Chloroplast biogenesis involves spatial coordination of nuclear and organellar gene expression in Chlamydomonas.

The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

Gut-associated lymphoid tissue attrition associates with response to anti-α4ß7 therapy in ulcerative colitis.

Vedolizumab (VDZ) is a first-line treatment in ulcerative colitis (UC) that targets the α4ß7- mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) axis. To determine the mechanisms of action of VDZ, we examined five distinct cohorts of patients with UC. A decrease in naïve B and T cells in the intestines and gut-homing (ß7+) plasmablasts in circulation of VDZ-treated patients suggested that VDZ targets gut-associated lymphoid tissue (GALT). Anti-α4ß7 blockade in wild-type and photoconvertible (KikGR) mice confirmed a loss of GALT size and cellularity because of impaired cellular entry. In VDZ-treated patients with UC, treatment responders demonstrated reduced intestinal lymphoid aggregate size and follicle organization and a reduction of ß7+IgG+ plasmablasts in circulation, as well as IgG+ plasma cells and FcγR-dependent signaling in the intestine. GALT targeting represents a previously unappreciated mechanism of action of α4ß7-targeted therapies, with major implications for this therapeutic paradigm in UC.

Effect of α-tubulin acetylation on the doublet microtubule structure.

Acetylation of α-tubulin at the lysine 40 residue (αK40) by αTAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage resistant. αK40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule (DMT) in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that αK40 acetylation exerts a small-scale effect on the DMT structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between αK40 acetylation and phosphorylation in cilia.

Experimental strategies to improve drug-target identification in mass spectrometry-based thermal stability assays.

Mass spectrometry (MS)-based thermal stability assays have recently emerged as one of the most promising solutions for the identification of protein-ligand interactions. Here, we have investigated eight combinations of several recently introduced MS-based advancements, including the Phase-Constrained Spectral Deconvolution Method, Field Asymmetric Ion Mobility Spectrometry, and the implementation of a carrier sample as improved MS-based acquisition approaches for thermal stability assays (iMAATSA). We used intact Jurkat cells treated with a commercially available MEK inhibitor, followed by heat treatment, to prepare a set of unfractionated isobarically-labeled proof-of-concept samples to compare the performance of eight different iMAATSAs. Finally, the best-performing iMAATSA was compared to a conventional approach and evaluated in a fractionation experiment. Improvements of up to 82% and 86% were demonstrated in protein identifications and high-quality melting curves, respectively, over the conventional approach in the proof-of-concept study, while an approximately 12% improvement in melting curve comparisons was achieved in the fractionation experiment.

Gut-associated lymphoid tissue attrition associates with response to anti-α4ß7 therapy in ulcerative colitis.

Targeting the α4ß7-MAdCAM-1 axis with vedolizumab (VDZ) is a front-line therapeutic paradigm in ulcerative colitis (UC). However, mechanism(s) of action (MOA) of VDZ remain relatively undefined. Here, we examined three distinct cohorts of patients with UC (n=83, n=60, and n=21), to determine the effect of VDZ on the mucosal and peripheral immune system. Transcriptomic studies with protein level validation were used to study drug MOA using conventional and transgenic murine models. We found a significant decrease in colonic and ileal naïve B and T cells and circulating gut-homing plasmablasts (ß7+) in VDZ-treated patients, pointing to gut-associated lymphoid tissue (GALT) targeting by VDZ. Murine Peyer's patches (PP) demonstrated a significant loss cellularity associated with reduction in follicular B cells, including a unique population of epithelium-associated B cells, following anti-α4ß7 antibody (mAb) administration. Photoconvertible (KikGR) mice unequivocally demonstrated impaired cellular entry into PPs in anti-α4ß7 mAb treated mice. In VDZ-treated, but not anti-tumor necrosis factor-treated UC patients, lymphoid aggregate size was significantly reduced in treatment responders compared to non-responders, with an independent validation cohort further confirming these data. GALT targeting represents a novel MOA of α4ß7-targeted therapies, with major implications for this therapeutic paradigm in UC, and for the development of new therapeutic strategies.

Scratch-AID, a deep learning-based system for automatic detection of mouse scratching behavior with high accuracy.

