A new family of bacterial ribosome hibernation factors.

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.

Morphological diversity of single neurons in molecularly defined cell types.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits.

Activation of the Endonuclease that Defines mRNA 3' Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex.

Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3' end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit "CPFcore" complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3' end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.

Structure of the Fanconi anaemia monoubiquitin ligase complex.

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.

Crumbs2 mediates ventricular layer remodelling to form the spinal cord central canal.

In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S-CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.

Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus.

The 2A protein of Theiler's murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed -1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.

Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

Attribution of Space-Time Variability in Global-Ocean Dissolved Inorganic Carbon.

The inventory and variability of oceanic dissolved inorganic carbon (DIC) is driven by the interplay of physical, chemical, and biological processes. Quantifying the spatiotemporal variability of these drivers is crucial for a mechanistic understanding of the ocean carbon sink and its future trajectory. Here, we use the Estimating the Circulation and Climate of the Ocean-Darwin ocean biogeochemistry state estimate to generate a global-ocean, data-constrained DIC budget and investigate how spatial and seasonal-to-interannual variability in three-dimensional circulation, air-sea CO2 flux, and biological processes have modulated the ocean sink for 1995-2018. Our results demonstrate substantial compensation between budget terms, resulting in distinct upper-ocean carbon regimes. For example, boundary current regions have strong contributions from vertical diffusion while equatorial regions exhibit compensation between upwelling and biological processes. When integrated across the full ocean depth, the 24-year DIC mass increase of 64 Pg C (2.7 Pg C year-1) primarily tracks the anthropogenic CO2 growth rate, with biological processes providing a small contribution of 2% (1.4 Pg C). In the upper 100 m, which stores roughly 13% (8.1 Pg C) of the global increase, we find that circulation provides the largest DIC gain (6.3 Pg C year-1) and biological processes are the largest loss (8.6 Pg C year-1). Interannual variability is dominated by vertical advection in equatorial regions, with the 1997-1998 El Niño-Southern Oscillation causing the largest year-to-year change in upper-ocean DIC (2.1 Pg C). Our results provide a novel, data-constrained framework for an improved mechanistic understanding of natural and anthropogenic perturbations to the ocean sink.

Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler's murine encephalomyelitis virus.

Many viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a 'slippery' sequence and a 3'-adjacent RNA structure. Recently, we showed that -1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.

Pharyngoplasty for Speech Disorders Following Brain Injury.

INTRODUCTION: Dysarthria is one of the commonest neurological speech disorders resulting from brain injury. However, hypernasality commonly co-exists in this subgroup of patients and is commonly overlooked. The authors aim to investigate the merit of surgery in improving hypernasality and speech intelligibility in patients with a mixed pattern of dysarthria and hypernasality secondary to brain injury. MATERIALS AND METHODS: Data was collected from the regional plastic surgery unit over a 10-year period. All patients who underwent a pharyngoplasty for speech improvement following total brain injury from either a traumatic injury or a cerebrovascular accident were included. Patients were followed up post-operatively to assess; improvement in speech rehabilitation, complications and the need for surgical revision. RESULTS: Six patients had a pharyngoplasty for speech improvement. Either a Hynes or Jackson pharyngoplasty was performed, with one patient requiring a hemi-pharyngoplasty. Post-operatively, 1 patient experienced self-limiting sleep apnea which resolved within 1 month. One patient developed obstructive symptoms and required revision. Overall, 83% of patients had clear improvement in speech intelligibility and articulation. CONCLUSIONS: The authors have shown that surgical intervention, in the form of a pharyngoplasty, is an effective method of improving speech intelligibility and articulation, by improving hypernasality and restoring communication in this cohort of patients. The aim of this paper is to highlight this option to colleagues and to heighten the awareness that many patients with a total brain injury have a mixed pattern of speech disturbance and not solely the dysarthria that is attributed to this condition.

Throat Packs in Cleft and Craniofacial Surgery.

ABSTRACT: Throat packs are used in a wide range of surgical and dental procedures. Indications for use include; airway stabilization, reducing the risk of aspiration, and postoperative nausea and vomiting. Despite these perceived benefits, a retained throat pack can be fatal. This has been highlighted since the 1970s but as of yet no effective fail-safe mechanisms exist to prevent the potential tragic consequences of a retained throat pack.A multifaceted questionnaire was distributed at the annual Craniofacial Society of Great Britain and Ireland 2017 to identify current views and practices amongst cleft surgeons. The questionnaire contained seventeen questions related to throat pack use. The responses to the questions were collated and discussed in the context of the current literature.Twenty-four cleft surgeons completed the questionnaire; 67% always use a throat pack, with 84% not securing the throat pack in any way. Almost half (48%) had encountered a serious adverse incident secondary to the use of a throat pack.This survey and review of the literature highlight ongoing discrepancies and a lack of standardization on a national basis. Adverse incidents and fatalities are still occurring. Overwhelming evidence now confirms the lack of benefit regarding postoperative nausea and vomiting and the authors urge both surgeons and anesthetists to strongly consider the need for a throat pack in routine elective procedures.

