Single-molecule tracking to determine the abundances and stoichiometries of freely-diffusing protein complexes in living cells: Past applications and future prospects.

Most biological processes in living cells rely on interactions between proteins. Live-cell compatible approaches that can quantify to what extent a given protein participates in homo- and hetero-oligomeric complexes of different size and subunit composition are therefore critical to advance our understanding of how cellular physiology is governed by these molecular interactions. Biomolecular complex formation changes the diffusion coefficient of constituent proteins, and these changes can be measured using fluorescence microscopy-based approaches, such as single-molecule tracking, fluorescence correlation spectroscopy, and fluorescence recovery after photobleaching. In this review, we focus on the use of single-molecule tracking to identify, resolve, and quantify the presence of freely-diffusing proteins and protein complexes in living cells. We compare and contrast different data analysis methods that are currently employed in the field and discuss experimental designs that can aid the interpretation of the obtained results. Comparisons of diffusion rates for different proteins and protein complexes in intracellular aqueous environments reported in the recent literature reveal a clear and systematic deviation from the Stokes-Einstein diffusion theory. While a complete and quantitative theoretical explanation of why such deviations manifest is missing, the available data suggest the possibility of weighing freely-diffusing proteins and protein complexes in living cells by measuring their diffusion coefficients. Mapping individual diffusive states to protein complexes of defined molecular weight, subunit stoichiometry, and structure promises to provide key new insights into how protein-protein interactions regulate protein conformational, translational, and rotational dynamics, and ultimately protein function.

Targeted AURKA degradation: Towards new therapeutic agents for neuroblastoma.

Aurora kinase A (AURKA) is a well-established target in neuroblastoma (NB) due to both its catalytic functions during mitosis and its kinase-independent functions, including stabilization of the key oncoprotein MYCN. We present a structure-activity relationship (SAR) study of MK-5108-derived PROTACs against AURKA by exploring different linker lengths and exit vectors on the thalidomide moiety. PROTAC SK2188 induces the most potent AURKA degradation (DC50,24h 3.9 nM, Dmax,24h 89%) and shows an excellent binding and degradation selectivity profile. Treatment of NGP neuroblastoma cells with SK2188 induced concomitant MYCN degradation, high replication stress/DNA damage levels and apoptosis. Moreover, SK2188 significantly outperforms the parent inhibitor MK-5108 in a cell proliferation screen and patient-derived organoids. Furthermore, altering the attachment point of the PEG linker to the 5-position of thalidomide allowed us to identify a potent AURKA degrader with a linker as short as 2 PEG units. With this, our SAR-study provides interesting lead structures for further optimization and validation of AURKA degradation as a potential therapeutic strategy in neuroblastoma.

Cost-effectiveness of home-based care of febrile neutropenia in children with cancer.

INTRODUCTION: Home-based treatment of febrile neutropenia (FN) in children with cancer with oral or intravenous antibiotics is safe and effective. There are limited data on the economic impact of this model of care. We evaluated the cost-effectiveness of implementing an FN programme, incorporating home-based intravenous antibiotics for carefully selected patients, in a tertiary paediatric hospital. METHODS: A decision analytic model was constructed to compare costs and outcomes of the home-based FN programme, with usual in-hospital treatment with intravenous antibiotics. The programme included a clinical decision rule to stratify patients by risk for severe infection and home-based eligibility criteria using disease, chemotherapy and patient-level factors. Health outcomes (quality of life) and probabilities of FN risk classification and home-based eligibility were based on prospectively collected data between 2017 and 2019. Patient-level costs were extracted from hospital administrative records. Cost-effectiveness was expressed as the incremental cost per quality-adjusted life year (QALY). FINDINGS: The mean health care cost of home-based FN treatment in low-risk patients was Australian dollars (A$) 7765 per patient compared to A$20,396 for in-hospital treatment (mean difference A$12,632 [95% CI: 12,496-12,767]). Overall, the home-based FN programme was the dominant strategy, being more effective (0.0011 QALY [95% CI: 0.0011-0.0012]) and less costly. Results of the model were most sensitive to proportion of children eligible for home-based care programme. CONCLUSION: Compared to in-hospital FN care, the home-based FN programme is cost-effective, with savings arising from cheaper cost of caring for children at home. These savings could increase as more patients eligible for home-based care are included in the programme.

Improved measurement of racial/ethnic disparities in COVID-19 mortality in the United States.

