Recovering Gene Interactions from Single-Cell Data Using Data Diffusion.

Single-cell RNA sequencing technologies suffer from many sources of technical noise, including under-sampling of mRNA molecules, often termed "dropout," which can severely obscure important gene-gene relationships. To address this, we developed MAGIC (Markov affinity-based graph imputation of cells), a method that shares information across similar cells, via data diffusion, to denoise the cell count matrix and fill in missing transcripts. We validate MAGIC on several biological systems and find it effective at recovering gene-gene relationships and additional structures. Applied to the epithilial to mesenchymal transition, MAGIC reveals a phenotypic continuum, with the majority of cells residing in intermediate states that display stem-like signatures, and infers known and previously uncharacterized regulatory interactions, demonstrating that our approach can successfully uncover regulatory relations without perturbations.

Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment.

Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.

High-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry.

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire-time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.

Improving the patient experience during musculoskeletal interventional procedures.

In the patient-centered practice of musculoskeletal interventional radiology, pre-procedure, intra-procedure, and post-procedure factors impact the patient's overall experience. Opportunities to improve the patient experience begin before the patient procedure starts, with smooth scheduling of the appointment, a positive office environment, and appropriate communication by the musculoskeletal interventional radiology staff before the procedure, including clear expectations regarding wait times. The initial conversation between the radiologist and the patient, including the informed consent process, is a crucial opportunity for creating rapport with the patient and generating trust in the radiologist. Being able to interpret a patient's verbal and non-verbal cues during this conversation can reduce anxiety and enhance relaxation. During the procedure, there are interventions which can decrease anxiety and reduce the perception of pain by the patient. These include psychological interventions such as self-induced hypnosis, communication-based interventions such as empathic communication to mold a patient's interpretation of anxiety and painful stimuli, and the use of ancillary tools such as music or video stimulation. Finally, clear post-procedure expectations regarding post-procedure pain and its management and assurances of access to the musculoskeletal interventional radiology team can facilitate an uncomplicated post-procedure recovery.

Discitis-osteomyelitis: optimizing results of percutaneous sampling.

Vertebral discitis-osteomyelitis is an infection of the spine that involves the intervertebral disc and the adjacent vertebral body but may also extend into the paraspinal and epidural soft tissues. If blood cultures and other culture data fail to identify a causative microorganism, percutaneous sampling is indicated to help guide targeted antimicrobial therapy. Despite limited supporting evidence, withholding antimicrobial therapy for up to 2 weeks is recommended to maximize microbiological yield, although literature supporting this recommendation is limited. During the procedure, technical factors that may improve yield include targeting of paraspinal fluid collections or soft tissue abnormalities for sampling, acquiring multiple core samples if possible, and use of larger gauge needles when available. Repeat sampling may be indicated if initial percutaneous biopsy is negative but should be performed no sooner than 72 h after the initial percutaneous biopsy to ensure adequate time for culture results to return.

Effects of COVID-19-related worry and rumination on mental health and loneliness during the pandemic: longitudinal analyses of adults in the UK COVID-19 mental health & wellbeing study.

BACKGROUND: The lasting effects of the coronavirus disease 2019 pandemic are likely to be significant. AIMS: This study tracked worry and rumination levels during the pandemic and investigated whether periods with higher COVID-related worry and rumination were associated with more negative mental health and loneliness. METHODS: A quota survey design and a sampling frame that permitted recruitment of a national sample were employed. Findings for waves 1 (March 2020) to 6 (November 2020) are reported (N = 1943). RESULTS: Covid-related worry and rumination levels were highest at the beginning of the first lockdown, then declined but increased when the UK returned to lockdown. Worry levels were higher than rumination levels throughout. High levels of COVID-related worry and rumination were associated with a five- and ten-fold increase in clinically meaningful rates of depression and anxiety (respectively) together with lower well-being and higher loneliness. The effects of COVID-related worry on depression and anxiety levels were most marked and clinically meaningful in individuals living with a pre-existing mental health condition. CONCLUSIONS: Psychological interventions should include components that specifically target COVID-related worry and rumination. Individuals with pre-existing mental health conditions should be prioritised as we emerge from the current pandemic and in any future public health crises.

