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β-Arrestins are classic attenuators of G-protein–coupled receptor signaling. However, they have multiple roles in cellular physiology, including carcinogenesis. This work shows for the first time that β-arrestins have prognostic significance for predicting metastasis and response to chemotherapy in bladder cancer. β-Arrestin-1 (ARRB1) and β-arrestin-2 (ARRB2) mRNA levels were measured by quantitative RT-PCR in two clinical specimen cohorts (n = 63 and 43). The role of ARRBs in regulating a stem cell-like phenotype and response to chemotherapy treatments was investigated. The consequence of forced expression of ARRBs on tumor growth and response to Gemcitabine in vivo were investigated using bladder tumor xenografts in nude mice. ARRB1 levels were significantly elevated and ARRB2 levels downregulated in cancer tissues compared with normal tissues. In multivariate analysis only ARRB2 was an independent predictor of metastasis, disease-specific-mortality, and failure to Gemcitabine + Cisplatin (G+C) chemotherapy; ∼80% sensitivity and specificity to predict clinical outcome. ARRBs were found to regulate stem cell characteristics in bladder cancer cells. Depletion of ARRB2 resulted in increased cancer stem cell markers but ARRB2 overexpression reduced expression of stem cell markers (CD44, ALDH2, and BMI-1), and increased sensitivity toward Gemcitabine. Overexpression of ARRB2 resulted in reduced tumor growth and increased response to Gemcitabine in tumor xenografts. CRISPR-Cas9–mediated gene-knockout of ARRB1 resulted in the reversal of this aggressive phenotype. ARRBs regulate cancer stem cell-like properties in bladder cancer and are potential prognostic indicators for tumor progression and chemotherapy response.

Supplementary Table S1: Specimen and patient characteristics; Supplementary Table S2: Determination of the association between adjuvant treatment failure and demographic/clinical parameters or marker levels; Supplementary Table S3: qPCR primers and ARRB1-specific CRISPR guide RNA sequences; Supplementary Table S4: Source of various antibodies used for immunofluorescence and Western blotting; Supplementary Figure S1: Kaplan Meier plot for stratification of patients into higher risk and lower risk for Disease-Specific Mortality (DSM) based on the ARRB2 arrestin level; Supplementary Figure S2: Expression of cytokeratins and Bmi-1 following modulation of ARRB2 levels in 5637 cells; Supplementary Figure S3: Expression of SOX2 following modulation of ARRB2 levels in bladder cancer cell lines; Supplementary Figure S4: Response of ARRB2 overexpressing transfectants to Cisplatin; Supplementary Figure S5: Expression ABC transporters in ARRB2-modulated bladder cancer cells; Supplementary Figure S6: Effect of ARRB2 overexpression on Cancer Stem Cell markers in vivo; Supplementary Figure S7: ARRB1 depletion affects clonogenic growth, spheroid formation and expression of CSC markers

Funder: International Helmholtz Research School of Biophysics and Soft Matter, Funder: Karolinska Institutet, The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S), in a manner that requires initiator fMet-tRNAMet binding. In addition, we demonstrate that in human cells and in vitro, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Furthermore, we show that mtIF2 is indispensable for mitochondrial protein synthesis. Our results demonstrate an important evolutionary divergence of the mitochondrial translation system and further our fundamental understanding of a process central to eukaryotic metabolism.

The MD Anderson Dysphagia Inventory (MDADI) is a 20-item dysphagia-specific QOL questionnaire with four subscales: global, emotional, functional, and physical. It is widely used in clinical practice and in research; however, its psychometric properties have been under-researched. We aim to evaluate the organisation of the MDADI subscales and identify any redundant items. The MDADI is a routinely collected outcome measure at two centres in northeast England. Questionnaires completed at three months following treatment were extracted from these existing databases. Factor analysis was carried out with the aim of reducing redundancy among the set of questionnaire items. Cases with missing values were excluded. A total of 196 complete patient questionnaires were used in factor analysis. A one-factor model accounted for around 50% of the total variance in item responses. The top five endorsed items (abbreviated by the questionnaire item keywords: Excluded, Irritate, Esteem, Social, and Why) in this one factor appeared in three (emotional, functional, and physical) of the four supposed MDADI subscales, i.e. global, emotional, functional, and physical. Our results suggest an overlap of three MDADI subscales across the top five endorsed items. The content of the top five questions all appear related to the psychosocial aspects of swallowing. This implies some redundancy of the items in the original subscales of the questionnaire. Using the most endorsed items, it appears feasible to abbreviate the 20-item MDADI questionnaire to a 5-item “MiniDADI” questionnaire, which is likely to have greater utility in routine clinical practice outside of research settings.