Different estimation methods produce diverging accounts of racial/ethnic disparities in COVID-19 mortality in the United States. The CDC's decision to present the racial/ethnic distribution of COVID-19 deaths at the state level alongside re-weighted racial/ethnic population distributions -- in effect, a geographic adjustment -- makes it seem that Whites have the highest death rates. Age adjustment procedures used by others, including the New York City Department of Health and Mental Hygiene, lead to the opposite conclusion that Blacks and Hispanics are dying from COVID-19 at higher rates than Whites. In this paper, we use indirect standardization methods to adjust per-capita death rates for both age and geography simultaneously, avoiding the one-sided adjustment procedures currently in use. Using CDC data, we find age-and-place- adjusted COVID-19 death rates are 80% higher for Blacks and more than 50% higher for Hispanics, relative to Whites, on a national level, while there is almost no disparity for Asians. State-specific estimates show wide variation in mortality disparities. Comparison with non- epidemic mortality reveals potential roles for pre-existing health disparities and differential rates of infection and care.

Improved measurement of racial/ethnic disparities in COVID-19 mortality in the United States

Different estimation methods produce diverging accounts of racial/ethnic disparities in COVID-19 mortality in the United States. The CDCs decision to present the racial/ethnic distribution of COVID-19 deaths at the state level alongside re-weighted racial/ethnic population distributions--in effect, a geographic adjustment--makes it seem that Whites have the highest death rates. Age adjustment procedures used by others, including the New York City Department of Health and Mental Hygiene, lead to the opposite conclusion that Blacks and Hispanics are dying from COVID-19 at higher rates than Whites. In this paper, we use indirect standardization methods to adjust per-capita death rates for both age and geography simultaneously, avoiding the one-sided adjustment procedures currently in use. Using CDC data, we find age-and-place-adjusted COVID-19 death rates are 80% higher for Blacks and more than 50% higher for Hispanics, relative to Whites, on a national level, while there is almost no disparity for Asians. State-specific estimates show wide variation in mortality disparities. Comparison with non-epidemic mortality reveals potential roles for pre-existing health disparities and differential rates of infection and care.

Author Correction: The association between physical activity and low back pain: a systematic review and meta-analysis of observational studies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Conservation of glucagon like peptide-1 level with liraglutide and linagilptin protects the kidney against angiotensin II-induced tissue fibrosis in rats.

This study tested the hypothesis that the enhancement of glucagon-like peptide-1 (GLP-1) level through either exogenous supply of GLP-1 agonist, liraglutide or prevention of endogenous GLP-1 degradation with dipeptidyl peptidease-4 inhibitor, lingaliptin ameliorates angiotensin II (Ang II)-induced renal fibrosis. Sprague-Dawley rats were randomly divided into four groups: 0.9% saline or Ang II (500 ng/kg/min) was infused with osmotic minipumps for 4 weeks, defined as sham and Ang II groups. In drug treated groups, liraglutide (0.3 mg/kg) was injected subcutaneously twice daily or linagliptin (8 mg/kg) was administered daily via oral gavage during Ang II infusion. Compared with Ang II stimulation, liraglutide or linagliptin comparatively down-regulated the protein level of the AT1 receptor, and up-regulated the AT2 receptor, as identified by a reduced AT1/AT2 ratio (all p < 0.05), consistent with less locally-expressed AT1 receptor and enhanced AT2 receptor in the glomerular capillaries and proximal tubules of the renal cortex. Furthermore, both drugs significantly increased the expression of GLP-1 receptor and attenuated the protein levels of TLR4, NOX4 and IL-6. The populations of macrophages and α-SMA expressing myofibroblasts decreased with treatment of liraglutide and linagliptin, in coincidence with the reduced expression of phosphor-Smad2/3, Smad4, TGFß1, and up-regulated Smad7. Along with these modulations, renal morphology was preserved and synthesis of fibronectin/collagen I was down-regulated, as identified by small collagen-rich area in the renal cortex. These results suggest that the preservation of GLP-1 level using liraglutide or linagliptin might be considered as an add-on therapeutic option for inhibiting Ang II induced renal fibrosis and failure.

The association between physical activity and low back pain: a systematic review and meta-analysis of observational studies.