Mice are the most commonly used model animals for itch research and for development of anti-itch drugs. Most laboratories manually quantify mouse scratching behavior to assess itch intensity. This process is labor-intensive and limits large-scale genetic or drug screenings. In this study, we developed a new system, Scratch-AID (Automatic Itch Detection), which could automatically identify and quantify mouse scratching behavior with high accuracy. Our system included a custom-designed videotaping box to ensure high-quality and replicable mouse behavior recording and a convolutional recurrent neural network trained with frame-labeled mouse scratching behavior videos, induced by nape injection of chloroquine. The best trained network achieved 97.6% recall and 96.9% precision on previously unseen test videos. Remarkably, Scratch-AID could reliably identify scratching behavior in other major mouse itch models, including the acute cheek model, the histaminergic model, and a chronic itch model. Moreover, our system detected significant differences in scratching behavior between control and mice treated with an anti-itch drug. Taken together, we have established a novel deep learning-based system that could replace manual quantification for mouse scratching behavior in different itch models and for drug screening.

The amyloid-ß1-42-oligomer interacting peptide D-AIP possesses favorable biostability, pharmacokinetics, and brain region distribution.

We have previously developed a unique 8-amino acid Aß42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer's disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aß42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid "seeding" in vitro (i.e., freshly monomerized versus preaggregated Aß42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aß42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer's disease.

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-EM.

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo-electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse-grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

Preparation of Doublet Microtubule Fraction for Single ParticleCryo-electron Microscopy.

Over the years, studying the ultrastructure of the eukaryotic cilia/flagella using electron microscopy (EM) has contributed significantly toward our understanding of ciliary function. Major complexes in the cilia, such as inner and outer dynein arms, radial spokes, and dynein regulatory complexes, were originally discovered by EM. Classical resin-embedding EM or cryo-electron tomography can be performed directly on the isolated cilia or in some cases, cilia directly attached to the cell body. Recently, single particle cryo-EM has emerged as a powerful structural technique to elucidate high-resolution structures of macromolecular complexes; however, single particle cryo-EM requires non-overlapping complexes, i.e., the doublet microtubule of the cilia. Here, we present a protocol to separate the doublet microtubule from the isolated cilia bundle of two species, Tetrahymena thermophila and Chlamydomonas reinhardtii, using ATP reactivation and sonication. Our approach produces good distribution and random orientation of the doublet microtubule fragments, which is suitable for single particle cryo-EM analysis.

Identification and mapping of central pair proteins by proteomic analysis.

Cilia or flagella of eukaryotes are small micro-hair like structures that are indispensable to single-cell motility and play an important role in mammalian biological processes. Cilia or flagella are composed of nine doublet microtubules surrounding a pair of singlet microtubules called the central pair (CP). Together, this arrangement forms a canonical and highly conserved 9+2 axonemal structure. The CP, which is a unique structure exclusive to motile cilia, is a pair of structurally dimorphic singlet microtubules decorated with numerous associated proteins. Mutations of CP-associated proteins cause several different physical symptoms termed as ciliopathies. Thus, it is crucial to understand the architecture of the CP. However, the protein composition of the CP was poorly understood. This was because the traditional method of identification of CP proteins was mostly limited by available Chlamydomonas mutants of CP proteins. Recently, more CP protein candidates were presented based on mass spectrometry results, but most of these proteins were not validated. In this study, we re-evaluated the CP proteins by conducting a similar comprehensive CP proteome analysis comparing the mass spectrometry results of the axoneme sample prepared from Chlamydomonas strains with and without CP complex. We identified a similar set of CP protein candidates and additional new 11 CP protein candidates. Furthermore, by using Chlamydomonas strains lacking specific CP sub-structures, we present a more complete model of localization for these CP proteins. This work has established a new foundation for understanding the function of the CP complex in future studies.

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications.

Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and ß-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.

Tubulin lattice in cilia is in a stressed form regulated by microtubule inner proteins.

Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic resolution map of the Tetrahymena doublet microtubule by cryoelectron microscopy. The map demonstrates that the network of microtubule inner proteins weaves into the tubulin lattice and forms an inner sheath. From mass spectrometry data and de novo modeling, we identified Rib43a proteins as the filamentous microtubule inner proteins in the protofilament ribbon region. The Rib43a-tubulin interaction leads to an elongated tubulin dimer distance every 2 dimers. In addition, the tubulin lattice structure with missing microtubule inner proteins (MIPs) by sarkosyl treatment shows significant longitudinal compaction and lateral angle change between protofilaments. These results are evidence that the MIPs directly affect and stabilize the tubulin lattice. It suggests that the doublet microtubule is an intrinsically stressed filament and that this stress could be manipulated in the regulation of ciliary waveforms.