Genome sequencing reveals underdiagnosis of primary ciliary dyskinesia in bronchiectasis.

BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.

Mental health and well-being during the COVID-19 pandemic: longitudinal analyses of adults in the UK COVID-19 Mental Health & Wellbeing study.

BACKGROUND: The effects of coronavirus disease 2019 (COVID-19) on the population's mental health and well-being are likely to be profound and long lasting. AIMS: To investigate the trajectory of mental health and well-being during the first 6 weeks of lockdown in adults in the UK. METHOD: A quota survey design and a sampling frame that permitted recruitment of a national sample was employed. Findings for waves 1 (31 March to 9 April 2020), 2 (10 April to 27 April 2020) and 3 (28 April to 11 May 2020) are reported here. A range of mental health factors was assessed: pre-existing mental health problems, suicide attempts and self-harm, suicidal ideation, depression, anxiety, defeat, entrapment, mental well-being and loneliness. RESULTS: A total of 3077 adults in the UK completed the survey at wave 1. Suicidal ideation increased over time. Symptoms of anxiety, and levels of defeat and entrapment decreased across waves whereas levels of depressive symptoms did not change significantly. Positive well-being also increased. Levels of loneliness did not change significantly over waves. Subgroup analyses showed that women, young people (18-29 years), those from more socially disadvantaged backgrounds and those with pre-existing mental health problems have worse mental health outcomes during the pandemic across most factors. CONCLUSIONS: The mental health and well-being of the UK adult population appears to have been affected in the initial phase of the COVID-19 pandemic. The increasing rates of suicidal thoughts across waves, especially among young adults, are concerning.

Impact of Patient-Reported Symptom Information on Agreement in the MRI Diagnosis of Presumptive Lumbar Spine Pain Generator.

BACKGROUND. Lumbar spine MRI is associated with a high prevalence of interpretive errors by radiologists. Treating physicians can obtain symptom information, correlate symptoms with MRI findings, and distinguish presumptive pain generators from incidental abnormalities. OBJECTIVE. The purpose of this study was to capture symptom information using a patient questionnaire, review lumbar spine MRI examinations with and without symptom information, diagnose pain generators, and compare MRI diagnoses with clinical reference diagnoses. METHODS. In this prospective study, 120 participants (70 men and 50 women; median age, 64 years; interquartile range, 49.5-74 years) were recruited from patients referred for lumbar spine injections between February and June 2019. Participants completed electronic questionnaires regarding their symptoms before receiving the injections. For three research arms, six radiologists diagnosed pain generators in MRI studies reviewed with symptom information from questionnaires, MRI studies reviewed without symptom information, and MRI reports. Interreading agreement was analyzed. Blinded to the questionnaire results, the radiologists who performed injections obtained patient histories, correlated symptoms with MRI findings, and diagnosed presumptive pain generators. These diagnoses served as clinical reference standards. Pain generators were categorized by type, level, and side and were compared using kappa statistics. Diagnostic certainty was recorded using numeric values (0-100) and was compared using Wilcoxon rank-sum test RESULTS. When compared with the reference standard, agreement for the type, level, and side of pain generator was almost perfect in MRI examinations reviewed with symptom information (κ = 0.82-0.90), fair to moderate in MRI examinations reviewed without symptom information (κ = 0.28-0.51) (all p < .001), and fair to moderate in MRI reports (κ = 0.27-0.45) (all p < .001). Interreading agreement was almost perfect when MRI examinations were reviewed with symptom information (κ = 0.82-0.90) but was only moderate without symptom information (κ = 0.42-0.56) (all p < .001). Diagnostic certainty levels were highest for radiologists performing injections (mean [± SD], 90.0 ± 9.9) and were significantly higher for MRI review with symptom information versus without symptom information (means for reading 1, 84.6 ± 13.1 vs 62.9 ± 20.7; p < .001). CONCLUSION. In lumbar spine MRI, presumptive pain generators diagnosed using symptom information from electronic questionnaires showed almost perfect agreement with pain generators diagnosed using symptom information from direct patient interviews. CLINICAL IMPACT. Patient-reported symptom information from a brief questionnaire can be correlated with MRI findings to distinguish presumptive pain generators from incidental abnormalities.