The aim of this review was to investigate the association between total and domain-specific physical activity (PA) and non-specific low back pain (LBP) in adults. Seven databases were searched for cohort and cross-sectional studies. Pooled estimates of the association of medium and high levels PA and LBP, using the generic inverse-variance method with fixed- and random-effects models were calculated. Twenty-four studies (15 cohort and nine cross-sectional; 95,796 participants) were included. The pooled fully adjusted risk ratios (RR) from cohort studies comparing medium with lowest activity levels were 0.90 (95%CI 0.85 to 0.96) for total PA, and 0.90 (95%CI 0.85 to 0.96) for leisure-time PA (LTPA). The pooled RR comparing highest with lowest activity levels were 1.00 (95%CI 0.92 to 1.08) for total PA, and 1.01 (95%CI 0.93 to 1.10) for LTPA. The pooled fully adjusted odds ratios (OR) from cross-sectional studies comparing medium with lowest activity levels were 0.93 (95%CI 0.65 to 1.32) for total PA, and 0.77 (95%CI 0.62 to 0.96) for LTPA. The pooled OR comparing highest with lowest activity levels were 1.05 (95%CI 0.89 to 1.23) for total PA, and 0.85 (95%CI 0.79 to 0.93) for LTPA. PA seems to be associated with lower prevalence of LBP.

Time for a Different Approach to Anterior Cruciate Ligament Injuries: Educate and Create Realistic Expectations.

Following an anterior cruciate ligament (ACL) injury, patients are often reassured that timely surgery followed by intensive physiotherapy will "fix their knee". Not only does this message create a false perception of uncomplicated return to sport (RTS), it also ignores the large body of evidence demonstrating a high RTS re-injury rate following ACL reconstruction. In this article, we propose an individualised approach to the management of ACL injuries that targets a shift away from early surgery and towards conservative management, with surgery 'as needed' and rehabilitation tailored to the patient's RTS goals. Education on the natural history of ACL injuries will ensure patients are not misguided into thinking surgery and intensive rehabilitation guarantees great outcomes. Further, understanding that conservative management is not inferior to surgery-and not more likely to cause knee osteoarthritis-will help the patient make an informed decision. For patients who opt for surgical management, rehabilitation must target strength and functional performance, avoid rapid increases in training load, and be guided by an RTS timeframe that is no shorter than 9 months. The content of rehabilitation should be similar for patients who opt for non-operative management, although the RTS timeframe will likely be shorter. All patients should receive education on the relationship between injury risk and training load, and understand that a home-exercise program is not inferior to intensive physiotherapist-led exercise.

Familial factors predicting recovery and maintenance of physical activity in people with low back pain: Insights from a population-based twin study.

BACKGROUND: It is unknown how familial factors influence the recovery from low back pain (LBP) and the maintenance activity behaviours. We aimed to investigate whether individual and within-family physical activity (PA) and sedentary behaviour influenced recovery from LBP, and maintenance of PA and sedentary behaviour in people with and without LBP. METHODS: Longitudinal logistic regression analyses were performed on adult twins from the Washington State Twin Registry. First, individual and within-family (based on co-twin data) sufficient PA (at least 75 min of vigorous-intensity PA, or 150 min of moderate-intensity PA per week) and high leisure sitting time (≥3 hr/day) were our exposure variables (baseline). LBP within the past 3 months at follow-up defined non-recovery (outcome). Second, within-family sufficient PA and high leisure sitting time were our exposure variables(baseline) and our outcomes were individual PA and sitting time at follow-up. All analyses were adjusted for follow-up length (range: 1-7 years) and confounding variables. RESULTS: Individual and within-family PA and sitting time were not associated with recovery. Within-family PA and sitting time predicted individual maintenance of PA (OR = 1.47, 95% CI: 1.17-1.84, n = 1,388 twins) and sitting time (OR = 1.41, 95% CI: 1.10-1.82, n = 1,534). Within-family PA and sitting time had the strongest effects on those without (OR = 1.79, 95% CI: 1.33-2.41, n = 788) and with LBP (OR = 1.90, 95% CI: 1.32-2.76, n = 698), respectively. CONCLUSION: Having active family members increased the likelihood of continuing to be active (particularly for those without LBP), while having sedentary family members increased the likelihood of maintaining sedentary behaviours (particularly for those with LBP). SIGNIFICANCE: This study was the first to investigate how familial activity behaviours influence recovery from LBP and ongoing activity behaviours. People with LBP living within a sedentary family likely require specific interventions to reduce their sedentary behaviours.

Pathologic and Radiologic Correlation of Adult Cystic Lung Disease: A Comprehensive Review.

