A machine learning‑based image segmentation method to quantify in vitro osteoclast culture endpoints

Quantification of in vitro osteoclast cultures (e.g. cell number) often relies on manual counting methods. These approaches are labour intensive, time consuming and result in substantial inter- and intra-user variability. This study aimed to develop and validate an automated workflow to robustly quantify in vitro osteoclast cultures. Using ilastik, a machine learning-based image analysis software, images of tartrate resistant acid phosphatase-stained mouse osteoclasts cultured on dentine discs were used to train the ilastik-based algorithm. Assessment of algorithm training showed that osteoclast numbers strongly correlated between manual- and automatically quantified values (r = 0.87). Osteoclasts were consistently faithfully segmented by the model when visually compared to the original reflective light images. The ability of this method to detect changes in osteoclast number in response to different treatments was validated using zoledronate, ticagrelor, and co-culture with MCF7 breast cancer cells. Manual and automated counting methods detected a 70% reduction (p < 0.05) in osteoclast number, when cultured with 10 nM zoledronate and a dose-dependent decrease with 1-10 μM ticagrelor (p < 0.05). Co-culture with MCF7 cells increased osteoclast number by ≥ 50% irrespective of quantification method. Overall, an automated image segmentation and analysis workflow, which consistently and sensitively identified in vitro osteoclasts, was developed. Advantages of this workflow are (1) significantly reduction in user variability of endpoint measurements (93%) and analysis time (80%); (2) detection of osteoclasts cultured on different substrates from different species; and (3) easy to use and freely available to use along with tutorial resources

Investigating diagnosis, treatment, and burden of disease in patients with ankylosing spondylitis in Central Eastern Europe and the United States: a real-world study.

INTRODUCTION/OBJECTIVES: Ankylosing spondylitis (AS) is a chronic inflammatory immune-mediated condition. We compared AS diagnosis, treatment, and burden in Central Eastern European countries (CEE), where this has been less researched, and the United States (US) from a real-world perspective. METHODS: Point-in-time survey of rheumatologists and their AS patients was conducted in the US (Apr-Oct 2018) and CEE (Aug-Nov 2019) via physician- and patient-completed record forms, including clinical and patient-reported outcomes. Statistical analysis included descriptive statistics, t-tests, Fisher's exact tests, and generalized linear models. RESULTS: In total, 487 patients were recruited from 88 rheumatologists in the US and 922 patients from 126 rheumatologists in CEE. Time from onset of symptoms to final AS diagnosis was longer in CEE than the US (4.2 vs 2.7 years, p < 0.05). At diagnosis, a greater use of conventional synthetic disease-modifying antirheumatic drugs (DMARDs) and injected steroids was reported in CEE vs the US (43.7% vs 27.6%, p < 0.05; 19.3% vs 8.7%, p < 0.05). 22.9% of US patients received a biologic DMARD at diagnosis vs 10% of CEE patients (p < 0.05). At current consultation, biologic DMARD use in CEE was lower vs the US (27.9% vs 71.0%, p < 0.05). CEE vs US patients had greater disease activity (mean Bath Ankylosing Spondylitis Disease Activity Index 4.2 vs 3.1, p < 0.05) and worse quality of life (QoL; mean Ankylosing Spondylitis Quality of Life Questionnaire score 6.2 vs 8.4, p < 0.05). CONCLUSIONS: AS patients in CEE vs the US faced slower diagnosis and worse access to biologics, disease activity, and QoL. Whether early access to biologics can improve symptoms, QoL, and daily activities in AS patients in CEE remains to be seen. Key Points • The study provided evidence on the real-world approach to the diagnosis, treatment, and burden of axSpA (axial spondyloarthritis) in CEE compared with the US. • The study reported patients in CEE experienced longer delays in diagnosis and poorer access to biologics than in the US. • This may have resulted in higher disease activity, greater levels of pain, and poorer outcomes, as reported by patients with axSpA in CEE

Peroxidases identified in a subtractive cDNA library approach show tissue-specific transcript abundance and enzyme activity during seed germination of Lepidium sativum