Direct Synthesis of Aluminosilicate SSZ-39 Zeolite Using Colloidal Silica as a Starting Source.

For the first time, SSZ-39 zeolite has been directly prepared using conventional colloidal silica and sodium aluminate instead of using FAU zeolite as the raw material in the alkaline media. The adjustment of the Si/Al ratios in the starting materials to the suitable values is a key factor to prepare the aluminosilicate SSZ-39 zeolite. Various characterizations (for instance, X-ray diffraction, scanning electron microscopy, nitrogen sorption, solid 27Al NMR, and NH3-temperature-programmed desorption) display that the aluminosilicate SSZ-39 zeolite owns high crystallinity, uniform cuboid morphology, large surface area, four-coordinated aluminum species, and strong acidic sites. Inductively coupled plasma analysis shows that the SiO2/Al2O3 ratios of the SSZ-39 products are ranged from 12.8 to 16.8. Considering the special framework of the SSZ-39 zeolite, the yield of this synthesis is not higher than 21.3%. Moreover, the catalytic performance of Cu-SSZ-39 catalyst synthesized from this route is excellent in the selective catalytic reduction of NO x with NH3 (NH3-SCR).

Morphometric analysis of lumbar pedicles in patients with spinal dysraphism.

BACKGROUND: In patients with open neural tube defects, the incidence of scoliosis and requirement for spinal fusions are increased. Historically, there has been no standardized measurement of vertebral morphometry in these patients. However, anecdotally, patients with open neural tube defects have a more medially oriented lumbar pedicle trajectory than the average population. METHODS: A single-institution retrospective review of patients with open neural tube defects was conducted. The demographic parameters and functional and anatomical levels of the defects were noted. CT and MRI scans of the lumbar spine were analyzed; the pedicles from L 1 to S 1 were measured for width (W), length (L) and midline angle (α). The measurements were compared bilaterally, at each level, and with data from previously published reports. RESULTS: 16 scans of pediatric patients (mean = 3.0 ・} 4.3; age range = 7 days to 14.4 years; 7 males, 9 females) with a diagnosis of either myelomeningocele or lipomyelomeningocele were assessed. Most defects occurred in the lumbar region, with L 2 and L 5 accounting for 37.5% each. All angles demonstrated a quadratic increase from L 1 to S 1 (means: L 1 = 28.3 ・} 5.24° ; L 2 = 29.1 ・} 6.2°; L 3 = 33.2 ・} 6.0°; L 4 = 36.8 ・} 5.6°; L 5 = 43.8 ・} 5.9°; S 1 = 52.0 ・} 3.6°) and were more medially angulated than those reported previously; no significant difference existed between right and left measurements (W = 0.65 ≤ p ≤ 0.94; L = 0.91 ≤ p ≤ 1; α = 0.24 ≤p ≤0.86). CONCLUSIONS: Patients with open neural tube defects had more medially angled pedicle trajectories in the lumbar spine when compared to previously reported values.

Magnetic resonance imaging appearance and changes on intracavitary Gliadel wafer placement: A pilot study.

AIM: To investigate changes on magnetic resonance imaging (MRI) which occur with intracavitary Gliadel wafer placement in patients with glioblastoma multiforme (GBM). METHODS: This retrospective Health Insurance Portability and Accountability Act-compliant study was approved by the institutional review board, with a waiver of informed consent. A total of eight patients aged 29-67 years with GBM underwent Gliadel wafer placement. T2-weighted/FLAIR images and post-contrast T1-weighted images both before and after wafer placement were retrospectively reviewed in consensus to determine changes in the following parameters: appearance of the pericavitary tissue, pattern of tumor recurrence or progression and appearance of the Gliadel wafer itself. RESULTS: Five out of the eight patients had a progressive increase in enhancement and pericavitary T2/ FLAIR hyperintensity within the first 2 mo and a subsequent decrease in these MRI findings. None of these patients had tumor recurrence within the first 6 mo. Three out of the eight patients demonstrated a progressive increase in enhancement and pericavitary T2 hyperintensity, which continued after the first 6 mo, and were subsequently diagnosed with true tumor progression. There was no increase in distant/nonlocal tumor recurrence. The Gliadel wafer appearance changed over time. CONCLUSION: Pseudoprogression is common after intracavitary Gliadel wafer placement and thus care should be taken before diagnosing tumor progression or recurrence within the first 2 mo.

