Modern approaches to improving phase contrast electron microscopy.

Although defocus can be used to generate partial phase contrast in transmission electron microscope images, cryo-electron microscopy (cryo-EM) can be further improved by the development of phase plates which increase contrast by applying a phase shift to the unscattered part of the electron beam. Many approaches have been investigated, including the ponderomotive interaction between light and electrons. We review the recent successes achieved with this method in high-resolution, single-particle cryo-EM. We also review the status of using pulsed or near-field enhanced laser light as alternatives, along with approaches that use scanning transmission electron microscopy (STEM) with a segmented detector rather than a phase plate.

Modern approaches to improving phase contrast electron microscopy.

Although defocus can be used to generate partial phase contrast in transmission electron microscope images, cryo-electron microscopy (cryo-EM) can be further improved by the development of phase plates which increase contrast by applying a phase shift to the unscattered part of the electron beam. Many approaches have been investigated, including the ponderomotive interaction between light and electrons. We review the recent successes achieved with this method in high-resolution, single-particle cryo-EM. We also review the status of using pulsed or near-field enhanced laser light as alternatives, along with approaches that use scanning transmission electron microscopy (STEM) with a segmented detector rather than a phase plate.

Chromatin binding by HORMAD proteins regulates meiotic recombination initiation.

The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.

Extended Rotational Coherence of Polar Molecules in an Elliptically Polarized Trap.

We demonstrate long rotational coherence of individual polar molecules in the motional ground state of an optical trap. In the present, previously unexplored regime, the rotational eigenstates of molecules are dominantly quantized by trapping light rather than static fields, and the main source of decoherence is differential light shift. In an optical tweezer array of NaCs molecules, we achieve a three-orders-of-magnitude reduction in differential light shift by changing the trap's polarization from linear to a specific "magic" ellipticity. With spin-echo pulses, we measure a rotational coherence time of 62(3) ms (one pulse) and 250(40) ms (up to 72 pulses), surpassing the projected duration of resonant dipole-dipole entangling gates by orders of magnitude.

Overcoming resolution loss due to thermal magnetic field fluctuations from phase plates in transmission electron microscopy.

We identify thermal magnetic field fluctuations, caused by thermal electron motion ("Johnson noise") in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.

Overcoming resolution loss due to thermal magnetic field fluctuations from phase plates in transmission electron microscopy.

We identify thermal magnetic field fluctuations, caused by thermal electron motion ("Johnson noise") in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.

Effect of a health system payment and quality improvement programme for tonsillectomy in Ontario, Canada: an interrupted time series analysis.

BACKGROUND: Tonsillectomy is among the most common and cumulatively expensive surgical procedures in children, with known variations in quality of care. However, evidence on health system interventions to improve quality of care is limited. The Quality-Based Procedures (QBP) programme in Ontario, Canada, introduced fixed episode hospital payment per tonsillectomy and disseminated a perioperative care pathway. We determined the association of this payment and quality improvement programme with tonsillectomy quality of care. METHODS: Interrupted time series analysis of children undergoing elective tonsillectomy at community and children's hospitals in Ontario in the QBP period (1 April 2014 to 31 December 2018) and the pre-QBP period (1 January 2009 to 31 January 2014) using health administrative data. We compared the age-standardised and sex-standardised rates for all-cause tonsillectomy-related revisits within 30 days, opioid prescription fills within 30 days and index tonsillectomy inpatient admission. RESULTS: 111 411 children underwent tonsillectomy: 51 967 in the QBP period and 59 444 in the pre-QBP period (annual median number of hospitals, 86 (range 77-93)). Following QBP programme implementation, revisit rates decreased for all-cause tonsillectomy-related revisits (0.48 to -0.18 revisits per 1000 tonsillectomies per month; difference -0.66 revisits per 1000 tonsillectomies per month (95% CI -0.97 to -0.34); p<0.0001). Codeine prescription fill rate continued to decrease but at a slower rate (-4.81 to -0.11 prescriptions per 1000 tonsillectomies per month; difference 4.69 (95% CI 3.60 to 5.79) prescriptions per 1000 tonsillectomies per month; p<0.0001). The index tonsillectomy inpatient admission rate decreased (1.12 to 0.23 admissions per 1000 tonsillectomies per month; difference -0.89 (95% CI -1.33 to -0.44) admissions per 1000 tonsillectomies per month; p<0.0001). CONCLUSIONS: The payment and quality improvement programme was associated with several improvements in quality of care. These findings may inform jurisdictions planning health system interventions to improve quality of care for tonsillectomy and other paediatric procedures.

Assembly of a Rovibrational Ground State Molecule in an Optical Tweezer.

We demonstrate the coherent creation of a single NaCs molecule in its rotational, vibrational, and electronic (rovibronic) ground state in an optical tweezer. Starting with a weakly bound Feshbach molecule, we locate a two-photon transition via the |c^{3}Σ_{1},v^{'}=26⟩ excited state and drive coherent Rabi oscillations between the Feshbach state and a single hyperfine level of the NaCs rovibronic ground state |X^{1}Σ,v^{''}=0,N^{''}=0⟩ with a binding energy of D_{0}=h×147044.63(11) GHz. We measure a lifetime of 3.4±1.6 s for the rovibronic ground state molecule, which possesses a large molecule-frame dipole moment of 4.6D and occupies predominantly the motional ground state. These long-lived, fully quantum-state-controlled individual dipolar molecules provide a key resource for molecule-based quantum simulation and information processing.