Single-cell RNA-sequencing (scRNA-seq) has emerged in recent years as a breakthrough technology to understand RNA metabolism at cellular resolution. In addition to allowing new cell types and states to be identified, scRNA-seq can permit cell-type specific differential gene expression changes, pre-mRNA processing events, gene regulatory networks and single-cell developmental trajectories to be uncovered. More recently, a new wave of multi-omic adaptations and complementary spatial transcriptomics workflows have been developed that facilitate the collection of even more holistic information from individual cells. These developments have unprecedented potential to provide penetrating new insights into the basic neural cell dynamics and molecular mechanisms relevant to the nervous system in both health and disease. In this review we discuss this maturation of single-cell RNA-sequencing over the past decade, and review the different adaptations of the technology that can now be applied both at different scales and for different purposes. We conclude by highlighting how these methods have already led to many exciting discoveries across neuroscience that have furthered our cellular understanding of the neurological disease.

BackgroundPatients with rheumatoid arthritis (RA) may have impaired immunogenicity to COVID-19 vaccines.ObjectivesTo investigate the incidence of COVID-19 infection and hospitalisation in unvaccinated and vaccinated patients with RA compared with matched individuals; and secondarily in patients with RA according to DMARD treatment.MethodsDanish nationwide matched cohort study from January to October 2021. Patients with RA were identified in DANBIO and matched 1:20 with individuals from the general population on age, sex, and vaccination status (month and exact type of vaccination). Primary and secondary outcomes were COVID-19 hospitalisation (Danish National Patient Register) and positive SARS-CoV2 PCR test (Danish COVID-19 Surveillance Register), respectively. Stratified by vaccination status, incidence rates (IRs) per 1000 person years (PY) and comorbidity-adjusted hazard ratios (aHRs) in cause-specific Cox models were calculated with 95% confidence intervals. Using the Aalen-Johansen estimator, the cumulative incidence of COVID-19 hospitalisations was visualised according to RA and vaccine exposure status.ResultsRegardless of vaccination status, patients with RA had increased incidence of COVID-19 hospitalisation compared to matched individuals (Table 1). However, the absolute risk was 0.20% for unvaccinated patients at 60 days and 0.08% for comparators, whereas it remained below 0.05% at 180 days of follow-up in both groups when fully vaccinated (Figure 1). Increased SARS-CoV2 infection rates were seen only among unvaccinated patients with RA (Table 1). Unadjusted analyses showed increased incidence of COVID-19 hospitalisation among rituximab-treated compared with conventional DMARD treated: unvaccinated HR 4.71 (1.98 to 11.18) and vaccinated HR 11.69 (2.07 to 66.06). However, the proportions of patients with previous cancer and treated with prednisolone were higher among the rituximab treated.Table 1.UnvaccinatedPartially vaccinatedFully vaccinatedRAControlsRAControlsRAControlsN28 447568 94027 154542 61026 217523 826Women, %71.371.371.271.271.071.0Age in years, median [IQR]67.7 [34.2 to 88.3]67.8 [34.2 to 88.4]68.4 [36.4 to 88.6]68.4 (36.5 to 88.6)68.9 [40.9 to 88.7]68.9 (41.0 to 88.7)Methotrexate /55.5 /0.5 /55.4 /1.2 /55.7 /1.3 /Sulfasalazine /14.2 /0.1 /13.7 /0.3 /13.5 /0.3 /Hydroxychloroquine /10.4 /0.1 /10.3 /0.0 /10.3 /0.0 /Other csDMARD,11.0 /0.2 /10.7 /0.3 /10.6 /0.3 /Prednisolone,all in %12.52.012.20.512.20.5TNFi /16.9 /0.1 /17.2 /2.9 /17.1 /3.1 /abatacept /1.5 /0.0 /1.5 /0.5 /1.5 /0.5 /tocilizumab /3.0 /0.0 /3.0 /0.0 /2.9 /0.0 /rituximab, all in %2.20.12.10.12.10.1COVID-19 hospitalisationN65727119511131Median [IQR] days of follow-up102 [62 to 137]115 [88 to 146]28 [22 to 35]30 (21 to 39)150 [111 to 189]150 (111 to 189)Rate per 1000 