Human and Doll's Hair in a Gastric Trichobezoar, Endoscopic Retrieval Hazards.

Trichobezoars are masses of ingested hair, usually the individual's own hair, that accumulate in the gastrointestinal tract, most commonly in the stomach. When extending into the small intestine, this is termed "Rapunzel syndrome." Removal has traditionally been by laparotomy; however, successful endoscopic removal has also been described. We report the case of a 9-year-old-girl with undiagnosed coeliac disease and Rapunzel syndrome who underwent endoscopic removal of a large trichobezoar, which was followed by unexpected multiple perforations of the small bowel and stomach. Argon plasma coagulation (APC) and snare electrocautery were employed during endoscopy to remove the trichobezoar piecemeal, and approximately 70% was removed without any clear signs of damage to the mucosa. It was discovered subsequently that about 20 of her dolls were found without hair. On investigating the composition of a specific doll hair from the manufacturer, it was discovered that it could be hazardous if burned. It was, therefore, hypothesized that a constellation of factors had conspired to lead to perforation, that is, the potentially hazardous gas produced from the electrical energy applied to the synthetic hair and possible mucosal damage by the physical abrasion of this hair. A review of the literature on endoscopic attempts to remove trichobezoars irrespective of the result reveals a success rate of 30.7%.

The Cleft Nurse Specialist: A Key Building Block in the Cleft Multidisciplinary Team.

INTRODUCTION: The cleft nurse specialist (CNS) plays a key role in counselling and supporting parents from the diagnosis onward. The CNS started in 2012 and we aimed to perform a qualitative study to determine the benefits this brought to the cleft community from the parents' perspective. METHODS: The cleft database was used to locate babies born in 2010/2011 and 2013/2014. Parents were contacted by phone by 2 authors and completed a questionnaire on the care and support they received following the diagnosis, in the early days and around the time of surgery. RESULTS: Parents of 38 babies completed the survey. In 2010/2011, only 21% had an antenatal diagnosis compared to 47% in 2013/2014.2011/2012: 3 unilateral cleft lip and palate (UCLP), 3 bilateral cleft lip and palate (BCLP), 4 CLO, 9 cleft palate only (CPO).2013/2014: 5 UCLP, 7 BCLP, 7 CPO.2011/2012: 68% were counselled by a surgeon, 42% were seen >1 week after birth, with some over a month.2013/2014: 84% were counselled by the CNS, 53% were seen within 48 hours, and 100% within 7 days.Parents in 2013/2014 felt more supported by the cleft team throughout pregnancy and the early days, with home visits being particularly advantageous. CONCLUSION: The introduction of the CNS to the cleft multidisciplinary team has significantly improved the pathway for parents and is a key link with the wider cleft team. With the improvement in antenatal diagnosis, counselling occurs at an earlier stage and prepares parents for the difficulties commonly experienced in the early days. Key themes included; home visits and direct contact with the CNS.

A low-complexity region in the YTH domain protein Mmi1 enhances RNA binding.

Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR, and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1-RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA-binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding.

Cranial subarachnoid and subdural haemorrhage caused by spinal melanoma metastasis.

A 73-year-old female with a history of metastatic melanoma presented with extensive intracranial subarachnoid haemorrhage. Cranial imaging failed to reveal any vascular anomaly or tumour. MRI of the neuroaxis revealed a melanoma metastasis at T10 with associated subarachnoid haemorrhage. We review the literature on this rare presentation of subarachnoid haemorrhage.

Molecular Mechanisms of Disease Pathogenesis Differ in Krabbe Disease Variants.

