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Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

OBJECTIVES Platelet-rich fibrin (PRF) has gained tremendous momentum in recent years as a natural autologous growth factor derived from blood capable of stimulating tissue regeneration. Owing to its widespread use, many companies have commercialized various centrifugation devices with various proposed protocols. The aim of the present study was to compare 3 different commercially available centrifuges at both high and low g-force protocols. MATERIALS AND METHODS PRF was produced on three commercially available centrifuges including the IntraSpin Device (IntraLock), the Duo Quattro (Process for PRF), and Salvin (Salvin Dental). Two separate protocols were tested on each machine including the original leukocyte and platelet-rich fibrin (L-PRF) protocol (~ 700 RCF max (~ 400 RCF clot) for 12 min) as well as the advanced platelet-rich fibrin (A-PRF+) protocol (~ 200 g RCF max (~ 130 g RCF clot) for 8 min). Each of the tested groups was compared for cell numbers, growth factor release, scanning electron microscopy (SEM) for morphological differences, and clot size (both weight and length/width). RESULTS The present study found that PRF clots produced utilizing the low-speed centrifugation speeds (~ 200 g for 8 min) produce clots that (1) contained a higher concentration of evenly distributed platelets, (2) secreted higher concentrations of growth factors over a 10 day period, and (3) were smaller in size. This was irrespective of the centrifugation device utilized and consistently observed on all 3 devices. The greatest impact was found between the protocols utilized (up to a 200%). Interestingly, it was further revealed that the centrifugation tubes used had a much greater impact on the final size outcome of PRF clots when compared to centrifugation devices. It was found that, in general, the Process for PRF tubes produced significantly greater-sized clots when compared to other commercially available tubes. The Salvin Dental tubes also produced significantly greater PRF clots when compared to the IntraLock tubes on each of the tested centrifugation devices. CONCLUSIONS The present study demonstrated the reproducibility of a scientific concept (reduction in RCF produces PRF clots with more evenly distributed cells and growth factors) utilizing different devices. Furthermore, (and until now overlooked), it was revealed for the first time that the centrifugation tubes are central to the quality production of PRF. Future research investigating tube characteristics thus becomes critically important for the future optimization of PRF. CLINICAL RELEVANCE This is the first study to reveal the marked impact of centrifugation tubes on the final production of PRF. Future study thus becomes markedly important to further optimize the quality of PRF-based matrices. It was further found that little variability existed between the centrifugation devices if optimized centrifugation protocols (lower centrifugation speeds) were utilized.

In order to adapt different scale land cover segmentation, an optimized approach under the guidance of k-means clustering for multi-scale segmentation is proposed. At first, small scale segmentation and k-means clustering are used to process the original images; then the result of k-means clustering is used to guide objects merging procedure, in which Otsu threshold method is used to automatically select the impact factor of k-means clustering; finally we obtain the segmentation results which are applicable to different scale objects. FNEA method is taken for an example and segmentation experiments are done using a simulated image and a real remote sensing image from GeoEye-1 satellite, qualitative and quantitative evaluation demonstrates that the proposed method can obtain high quality segmentation results.