PY10.4 (8.0 to 13.4)4.7 (4.3 to 5.1)5.5 (3.0 to 10.0)2.2 (1.8 to 2.7)0.9 (0.5 to 1.6)0.5 (0.4 to 0.6)Adjusted HRa1.88 (1.44 to 2.46)1 (Ref.)2.47 (1.25 to 4.89)1 (Ref.)1.94 (1.03 to 3.66)1 (Ref.)SARS-CoV2 infectionRate per 1000 PY37.8 (33.6 to 42.6)33.9 (33.1 to 34.8)27 (20.7 to 35.1)28.5 (27 to 30.2)11.3 (9.2 to 13.9)10.4 (9.9 to 10.9)Adjusted HRa1.22 (1.09 to 1.57)1 (Ref.)0.87 (0.95 to 1.74)1 (Ref.)1.09 (0.92 to 1.14)1 (Ref.)IQR, Interquartile range. a Adjusted for cancer history, cardiovascular disease, diabetes mellitus, chronic kidney disease, and chronic lung disease.Figure 1.Cumulative incidence of COVID-19 hospitalisation (%) as a function of follow-up time (days) for (A) unvaccinated, (B) partially vaccinated and (C) fully vaccinated patients and comparators.ConclusionThe incidence of COVID-19 hospitalisation was increased for both unvaccinated and vaccinated patients with RA compared with controls. Importantly, the parallel decreasing risk for patients with RA suggests a comparable relative benefit of vaccination. Less favourable outcomes among rituximab-treated warrant that this drug should be considered with extra care.AcknowledgementsThe authors wish to acknowledge The Danish Departments of Clinical Microbiology and Statens Serum Institut for carrying out laboratory analysis, registration, and release of the national SARS-CoV-2 surveillance data use in the present study. Further, the authors wish to thank all the Danish departments of rheumatology for reporting to the DANBIO register.Disclosure of InterestsRené Cordtz: None declared, Salome Kristensen: None declared, Rasmus Westermann: None declared, Kirsten Duch: None declared, Fiona Pearce Grant/research support from: Pearce reports a grant from Vifor Pharma outside the submitted work., Jesper Lindhardsen: None declared, Christian Torp-Pedersen Grant/research support from: Torp-Pedersen reports grants from Bayer and Novo Nordisk outside the submitted work., Mikkel Porsborg Andersen: None declared, Lene Dreyer Speakers bureau: Dreyer has received speakers bureau from Eli Lilly and Galderma., Grant/research support from: Dreyer has received research grant/support from BMS.

Summary Mitochondria harbor specialized ribosomes (mitoribosomes) necessary for the synthesis of key membrane proteins of the oxidative phosphorylation (OXPHOS) machinery located in the mitochondrial inner membrane. To date, no animal model exists to study mitoribosome composition and mitochondrial translation coordination in mammals in vivo. Here, we create MitoRibo-Tag mice as a tool enabling affinity purification and proteomics analyses of mitoribosomes and their interactome in different tissues. We also define the composition of an assembly intermediate formed in the absence of MTERF4, necessary for a late step in mitoribosomal biogenesis. We identify the orphan protein PUSL1, which interacts with a large subunit assembly intermediate, and demonstrate that it is an inner-membrane-associated mitochondrial matrix protein required for efficient mitochondrial translation. This work establishes MitoRibo-Tag mice as a powerful tool to study mitoribosomes in vivo, enabling future studies on the mitoribosome interactome under different physiological states, as well as in disease and aging., Graphical Abstract, Highlights • MitoRibo-Tag mice with a tag on mL62 were generated to study mitoribosomes in vivo • The mitoribosome interactome of different mouse tissues was defined with proteomics • PUSL1 was identified as a mitoribosome-interacting protein using MitoRibo-Tag mice • MitoRibo-Tag mice allow mitoribosome analysis under different conditions and setups, Busch et al. generated MitoRibo-Tag mice to study mitoribosome composition in vivo in different tissues. Proteomics of MitoRibo-Tag mice with defective assembly identified PUSL1 as a mitoribosome-interacting protein required for efficient mitochondrial translation. MitoRibo-Tag mice provide a tool to study mitoribosomes under different physiological conditions and in disease and aging.

