity is disrupted, the apically directed collagen offset is no longer observed. These results highlight the importance of the tectorin-based matrix and epithelial signals for precise collagen organisation in the tectorial membrane.