Krabbe disease is a severe, fatal neurodegenerative disorder caused by defects in the lysosomal enzyme galactocerebrosidase (GALC). The correct targeting of GALC to the lysosome is essential for the degradation of glycosphingolipids including the primary lipid component of myelin. Over 100 different mutations have been identified in GALC that cause Krabbe disease but the mechanisms by which they cause disease remain unclear. We have generated monoclonal antibodies against full-length human GALC and used these to monitor the trafficking and processing of GALC variants in cell-based assays and by immunofluorescence microscopy. Striking differences in the secretion, processing and endosomal targeting of GALC variants allows the classification of these into distinct categories. A subset of GALC variants are not secreted by cells, not proteolytically processed, and remain trapped in the ER; these are likely to cause disease due to protein misfolding and should be targeted for pharmacological chaperone therapies. Other GALC variants can be correctly secreted by cells and cause disease due to catalytic defects in the enzyme active site, inappropriate post-translational modification or a potential inability to bind essential cofactors. The classification of disease pathogenesis presented here provides a molecular framework for appropriate targeting of future Krabbe disease therapies.

Structural snapshots illustrate the catalytic cycle of ß-galactocerebrosidase, the defective enzyme in Krabbe disease.

Glycosphingolipids are ubiquitous components of mammalian cell membranes, and defects in their catabolism by lysosomal enzymes cause a diverse array of diseases. Deficiencies in the enzyme ß-galactocerebrosidase (GALC) cause Krabbe disease, a devastating genetic disorder characterized by widespread demyelination and rapid, fatal neurodegeneration. Here, we present a series of high-resolution crystal structures that illustrate key steps in the catalytic cycle of GALC. We have captured a snapshot of the short-lived enzyme-substrate complex illustrating how wild-type GALC binds a bona fide substrate. We have extensively characterized the enzyme kinetics of GALC with this substrate and shown that the enzyme is active in crystallo by determining the structure of the enzyme-product complex following extended soaking of the crystals with this same substrate. We have also determined the structure of a covalent intermediate that, together with the enzyme-substrate and enzyme-product complexes, reveals conformational changes accompanying the catalytic steps and provides key mechanistic insights, laying the foundation for future design of pharmacological chaperones.

Glioblastoma and radiotherapy: A multicenter AI study for Survival Predictions from MRI (GRASP study).

BACKGROUND: The aim was to predict survival of glioblastoma at 8 months after radiotherapy (a period allowing for completing a typical course of adjuvant temozolomide), by applying deep learning to the first brain MRI after radiotherapy completion. METHODS: Retrospective and prospective data were collected from 206 consecutive glioblastoma, isocitrate dehydrogenase -wildtype patients diagnosed between March 2014 and February 2022 across 11 UK centers. Models were trained on 158 retrospective patients from 3 centers. Holdout test sets were retrospective (n = 19; internal validation), and prospective (n = 29; external validation from 8 distinct centers). Neural network branches for T2-weighted and contrast-enhanced T1-weighted inputs were concatenated to predict survival. A nonimaging branch (demographics/MGMT/treatment data) was also combined with the imaging model. We investigated the influence of individual MR sequences; nonimaging features; and weighted dense blocks pretrained for abnormality detection. RESULTS: The imaging model outperformed the nonimaging model in all test sets (area under the receiver-operating characteristic curve, AUC P = .038) and performed similarly to a combined imaging/nonimaging model (P > .05). Imaging, nonimaging, and combined models applied to amalgamated test sets gave AUCs of 0.93, 0.79, and 0.91. Initializing the imaging model with pretrained weights from 10 000s of brain MRIs improved performance considerably (amalgamated test sets without pretraining 0.64; P = .003). CONCLUSIONS: A deep learning model using MRI images after radiotherapy reliably and accurately determined survival of glioblastoma. The model serves as a prognostic biomarker identifying patients who will not survive beyond a typical course of adjuvant temozolomide, thereby stratifying patients into those who might require early second-line or clinical trial treatment.

Structural and Functional Insights into Viral Programmed Ribosomal Frameshifting.

Protein synthesis by the ribosome is the final stage of biological information transfer and represents an irreversible commitment to gene expression. Accurate translation of messenger RNA is therefore essential to all life, and spontaneous errors by the translational machinery are highly infrequent (∼1/100,000 codons). Programmed -1 ribosomal frameshifting (-1PRF) is a mechanism in which the elongating ribosome is induced at high frequency to slip backward by one nucleotide at a defined position and to continue translation in the new reading frame. This is exploited as a translational regulation strategy by hundreds of RNA viruses, which rely on -1PRF during genome translation to control the stoichiometry of viral proteins. While early investigations of -1PRF focused on virological and biochemical aspects, the application of X-ray crystallography and cryo-electron microscopy (cryo-EM), and the advent of deep sequencing and single-molecule approaches have revealed unexpected structural diversity and mechanistic complexity. Molecular players from several model systems have now been characterized in detail, both in isolation and, more recently, in the context of the elongating ribosome. Here we provide a summary of recent advances and discuss to what extent a general model for -1PRF remains a useful way of thinking.