The presence of pulmonary parenchymal cysts on computed tomography (CT) imaging presents a significant diagnostic challenge. The diverse range of possible etiologies can usually be differentiated based on the clinical setting and radiologic features. In fact, the advent of high-resolution CT has facilitated making a diagnosis solely on analysis of CT image patterns, thus averting the need for a biopsy. While it is possible to make a fairly specific diagnosis during early stages of disease evolution by its characteristic radiological presentation, distinct features may progress to temporally converge into relatively nonspecific radiologic presentations sometimes necessitating histological examination to make a diagnosis. The aim of this review study is to provide both the pathologist and the radiologist with an overview of the diseases most commonly associated with cystic lung lesions primarily in adults by illustration and description of pathologic and radiologic features of each entity. Brief descriptions and characteristic radiologic features of the various disease entities are included and illustrative examples are provided for the common majority of them. In this article, we also classify pulmonary cystic disease with an emphasis on the pathophysiology behind cyst formation in an attempt to elucidate the characteristics of similar cystic appearances seen in various disease entities.

Are people with chronic low back pain meeting the physical activity guidelines? A co-twin control study.

BACKGROUND: Despite a large amount of research investigating physical activity (PA) levels in people with chronic low back pain (LBP), no study has investigated whether people with chronic LBP are meeting the World Health Organization (WHO) PA guidelines. Furthermore, with genetics and the early shared environment substantially influencing the presence of LBP and PA engagement, these factors could confound the association between LBP and PA and need to be controlled for. PURPOSE: This study aimed to investigate the association between chronic LBP and meeting the PA guidelines, while controlling for the effects of genetics and early shared environment. DESIGN: This is a cross-sectional co-twin control study. PATIENT SAMPLE: A cross-sectional analysis was performed on 1,588 twins from the Murcia Twin Registry in Spain with available data on LBP and PA from the 2013 data collection wave. OUTCOME MEASURES: The exposure and outcome variables in our study were self-reported. Twins reporting a history of chronic LBP were asked follow-up questions to inform on the presence of recent LBP (within the past 4 weeks), previous LBP (no pain within the past 4 weeks), and persistent LBP (no pain-free month in the last 6 months). These were our exposure variables. Our outcome variable was meeting the WHO PA guidelines, which involved at least 75 minutes of vigorous-intensity PA, or at least 150 minutes of moderate-intensity PA per week. METHODS: To investigate the association between chronic LBP and meeting the PA guidelines, we first performed a multivariate logistic regression on the total sample of twins. Co-variables entered the model if the univariate association between the co-variable, and both the exposure and the outcome reached a significance of p<.2. Second, to adjust for the influence of genetics and early shared environment, we performed a conditional multivariate logistic regression on complete twin pairs discordant for LBP. The Murcia Twin Registry is supported by Fundación Séneca, Regional Agency for Science and Technology, Murcia, Spain (08633/PHCS/08 and 15302/PHCS/10) and the Ministry of Science and Innovation, Spain (PSI11560-2009). Funding for this project has also been received from Fundación MAPFRE (2012). The authors declare that there are no conflicts of interest. RESULTS: There was a significant inverse association between recent LBP and meeting the PA guidelines (odds ratio [OR]=0.71, p=.034). When controlling for genetics and early shared environment, this association disappeared. There was no association between previous (OR=0.95, p=.779) or persistent LBP (OR=0.78, p=.192) and meeting the PA guidelines. CONCLUSION: Twins with recent LBP are less likely to meet the PA guidelines than those with no history of chronic LBP, highlighting the importance of incorporating PA promotion in the treatment of these individuals. Genetics and early shared environment appear to be confounding the association between LBP and PA, although this needs to be further tested in larger twin samples.

Video-game based exercises for older people with chronic low back pain: a protocol for a feasibility randomised controlled trial (the GAMEBACK trial).

OBJECTIVES: To investigate the feasibility of implementing a video-game exercise programme for older people with chronic low back pain (LBP). DESIGN: Single-centred single-blinded randomised controlled trial (RCT). SETTING: Physiotherapy outpatient department in a public hospital in Western Sydney, Australia. PARTICIPANTS: We will recruit 60 participants over 55 years old with chronic LBP from the waiting list. INTERVENTIONS: Participants will be randomised to receive video-game exercise (n=30) or to remain on the waiting list (n=30) for 8 weeks, with follow up at 3 and 6 months. Participants engaging in video-game exercises will be unsupervised and will complete video-game exercise for 60minutes, 3 times per week. Participants allocated to remain on the waiting list will be encouraged to maintain their usual levels of physical activity. MAIN OUTCOME MEASURE: The primary outcomes for this feasibility study will be study processes (recruitment and response rates, adherence to and experience with the intervention, and incidence of adverse events) relevant to the future design of a large RCT. Estimates of treatment efficacy (point estimates and 95% confidence intervals) on pain self-efficacy, care seeking, physical activity, fear of movement/re-injury, pain, physical function, disability, falls-efficacy, strength, and walking speed, will be our secondary outcome measures. RESULTS: Recruitment for this trial began in November 2015. CONCLUSION: This study describes the rationale and processes of a feasibility study investigating a video-game exercise programme for older people with chronic LBP. Results from the feasibility study will inform on the design and sample required for a large multicentre RCT. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry: ACTRN12615000703505.

