A major frontier in single cell biology is decoding how transcriptional states result in cellular-level architectural changes, ultimately driving function. A remarkable example of this cellular remodelling program is the differentiation of airway stem cells into the human respiratory multiciliated epithelium, a tissue barrier protecting against bacteria, viruses and particulate matter. Here, we present the first isotropic three-dimensional map of the airway epithelium at the nanometre scale unveiling the coordinated changes in cellular organisation, organelle topology and contacts, occurring during multiciliogenesis. This analysis led us to discover a cellular mechanism of communication whereby motile cilia relay mechanical information to mitochondria through striated cytoskeletal fibres, the rootlets, to promote effective ciliary motility and ATP generation. Altogether, this study integrates nanometre-scale structural, functional and dynamic insights to elucidate fundamental mechanisms responsible for airway defence.

PURPOSE: This paper describes an approach for the three-dimensional (3D) shape and pose reconstruction of the human rib cage from few segmented two-dimensional (2D) projection images. Our work is aimed at supporting temporal subtraction techniques of subsequently acquired radiographs by establishing a method for the assessment of pose differences in sequences of chest radiographs of the same patient.

METHODS: The reconstruction method is based on a 3D statistical shape model (SSM) of the rib cage, which is adapted to binary 2D projection images of an individual rib cage. To drive the adaptation we minimize a distance measure that quantifies the dissimilarities between 2D projections of the 3D SSM and the projection images of the individual rib cage. We propose different silhouette-based distance measures and evaluate their suitability for the adaptation of the SSM to the projection images.

RESULTS: An evaluation was performed on 29 sets of biplanar binary images (posterior-anterior and lateral). Depending on the chosen distance measure, our experiments on the combined reconstruction of shape and pose of the rib cages yield reconstruction errors from 2.2 to 4.7 mm average mean 3D surface distance. Given a geometry of an individual rib cage, the rotational errors for the pose reconstruction range from 0.1 degrees to 0.9 degrees.

CONCLUSIONS: The results show that our method is suitable for the estimation of pose differences of the human rib cage in binary projection images. Thus, it is able to provide crucial 3D information for registration during the generation of 2D subtraction images.

Cryogenic electron tomography (cryo-ET) has gained increasing interest in recent years due to its ability to image whole cells and subcellular structures in 3D at nanometer resolution in their native environment. However, due to dose restrictions and the inability to acquire high tilt angle images, the reconstructed volumes are noisy and have missing information. Thus, features are unreliable, and precision extraction of the cell boundary is difficult, manual and time intensive. This paper presents an efficient recursive algorithm called BLASTED (Boundary Localization using Adaptive Shape and Texture Discovery) to automatically extract the cell boundary using a conditional random field (CRF) framework in which boundary points and shape are jointly inferred. The algorithm learns the texture of the boundary region progressively, and uses a global shape model and shape-dependent features to propose candidate boundary points on a slice of the membrane. It then updates the shape of that slice by accepting the appropriate candidate points using local spatial clustering, the global shape model, and trained boosted texture classifiers. The BLASTED algorithm segmented the cell membrane over an average of 93% of the length of the cell in 19 difficult cryo-ET datasets.

New methods in stem cell 3D organoid tissue culture, advanced imaging and big data image analytics now allow tissue scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice Light-Sheet Imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70µm x 60µm x 40µm xyz) at 5.7s/frame. We developed an open source data analysis package termed pyLattice to process the resulting large (∼60Gb) movie datasets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. Based on their localization in the organoid, we classified CME tracks into apical, lateral and basal events and found that CME dynamics are similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative, high temporal and spatial resolution analysis of subcellular events within tissues. Movie S1 Movie S1 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid showing clathrin (red) and dynamin2 (green) puncta in surface depiction. The movie zooms out from a single clathrin mediated endocytosis event that shows both clathrin and dynamin2 at the same location to eventually show the whole AO-LLSM field of view. Nuclear envelopes and the outer membranes of the tissue are depicted in transparent white. Movie S2 Movie S2 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid showing clathrin (red) and dynamin2 (green) puncta in surface depiction. The movie rotates the AO-LLSM field of view. Nuclear envelopes and the outer membranes of the tissue are depicted in transparent white. Movie S3 Movie S3 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid. The curved surface is of the spherical organoid is visible as the movie rotates. Clathrin puncta are visible throughout the tissue (white). Movie S4 Movie S4 The detection step in the data processing pipeline retrieves all clathrin puncta in the volume. Detected puncta are marked with a cube (blue). Movie S5 Movie S5 Zoom on one clathrin puncta in the thresholded 3D dataset. The punctum of interest is marked with a blue cube. Other puncta are also visible. Movie S6 Movie S6 Zoom on the same clathrin puncta as in M3 in non-thresholded 3D data. The surrounding fluorescence is visible as a transparent cloud.