Ribosome profiling (Ribo-Seq) is a technique that allows genome-wide, quantitative analysis of translation. In recent years, it has found multiple applications in studies of translation in diverse organisms, tracking protein synthesis with single codon resolution. Traditional protocols applied for generating Ribo-Seq libraries from mammalian cell cultures are not suitable to study mitochondrial translation due to differences between eukaryotic cytosolic and mitochondrial ribosomes. Here, we present an adapted protocol enriching for mitoribosome footprints. In addition, we describe the preparation of small RNA sequencing libraries from the resultant mitochondrial ribosomal protected fragments (mtRPFs).

Ribosome profiling (Ribo-Seq) is a technique that allows genome-wide, quantitative analysis of translation. In recent years, it has found multiple applications in studies of translation in diverse organisms, tracking protein synthesis with single codon resolution. Traditional protocols applied for generating Ribo-Seq libraries from mammalian cell cultures are not suitable to study mitochondrial translation due to differences between eukaryotic cytosolic and mitochondrial ribosomes. Here, we present an adapted protocol enriching for mitoribosome footprints. In addition, we describe the preparation of small RNA sequencing libraries from the resultant mitochondrial ribosomal protected fragments (mtRPFs).

Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6‐months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease., Heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu induce the integrated stress response and metabolic rearrangements, reducing penetrance and promoting spontaneous recovery in a rare mitochondrial myopathy.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6 months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation, however only ∼1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGluincludingEARS2andTRMUin the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. The spontaneous recovery in infants with digenic mutations is modulated by changes in amino acid availability in a multi-step process. First, the integrated stress-response associated with increasedFGF21andGDF15expression enhances catabolism via β-oxidation and the TCA cycle increasing the availability of amino acids. In the second phase mitochondrial biogenesis increases via mTOR activation, leading to improved mitochondrial translation and recovery. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

The SIGMA Germanium detector has the potential to revolutionise γ -ray spectroscopy, providing superior energy and position resolving capabilities compared with current large volume state-of-the-art Germanium detectors. The theoretical position resolution of the detector as a function of γ -ray interaction position has been studied using simulated detector signals. A study of the effects of RMS noise at various energies has been presented with the position resolution ranging from 0.33 mm FWHM at E γ = 1 MeV , to 0.41 mm at E γ = 150 keV. An additional investigation into the effects pulse alignment have on pulse shape analysis and in turn, position resolution has been performed. The theoretical performance of SIGMA operating in an experimental setting is presented for use as a standalone detector and as part of an ancillary system.

Nocturnin (NOCT) is a eukaryotic enzyme that belongs to a superfamily of exoribonucleases, endonucleases, and phosphatases. In this study, we analyze the expression, processing, localization, and cellular functions of human NOCT. We demonstrate that the NOCT protein is differentially expressed and processed in a cell and tissue type specific manner as a means to control its localization to the cytoplasm or mitochondria. Our studies also show that the N-terminus of NOCT is necessary and sufficient to confer mitochondrial localization. We then measured the impact of cytoplasmic NOCT on the transcriptome and report that it regulates the levels of hundreds of mRNAs that are enriched for components of signaling pathways, neurological functions, and regulators of osteoblast differentiation. Recent biochemical data indicate that NOCT dephosphorylates nicotinamide adenine dinucleotide (NAD) metabolites, and thus we measured the effect of NOCT on these cofactors in cells. We find that NOCT increases NAD(H) and decreases NADP(H) levels in a manner dependent on its intracellular localization. Collectively, our data indicate that NOCT can regulate levels of both mRNAs and NADP(H) cofactors in manner specified by its intracellular localization.

This article reviews the traditional Subjective, Objective, Assessment, and Plan (SOAP) note documentation format. The information in the SOAP note is useful to both providers and students for history taking and physical exam, and highlights the importance of including critical documentation details with or without an electronic health record.