Observer agreement in the choice of lumbar spine injection for pain management.

BACKGROUND AND PURPOSE: Lumbar spine MRI can help guide the choice of corticosteroid injection in pain management. We investigated whether patient-reported symptom information from a questionnaire could improve agreement in the choice of type, level, and side of injection. MATERIALS AND METHODS: In this prospective observational study, 120 patients (median age 64, 70 men) were recruited from patients referred for pain management. After informed consent, they completed electronic questionnaires that obtained symptom information for later use during MRI reviews. In 3 research arms, 6 radiologists chose injections that would ideally deliver corticosteroid to the presumed sources of pain in (1) MRI studies reviewed with symptom information from questionnaires, (2) MRI studies reviewed without symptom information, and (3) MRI reports. Blinded to questionnaire results, radiologists providing clinical care and interviewing patients chose ideal therapeutic injections to establish reference standards. Injections were categorized by type, level, and side and compared using percent agreement and kappa statistics. Interreading agreement was analyzed. RESULTS: Compared to the reference standard, kappa agreements for injection types, levels, and sides were almost perfect when MRIs were reviewed knowing symptoms (0.85-0.93), fair without symptoms (0.23-0.35) (all P < .001) and fair in MRI reports (0.24-0.36) (all P < .001). Interreading kappa agreements were almost perfect knowing symptoms (0.82-0.90), but only moderate without symptoms (0.42-0.49) (all P < .001). CONCLUSIONS: Radiologists reviewing lumbar spine MRI converged on the type, level, and side of ideal therapeutic injection whether they obtained symptom information from direct patient interview or electronic questionnaire. Observer agreement was significantly lower without symptom information.

Understanding and managing suicide risk.

BACKGROUND: Suicidal behaviours and non-suicidal self-harm (NSSH) are global public health concerns that affect millions of lives. SOURCES OF DATA: This review is a narrative synthesis of systematic reviews, meta-analyses of randomized control trials (RCTs) and landmark studies published in scientific journals. AREAS OF AGREEMENT: Restricting access to lethal means reduces the likelihood of future suicide deaths. AREAS OF CONTROVERSY: Our ability to predict future suicidal behaviour is no better than chance. No individual risk prediction instrument offers sufficient sensitivity and specificity to inform clinically useful decision-making. GROWING POINTS: Different types of psychosocial interventions may be effective in preventing future suicide attempts; such interventions include clinical assessment, tailored crisis response and safety plans and follow-up contact. AREAS TIMELY FOR DEVELOPING RESEARCH: Whilst some psychosocial interventions can be effective in reducing suicide risk, little is known about the mechanisms of recovery from suicidal thoughts and behaviours.

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells.

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.

Percutaneous Cryoablation: Safety and Efficacy for Pain Palliation of Metastases to Pleura and Chest Wall.