Understanding how neural circuits control behavior requires monitoring a large population of neurons with high spatial resolution and volume rate. Here we report an axicon-based Bessel beam module with continuously adjustable depth of focus (CADoF), that turns frame rate into volume rate by extending the excitation focus in the axial direction while maintaining high lateral resolutions. Cost-effective and compact, this CADoF Bessel module can be easily integrated into existing two-photon fluorescence microscopes. Simply translating one of the relay lenses along its optical axis enabled continuous adjustment of the axial length of the Bessel focus. We used this module to simultaneously monitor activity of spinal projection neurons extending over 60 µm depth in larval zebrafish at 50 Hz volume rate with adjustable axial extent of the imaged volume.

Direct identification of macromolecular complexes in their native context remains a major barrier to unbiased biological discovery. This challenge is particularly acute in mammalian sperm nuclei, in which condensed chromatin is interspersed with poorly understood phase-separated compartments termed nuclear vacuoles. Vacuoles are associated with reduced fertilization efficiency, yet their composition remains unclear. Here we combine high-resolution in situ cryo-electron tomography (cryo-ET) with AlphaFold docking to identify vacuole components as proteasomes, the proteasome activator PA200, and ferritin. In situ structures at resolutions up to 3.8 Å reveal distinct proteasome-PA200 associations and gating states, consistent with a stepwise activation mechanism. Ferritin assemblies exhibit heterogeneous mineralization states and directly contact chromatin. Together, these findings establish the molecular organization of sperm nuclear vacuoles and implicate protein turnover and metal homeostasis in shaping the nuclear landscape, while demonstrating the power of in situ cryo-ET to resolve protein identity and conformational dynamics in native cellular environments.

Sleep is regulated by a homeostatic process and associated with an increased arousal threshold, but the genetic and neuronal mechanisms that implement these essential features of sleep remain poorly understood. To address these fundamental questions, we performed a zebrafish genetic screen informed by human genome-wide association studies. We found that mutation of serine/threonine kinase 32a (stk32a) results in increased sleep and impaired sleep homeostasis in both zebrafish and mice, and that stk32a acts downstream of neurotensin signaling and the serotonergic raphe in zebrafish. stk32a mutation reduces phosphorylation of neurofilament proteins, which are co-expressed with stk32a in neurons that regulate motor activity and in lateral line hair cells that detect environmental stimuli, and ablating these cells phenocopies stk32a mutation. Neurotensin signaling inhibits specific sensory and motor populations, and blocks stimulus-evoked responses of neurons that relay sensory information from hair cells to the brain. Our work thus shows that stk32a is an evolutionarily conserved sleep regulator that links neuropeptidergic and neuromodulatory systems to homeostatic sleep drive and changes in arousal threshold, which are implemented through suppression of specific sensory and motor systems.

To grasp the international developing tendency of acupuncture research and provide some references for promoting acupuncture and moxibustion internationalization process, the articles about acupuncture in Science Citation Index (SCI) periodicals in 2007 were retrieved by adopting the retrieval tactics on line in combination with database searching. Results indicate that 257 articles about acupuncture had been retrived from the SCI Web databases. These articles were published in 125 journals respectively, most of which were Euramerican journals. Among these journals, the impact factor of the Journal of the American Medical Association (JAMA), 25. 547, is the highest one. It is shown that the impact factors of the SCI periodicals, in which acupuncture articles embodied are increased, the quality of these articles are improved obviously and the types of the articles are various in 2007, but there is obvious difference in the results of these studies due to the difference of experimental methods, the subjects of these experiments and acupuncture manipulations. Therefore, standardization of many problems arising from the researches on acupuncture is extremely imminent.

Under the situation of the rapid expansion of hospital, the dilemma of acupuncture-moxibustion department, as well as the relevant solutions are explored. The main reasons for the shrinking situation of the service in acupuncture-moxibustion department include: the disease-based department division trends to divert many diseases suitably treated in acupuncture-moxibustion department; the environment pursuing economic benefits restricts the development of acupuncture-moxibustion therapy characterized by "simple and low-cost operation". There are three important approaches for breaking through the dilemma of acupuncture and moxibustion therapy. First, modifying the traditional service mode as waiting for patients in acupuncture-moxibustion department and promoting acupuncture and moxibustion technology to be adopted in other departments rather than limited only in acupuncture-moxibustion department. Second, increasing the charges of acupuncture and moxibustion technology rationally. Third, positioning accurately the role of acupuncture and moxibustion technology in health services based on its own characteristics and advantages and promoting it in community medical institutions. All of these solutions require the guidance of supporting policies.

To explore the role of Bid protein in the mitochondria and endoplasmic reticulum (ER) associated apoptotic pathway.