Single dose GLP toxicity and biodistribution study of a conditionally replicative adenovirus vector, CRAd-S-pk7, administered by intracerebral injection to Syrian hamsters.

BACKGROUND: CRAd-S-pk7 is a conditionally replicative oncolytic adenoviral vector that contains a survivin promoter and a pk7 fiber modification that confer tumor-specific transcriptional targeting and preferential replication in glioma while sparing the surrounding normal brain parenchyma. METHODS: This IND-enabling study performed under GLP conditions evaluated the toxicity and biodistribution of CRAd-S-pk7 administered as a single intracerebral dose to Syrian hamsters, a permissive model of adenoviral replication. Two hundred and forty animals were stereotactically administered either vehicle (n = 60) or CRAd-S-pk7 at 2.5 × 10(7), 2.5 × 10(8), or 2.5 × 10(9) viral particles (vp)/animal (each n = 60) on day 1. The animals were closely monitored for toxicology evaluation, assessment of viral distribution, and immunogenicity of CRAd-S-pk7. RESULTS: Changes in hematology, clinical chemistry, and coagulation parameters were minor and transient, and consistent with the inflammatory changes observed microscopically. These changes were considered to be of little toxicological significance. The vector remained localized primarily in the brain and to some degree in the tissues at the incision site. Low levels of vector DNA were detected in other tissues in a few animals suggesting systemic circulation of the virus. Viral DNA was detected in brains of hamsters for up to 62 days. However, microscopic changes and virus-related toxicity to the central nervous system were considered minor and decreased in incidence and severity over time. Such changes are not uncommon in studies using adenoviral vectors. CONCLUSION: This study provides safety and toxicology data justifying a clinical trial of CRAd-S-pk7 loaded in FDA-approved HB1.F3.CD neural stem cell carriers administered at the tumor resection bed in humans with recurrent malignant glioma.

Plexiform fibromyxoma with cotyledon-like serosal growth: A case report of a rare gastric tumor and review of the literature.

Plexiform fibromyxoma is a rare, benign mesenchymal neoplasm that predilects the gastric antrum and has potential for misdiagnosis as a gastrointestinal stromal tumor (GIST). The histology of the tumor is characterized by interwoven fascicular growth of cytologically bland spindled cells within a variably myxoid stroma. The current study reports the clinicopathological and immunohistochemical findings of a plexiform fibromyxoma resected from a 28-year-old Vietnamese female. The patient presented with acute, severe abdominal pain and worsening anemia. The initial fine-needle aspiration and needle core biopsy of the gastric antral mass led to an initial diagnosis of GIST. The subsequent distal partial gastrectomy revealed a 5.5-cm transmural antral mass that ulcerated the overlying mucosa and grew as variably elongated, myxoedematous, polypoid (cotyledon-like) excrescences from the serosal surface. Microscopically, the tumor demonstrated plexiform and multinodular growth of cytologically bland spindled cells proliferating in an abundant myxocollagenous stroma with a prominent capillary network. Tumor cells immunohistochemically expressed smooth muscle actin and CD10, but did not express CD117, Discovered on GIST-1 or nuclear ß-catenin. Follow-up evaluation 23 months post surgery revealed no evidence of residual tumor. A review the cases of this rare entity reported in the English language literature is also provided.

Sarcomatoid (spindle cell) carcinoma of the pancreas: A case report and review of the literature.

Sarcomatoid (spindle cell) carcinoma of the pancreas is a rare, high-grade epithelial malignancy composed predominantly or exclusively of spindle cells demonstrating evidence of epithelial derivation, but no features indicative of a specific line of mesenchymal differentiation. The current study presents the case of an 85-year-old Caucasian male with a tumor mass in the body of the pancreas. The individual subsequently underwent a distal pancreatectomy, splenectomy and partial gastrectomy. Microscopic examination of the 3.3-cm mass located in the body of the pancreas revealed a small, but high-grade, adenocarcinomatous component that blended imperceptibly with malignant spindle cells. No light microscopic or immunohistochemical evidence of specific mesenchymal differentiation was identified, and the spindle cells, as in the case of the carcinoma, were diffusely keratin-positive. Sarcomatoid (spindle cell) carcinoma defined in this way rarely presents in the pancreas, with, to the best of our knowledge, only six cases reported in the English literature.