Critical Role for Cold Shock Protein YB-1 in Cytokinesis.

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, ß-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division.

Forming a Single Molecule by Magnetoassociation in an Optical Tweezer.

We demonstrate the formation of a single NaCs molecule in an optical tweezer by magnetoassociation through an s-wave Feshbach resonance at 864.11(5) G. Starting from single atoms cooled to their motional ground states, we achieve conversion efficiencies of 47(1)%, and measure a molecular lifetime of 4.7(7) ms. By construction, the single molecules are predominantly [77(5)%] in the center-of-mass motional ground state of the tweezer. Furthermore, we produce a single p-wave molecule near 807 G by first preparing one of the atoms with one quantum of motional excitation. Our creation of a single weakly bound molecule in a designated internal state in the motional ground state of an optical tweezer is a crucial step towards coherent control of single molecules in optical tweezer arrays.

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.

To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia-telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.

Oligodendrocyte-specific loss of Cdk5 disrupts the architecture of nodes of Ranvier as well as learning and memory.

Myelination of the central nervous system is important for normal motor and sensory neuronal function and recent studies also link it to efficient learning and memory. Cyclin-dependent kinase 5 (Cdk5) is required for normal oligodendrocyte development, myelination and myelin repair. Here we show that conditional deletion of Cdk5 by targeting with CNP (CNP;Cdk5 CKO) results in hypomyelination and disruption of the structural integrity of Nodes of Ranvier. In addition, CNP;Cdk5 CKO mice exhibited a severe impairment of learning and memory compared to controls that may reflect perturbed neuron-glial interactions. Co-culture of cortical neurons with CNP;Cdk5 CKO oligodendrocyte lineage cells resulted in a significant reduction in the density of neuronal dendritic spines. In short term fear-conditioning studies, CNP;Cdk5 CKO mice had decreased hippocampal levels of immediate early genes such as Arc and Fos, and lower levels of p-CREB and p-cofilin suggested these pathways are affected by the levels of myelination. The novel roles of Cdk5 in oligodendrocyte lineage cells may provide insights for helping understand the cognitive changes sometimes seen in demyelinating diseases such as multiple sclerosis.

Rpl13a small nucleolar RNAs regulate systemic glucose metabolism.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that form ribonucleoproteins to guide covalent modifications of ribosomal and small nuclear RNAs in the nucleus. Recent studies have also uncovered additional non-canonical roles for snoRNAs. However, the physiological contributions of these small RNAs are largely unknown. Here, we selectively deleted four snoRNAs encoded within the introns of the ribosomal protein L13a (Rpl13a) locus in a mouse model. Loss of Rpl13a snoRNAs altered mitochondrial metabolism and lowered reactive oxygen species tone, leading to increased glucose-stimulated insulin secretion from pancreatic islets and enhanced systemic glucose tolerance. Islets from mice lacking Rpl13a snoRNAs demonstrated blunted oxidative stress responses. Furthermore, these mice were protected against diabetogenic stimuli that cause oxidative stress damage to islets. Our study illuminates a previously unrecognized role for snoRNAs in metabolic regulation.

Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells.

Niemann-Pick type C disease (NP-C) is a progressive lysosomal lipid storage disease caused by mutations in the NPC1 and NPC2 genes. NPC1 is essential for transporting cholesterol and other lipids out of lysosomes, but little is known about the mechanisms that control its cellular abundance and localization. Here we show that a reduction of TMEM97, a cholesterol-responsive NPC1-binding protein, increases NPC1 levels in cells through a post-transcriptional mechanism. Reducing TMEM97 through RNA-interference reduces lysosomal lipid storage and restores cholesterol trafficking to the endoplasmic reticulum in cell models of NP-C. In TMEM97 knockdown cells, NPC1 levels can be reinstated with wild type TMEM97, but not TMEM97 missing an ER-retention signal suggesting that TMEM97 contributes to controlling the availability of NPC1 to the cell. Importantly, knockdown of TMEM97 also increases levels of residual NPC1 in NPC1-mutant patient fibroblasts and reduces cholesterol storage in an NPC1-dependent manner. Our findings propose TMEM97 inhibition as a novel strategy to increase residual NPC1 levels in cells and a potential therapeutic target for NP-C.

The Activators of Cyclin-Dependent Kinase 5 p35 and p39 Are Essential for Oligodendrocyte Maturation, Process Formation, and Myelination.