Background It is unclear on how children with compromised first permanent molars (cFPMs) are currently managed in the UK by either general dental practitioners (GDP) or specialists in paediatric dentistry (SPD). Aim Explore the current attitudes to the management of compromised first permanent molars amongst UK general dental practitioners and specialists in paediatric dentistry. Design Self-completed online questionnaire including three clinical vignettes of 7, 9, and 15 years old with cFPM. All registered SPDs (n = 236), as of May 2017, 500 randomly selected GDPs from England, selected from a national performers list, and 52 Scottish GDPs, part of Scottish dental practice research network, were invited to complete the questionnaire. Results About 71.6% (n = 53) of SPDs agreed that children with cFPM should be referred to a paediatric specialist for treatment planning, whereas the reverse for GDPs is true, as 86.8% (n = 138) believe they have a responsibility to treat these teeth. Responses to clinical vignettes suggest a slight preference amongst GDPs to restore cFPM, including root canal treatment, whereas SPDs have a slight preference towards extraction. Conclusion Current pathways for cFPM, amongst UK general dental practitioners and specialists in paediatric dentistry, vary greatly between and within each professional group.

Anxiety is an important comorbidity in chronic obstructive pulmonary disease (COPD). We investigated if cognitive behavioural therapy (CBT), delivered by respiratory nurses, reduced symptoms of anxiety and was cost-effective.Patients with COPD and anxiety were randomised to CBT or self-help leaflets. Anxiety, depression and quality of life were measured at baseline, 3, 6 and 12 months. A cost-effectiveness analysis was conducted from a National Health Service hospital perspective and quality-adjusted life-years estimated using the EuroQol-5D questionnaire.In total, 279 patients were recruited. Group mean change from baseline to 3 months in the Hospital Anxiety and Depression Anxiety Subscale was 3.4 (95% CI 2.62–4.17, pCBT delivered by respiratory nurses is a clinically and cost-effective treatment for anxiety in patients with COPD relative to self-help leaflets.

The optimal method for monitoring antiplatelet therapy in children supported with ventricular assist devices (VADs) is unknown. We conducted a retrospective study to compare Thromboelastography Platelet Mapping (TEG/PM) with multiple electrode platelet aggregometry (MEA) on a Multiplate analyzer (Roche Diagnostics, Mannheim, Germany). We analyzed data from 66 paired blood samples from 9 patients

The purposes of this study were to determine the level of engagement of registered nurse (RN) to Bachelor of Science in Nursing (BSN), Master of Science in Nursing (MSN), and Doctor of Nursing Practice (DNP) nursing students enrolled in online nursing degree programs and to understand whether there are generational differences in level of student engagement. Significant differences were noted for engagement level between generations of students, but no significant difference was noted in the engagement level of students from RN to BSN, MSN, or DNP programs.

Graft-versus-host disease (GVHD) can be a fatal complication of allogeneic stem cell transplantation (allo-HSCT). GVHD can be classified as acute (aGVHD: up to 100 days) or chronic (cGVHD: after 100 days) based on the time-point of disease occurrence. At present there are a limited number of biomarkers available for use in the clinic. Thus, the aim of this research was to evaluate the biomarker potential of the extensively studied Heat Shock Protein 70 family members (HSPA1A/HSPA1B and HSPA1L) at the messenger RNA (mRNA) level in acute and cGVHD patient cohorts. In the skin biopsies, HSPA1L mRNA expression was lower in patients with severe aGVHD (grades II-III) when compared to those with none or low grade aGVHD (grades 0-I) and normal controls. In whole blood, HSPA1L mRNA expression level was significantly (p = 0.008) up-regulated at 28 days post-transplant in cGVHD patients with a significant area under the curve (AUC = 0.773). In addition, HSPA1B expression in whole blood was significantly higher at 3 months post-transplant in both the aGVHD grade II-III (p = 0.012) and cGVHD (p = 0.027) patients. Our initial results in this small cohort show that quantifying HSPA1L mRNA expression in the whole blood of allo-HSCT patients at day 28 post-allo-HSCT may be a useful predictive biomarker for cGVHD.