Analysis of visual toxicity after gamma knife radiosurgery for treatment of choroidal melanoma: identification of multiple targets and mechanisms of toxicity.

OBJECTIVES: Identification of the targets of radiation damage after radiosurgical treatment of ocular melanoma will potentially allow for sparing of vision with improved treatment planning. MATERIALS AND METHODS: Six patients with ocular melanoma, who had useful vision before therapy, were treated with gamma knife stereotactic radiosurgery with curative intent. Dosimetric analysis of functional targets of radiation damage including the fovea, optic nerve, lens, and iris was carried out. Serial testing of visual acuity and fundoscopic examination were carried out after treatment. RESULTS: Visual sparing was achieved in 3 of 6 patients at last followup with a median follow-up of 2 years. The causes of loss of vision in those patients who lost useful vision were retinal detachment, neovascular glaucoma, and optic neuropathy. CONCLUSIONS: Preradiosurgical size and location are likely predictors of posttreatment visual outcomes.

Crescent sign on magnetic resonance angiography revealing incomplete stent apposition: correlation with diffusion-weighted changes in stent-mediated coil embolization of aneurysms.

OBJECT: Few data are available on how closely stents appose the luminal vessel wall in stent-mediated coil embolization of intracranial aneurysms and on the effect of incomplete stent apposition on procedural thromboembolic complications. METHODS: Postprocedural 3-T MR diffusion-weighted imaging and time-of-flight angiography were obtained in 58 patients undergoing stent-mediated coil embolization of aneurysms using the Enterprise closed-cell and Neuroform open-cell self-expanding intracranial microstents. RESULTS: A distinctive semilunar signal pattern, identified using 3-T MR angiography, represented flow outside the confines of the stent struts in patients in whom Enterprise but not Neuroform devices were used. This pattern, designated as the crescent sign, was confirmed to correspond to incomplete stent apposition by use of high-resolution angiographic flat-panel CT scanning revealing flow ingress into and egress out of the isolated luminal wedge. The presence of the crescent sign was seen in 18 of 33 Enterprise-treated but in 0 of 25 Neuroform-treated cases, and was more likely in stents delivered in the tortuous internal carotid artery (p = 0.034). The crescent sign was strongly predictive of ipsilateral postprocedural lesions seen on diffusion-weighted imaging in the entire population (OR 18, 95% CI 4.33-74.8; p < 0.0001). In the Enterprise stent subset, ipsilateral lesions were detected on diffusion-weighted imaging in 15 (45%) of 33 cases; the crescent sign was seen in 12 (80%) of 15 patients with ipsilateral lesions on diffusion-weighted imaging, but in only 6 of 18 patients without lesions (OR 8, 95% CI 1.61-39.6; p = 0.006). CONCLUSIONS: Incomplete stent apposition is detectable on 3-T MR angiography as a crescent sign, and was found to be highly prevalent in Enterprise closed-cell design stents used to assist coil embolization of aneurysms. Incomplete stent apposition was also associated with periprocedural ipsilateral hyperintense lesions on diffusion-weighted imaging. These results identify an association between incomplete stent apposition and thromboembolic complications in stent-mediated coil embolization of intracranial aneurysms.

Magnetic resonance imaging of intramedullary spinal cord lesions: a pictorial review.

Magnetic resonance imaging is the current imaging modality of choice in the evaluation of patients presenting with myelopathic symptoms in the search for spinal cord lesions. It is important for the radiologist to recognize and differentiate nonneoplastic from the neoplastic process of the spinal cord as the differentiation of the 2 entities is extremely crucial to the neurosurgeon. This article presents a broad spectrum of benign intramedullary spinal abnormalities including syrinx, contusion, abscess, infarction, myelitis, multiple sclerosis, sarcoid, cavernoma, and arteriovenous malformation. Rare intramedullary neoplasms including dermoid tumor, astrocytoma, ependymoma, hemangioblastoma, lymphoma, ganglioneuroblastoma, and metastases are also illustrated. The clinical presentation and magnetic resonance signal characteristics as well as the differential diagnosis of the intramedullary lesions are discussed. The potential pitfalls in the differentiation of tumors from nonneoplastic disease of the spinal cord are also elucidated.