The regulation of oligodendrocyte development and myelin formation in the CNS is poorly defined. Multiple signals influence the rate and extent of CNS myelination, including the noncanonical cyclin-dependent kinase 5 (Cdk5) whose functions are regulated by its activators p35 and p39. Here we show that selective loss of either p35 or p39 perturbed specific aspects of oligodendrocyte development, whereas loss of both p35 and p39 completely inhibited the development of mature oligodendrocytes and myelination. In the absence of p35, oligodendrocyte differentiation was delayed, process outgrowth was truncated in vitro, and the patterning and extent of myelination were perturbed in the CNS of p35(-/-) mice. In the absence of p39, oligodendrocyte maturation was transiently affected both in vitro and in vivo. However, loss of both p35 and p39 in oligodendrocyte lineage cells completely inhibited oligodendrocyte progenitor cell differentiation and myelination both in vitro and after transplantation into shiverer slice cultures. Loss of p35 and p39 had a more profound effect on oligodendrocyte development than simply the loss of Cdk5 and could not be rescued by Cdk5 overexpression. These data suggest p35 and p39 have specific and overlapping roles in oligodendrocyte development, some of which may be independent of Cdk5 activation.

A murine Niemann-Pick C1 I1061T knock-in model recapitulates the pathological features of the most prevalent human disease allele.

Niemann-Pick Type C1 (NPC1) disease is a rare neurovisceral, cholesterol-sphingolipid lysosomal storage disorder characterized by ataxia, motor impairment, progressive intellectual decline, and dementia. The most prevalent mutation, NPC1(I1061T), encodes a misfolded protein with a reduced half-life caused by ER-associated degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce cholesterol storage in fibroblasts but have not been tested in vivo because of lack of a suitable animal model. Whereas the prominent features of human NPC1 disease are replicated in the null Npc1(-/-) mouse, this model is not amenable to examining proteostatic therapies. The objective of the present study was to develop an NPC1 I1061T knock-in mouse in which to test proteostatic therapies. Compared with the Npc1(-/-) mouse, this Npc1(tm(I1061T)Dso) model displays a less severe, delayed form of NPC1 disease with respect to weight loss, decreased motor coordination, Purkinje cell death, lipid storage, and premature death. The murine NPC1(I1061T) protein has a reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated degradation, and can be stabilized by histone deacetylase inhibition. This novel mouse model faithfully recapitulates human NPC1 disease and provides a powerful tool for preclinical evaluation of therapies targeting NPC1 protein variants with compromised stability.

Cholesterol homeostatic responses provide biomarkers for monitoring treatment for the neurodegenerative disease Niemann-Pick C1 (NPC1).

Niemann-Pick C1 (NPC1) disease is a rare, neurodegenerative lysosomal cholesterol storage disorder, typified by progressive cognitive and motor function impairment. Affected individuals usually succumb to the disease in adolescence. 2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) has emerged as a promising intervention that reduces lipid storage and prolongs survival in NPC1 disease animal models. A barrier to the development of HP-ß-CD and other treatments for NPC disease has been the lack of validated biochemical measures to evaluate efficacy. Here we explored whether cholesterol homeostatic responses resulting from HP-ß-CD-mediated redistribution of sequestered lysosomal cholesterol could provide biomarkers to monitor treatment. Upon direct CNS delivery of HP-ß-CD, we found increases in plasma 24(S)-HC in two independent NPC1 disease animal models, findings that were confirmed in human NPC1 subjects receiving HP-ß-CD. Since circulating 24(S)-HC is almost exclusively CNS-derived, the increase in plasma 24(S)-HC provides a peripheral, non-invasive measure of the CNS effect of HP-ß-CD. Our findings suggest that plasma 24(S)-HC, along with the other cholesterol-derived markers examined in this study, can serve as biomarkers that will accelerate development of therapeutics for NPC1 disease.

Identification of Niemann-Pick C1 disease biomarkers through sphingolipid profiling.

Niemann-Pick type C (NPC)1 is a rare neurodegenerative disease for which treatment options are limited. A major barrier to development of effective treatments has been the lack of validated biomarkers to monitor disease progression or serve as outcome measures in clinical trials. Using targeted metabolomics to exploit the complex lipid storage phenotype that is the hallmark of NPC1 disease, we broadly surveyed Npc1(-/-) mouse tissues and identified elevated species across multiple sphingolipid classes that increased with disease progression. There was a striking accumulation of sphingoid bases, monohexosylceramides (MCs), and GM2 gangliosides in liver, and sphingoid bases and GM2 and GM3 gangliosides in brain. These lipids were modestly decreased following miglustat treatment, but markedly decreased in response to treatment with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), two drugs that have shown efficacy in NPC1 animal models. Extending these studies to human subjects led to identification of sphingolipid classes that were significantly altered in the plasma of NPC1 patients. Plasma MCs and ceramides were elevated, whereas sphingoid bases were reduced in NPC1 subjects. Intervention with miglustat in NPC1 patients was accompanied by striking alterations in plasma (reductions in GM1 and GM3 gangliosides) and cerebrospinal fluid (CSF) (increased MCs) sphingolipids. Similar alterations were observed in the CSF from the NPC1 feline model following HP-ß-CD treatment. Our findings suggest that these lipid biomarkers may prove useful as outcome measures for monitoring efficacy of therapy in clinical trials.

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma.

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3ß,5α,6ß-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3ß,5α,6ß-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3ß,5α,6ß-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.