PURPOSE: To assess safety and efficacy of percutaneous cryoablation for pain palliation of metastases to pleura and chest wall. MATERIALS AND METHODS: This retrospective single-center cohort study included 22 patients (27% female, mean age 63 y ± 11.4) who underwent 25 cryoablation procedures for pain palliation of 39 symptomatic metastases measuring 5.1 cm ± 1.9 (range, 2.0-8.0 cm) in pleura and chest wall between June 2012 and December 2017. Pain intensity was assessed using a numerical scale (0-10 points). Statistical tests t test, χ2, and Wilcoxon signed rank were performed. RESULTS: Patients were followed for a median of 4.1 months (interquartile range [IQR], 2.3-10.1; range, 0.1-36.7 mo) before death or loss to follow-up. Following cryoablation, pain intensity decreased significantly by a median of 4.5 points (IQR, 2.8-6; range, 0-10 points; P = .0002 points, Wilcoxon signed rank). Pain relief of at least 3 points was documented following 18 of 20 procedures. Pain relief occurred within a median of 1 day following cryoablation (IQR, 1-2; range, 1-4 d) and lasted for a median of 5 weeks (IQR, 3-17; range, 1-34 wk). Systemic opioid requirements decreased in 11 of 22 patients (50%) by an average of 56% ± 34. Difference in morphine milligram equivalents was not significant (P = .73, Wilcoxon signed rank). No procedure-related complications occurred despite previous radiation of 7 tumors. Of 25 procedures, 22 (88%) were performed on an outpatient basis. CONCLUSIONS: Percutaneous cryoablation for metastases to pleura and chest wall can safely provide significant pain relief within days following a single session.

The effect of music on pain and subjective experience in image-guided musculoskeletal corticosteroid injections: a randomized controlled trial.

PURPOSE: To investigate the role of music on subjects undergoing routine image-guided musculoskeletal corticosteroid injections and its effect on post-procedure pain and subjective overall experience. MATERIALS AND METHODS: This prospective study was IRB-approved and HIPAA-compliant. A total of 126 subjects referred for outpatient image-guided musculoskeletal corticosteroid injections were enrolled in the study and randomized into a music offered group ((+)MO) and a no music offered group ((-)MO). (+)MO subjects were given the opportunity to listen to music during their corticosteroid injection. All subjects were then given an anonymous survey on which they recorded their pre-procedural and post-procedural pain on a scale from 0 to 9 and rated their overall experience and how likely they were to recommend our department for musculoskeletal procedures on scales from 1 to 5. RESULTS: (+)MO subjects had significantly lower post-procedural pain (p = 0.013) and significantly greater decrease in pain (p = 0.031) compared to (-)MO subjects. Among the (+)MO subjects, there was no statistically significant difference in post-procedure pain (p = 0.34) or change in pain (p = 0.62) if music was accepted or declined. However, subjects who listened to music did have lower post-procedural pain compared to those who did not listen to music (p = 0.012), although the differences in the decrease of pain between the two groups did not quite reach statistical significance (p = 0.062). CONCLUSIONS: Playing music during image-guided musculoskeletal corticosteroid injections may reduce patients' post-procedure pain. Offering patients some measure of control over their procedure may be a factor that contributes to decreased post-procedure pain as well.

Differentiating adults who think about self-harm from those who engage in self-harm: the role of volitional alcohol factors.

BACKGROUND: Self-harm, an act of self-poisoning or self-injury irrespective of motivation, is a major public health concern. Use of alcohol prior to or alongside acts of self-harm is common but little is known about the alcohol-related mechanisms of self-harm enaction. We utilised an ideation-to-action approach to clarify the extent to which volitional alcohol factors differentiated those who have thoughts of self-harm but do not act on them (self-harm ideation) and those who engage in self-harm (self-harm enaction). METHODS: Cross-sectional analyses of the baseline phase of the Health Lifestyle and Wellbeing study: 1546 adults (1079 female; Mean age = 34 y; 92% White) resident in Scotland completed measures of demographics, lifetime self-harm, volitional alcohol factors and psychosocial factors. Multinomial logistic regression compared those with a history of self-harm thoughts ('ideation', n = 297), self-harm acts ('enaction', n = 346) and 'controls' (n = 897) to identify volitional alcohol factors associated with self-harm enaction. RESULTS: Volitional alcohol factors differentiated those with a history of self-harm enaction from those with a history of self-harm ideation (as well as those with no history) in initial models adjusted for demographics and depressive symptoms: the self-harm enaction group reported stronger alcohol-related negative urgency (OR = 1.74, 95% CI 1.41-2.16, p < .001), more frequent heavy drinking (OR = 1.46, 95% CI 1.24-1.72, p < .001) and stronger expectancies that drinking alcohol leads to negative self-perceptions (OR = 1.33, 95% CI 1.03-1.72, p = 0.03) and markers of self-harm risk (OR = 1.64, 95% CI 1.18-2.30, p = 0.004). Alcohol-related negative urgency and heavy-drinking frequency continued to differentiate those in the self-harm enaction group from those in ideation group in multivariate models. Consistent with theoretical models positing phase-specific moderators of self-harm ideation and enaction, psychosocial factors (perceived stress, support, negative mood regulation expectancies) differentiated those with a history of self-harm ideation from those without but not those in the ideation and enaction groups. CONCLUSIONS: Management of self-harm risk requires better understanding of alcohol-related mechanisms of self-harm enaction. Volitional alcohol factors may play a role in governing the translation of self-harm thoughts into self-harm acts.

Ultrasound-guided injection for the diagnosis and treatment of posteromedial knee friction syndrome.

OBJECTIVE: To describe an ultrasound guided injection technique for diagnosing and treating posteromedial knee friction syndrome, which occurs between the sartorius/gracilis tendons and medial femoral condyle (MFC). MATERIALS AND METHODS: Our study was IRB-approved and HIPAA-compliant. We identified patients via a retrospective review of medical records and MRI with posteromedial knee pain and isolated edema between MFC and sartorius/gracilis tendons and no evidence for meniscal tear, ruptured Baker's cyst or degenerative joint disease. Patients were referred for an ultrasound-guided procedure to inject anesthetic and corticosteroid at the site of edema. Procedures were evaluated for technical success, which was defined as satisfactory identification of the injection site and adequate delivery of medication. Follow-up was available up to 8 weeks after the procedure to determine the response and any potential complications. RESULTS: Fourteen subjects with MRI and symptoms of posteromedial knee friction syndrome underwent 14 injections. Technical success was achieved in all procedures, with no complications. At 8 weeks' follow-up, 92% of patients had symptom improvement. VAS before and 8 weeks after the procedure changed from 5.2 ± 2.7 to 0.9 ± 2.1 (p = 0.0002), respectively. CONCLUSION: Ultrasound-guided injection of edema between the MFC and sartorius/gracilis tendons supports the diagnosis of a posteromedial knee friction syndrome and successfully treats its associated symptoms.

Percutaneous CT-guided sacroiliac joint sampling for infection: aspiration, biopsy, and technique.

OBJECTIVE: To evaluate methods of CT-guided sacroiliac joint sampling in patients with suspected infection. MATERIALS AND METHODS: All CT-guided sacroiliac joint sampling procedures for suspected infection were reviewed for sampling type (aspiration, lavage aspiration, biopsy), microbiology results, and clinical and imaging follow-up. The primary gold standard was anatomic pathology. If pathology was not available, then positive blood culture with the same organism as SIJ sampling, imaging and clinical follow-up, or clinical follow-up only were used. Anterior and posterior joint distention was evaluated by MRI within 7 days of the procedure. RESULTS: A total of 34 patients (age 39 ± 20 (range, 6-75) years; 21 F, 13 M) were included. Aspiration samples only were obtained in 13/34 (38%) cases, biopsy samples only in 9/34 (26%) cases, and both samples in 12/34 (35%) cases. There was an overall 54% sensitivity and 86% specificity. For the aspiration samples, sensitivity and specificity were 60 and 81%, respectively, compared to 45 and 90% for the biopsy samples. In cases with both samples, biopsy did not add additional microbial information. Seventeen (17/34, 50%) patients had an MRI. The anterior joint was more distended than the posterior joint in 15/17 (88%) of patients, and this difference was significant (P = 0.0003). All of these 17 patients had an attempted aspiration by a posterior approach; 6/17 (35%) resulted in a successful aspiration. CONCLUSIONS: Aspiration of the sacroiliac joint has a higher sensitivity than biopsy and should always be attempted first. MRI may be helpful for procedure planning.