The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed

<em>TESS</em> Cycle 2 observations of roAp stars with 2-min cadence data

\ua9 The Author(s) 2023.We present the results of a systematic search of the Transiting Exoplanet Survey Satellite (TESS) 2-min cadence data for new rapidly oscillating Ap (roAp) stars observed during the Cycle 2 phase of its mission. We find seven new roAp stars previously unreported as such and present the analysis of a further 25 roAp stars that are already known. Three of the new stars show multiperiodic pulsations, while all new members are rotationally variable stars, leading to almost 70 per cent (22) of the roAp stars presented being α2 CVn-type variable stars. We show that targeted observations of known chemically peculiar stars are likely to overlook many new roAp stars, and demonstrate that multiepoch observations are necessary to see pulsational behaviour changes. We find a lack of roAp stars close to the blue edge of the theoretical roAp instability strip, and reaffirm that mode instability is observed more frequently with precise, space-based observations. In addition to the Cycle 2 observations, we analyse TESS data for all-known roAp stars. This amounts to 18 further roAp stars observed by TESS. Finally, we list six known roAp stars that TESS is yet to observe. We deduce that the incidence of roAp stars amongst the Ap star population is just 5.5 per cent, raising fundamental questions about the conditions required to excite pulsations in Ap stars. This work, coupled with our previous work on roAp stars in Cycle 1 observations, presents the most comprehensive, homogeneous study of the roAp stars in the TESS nominal mission, with a collection of 112 confirmed roAp stars in total

ANCA-associated vasculitis.

The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) are a group of disorders involving severe, systemic, small-vessel vasculitis and are characterized by the development of autoantibodies to the neutrophil proteins leukocyte proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA). The three AAV subgroups, namely granulomatosis with polyangiitis (GPA), microscopic polyangiitis and eosinophilic GPA (EGPA), are defined according to clinical features. However, genetic and other clinical findings suggest that these clinical syndromes may be better classified as PR3-positive AAV (PR3-AAV), MPO-positive AAV (MPO-AAV) and, for EGPA, by the presence or absence of ANCA (ANCA+ or ANCA-, respectively). Although any tissue can be involved in AAV, the upper and lower respiratory tract and kidneys are most commonly and severely affected. AAVs have a complex and unique pathogenesis, with evidence for a loss of tolerance to neutrophil proteins, which leads to ANCA-mediated neutrophil activation, recruitment and injury, with effector T cells also involved. Without therapy, prognosis is poor but treatments, typically immunosuppressants, have improved survival, albeit with considerable morbidity from glucocorticoids and other immunosuppressive medications. Current challenges include improving the measures of disease activity and risk of relapse, uncertainty about optimal therapy duration and a need for targeted therapies with fewer adverse effects. Meeting these challenges requires a more detailed knowledge of the fundamental biology of AAV as well as cooperative international research and clinical trials with meaningful input from patients

Like mother, like child : investigating perinatal and maternal health stress in post-medieval London.

Post-Medieval London (sixteenth-nineteenth centuries) was a stressful environment for the poor. Overcrowded and squalid housing, physically demanding and risky working conditions, air and water pollution, inadequate diet and exposure to infectious diseases created high levels of morbidity and low life expectancy. All of these factors pressed with particular severity on the lowest members of the social strata, with burgeoning disparities in health between the richest and poorest. Foetal, perinatal and infant skeletal remains provide the most sensitive source of bioarchaeological information regarding past population health and in particular maternal well-being. This chapter examined the evidence for chronic growth and health disruption in 136 foetal, perinatal and infant skeletons from four low-status cemetery samples in post-medieval London. The aim of this study was to consider the impact of poverty on the maternal-infant nexus, through an analysis of evidence of growth disruption and pathological lesions. The results highlight the dire consequences of poverty in London during this period from the very earliest moments of life

Cardiovascular disease, chronic kidney disease, and diabetes mortality burden of cardiometabolic risk factors from 1980 to 2010: a comparative risk assessment

Background High blood pressure, blood glucose, serum cholesterol, and BMI are risk factors for cardiovascular diseases and some of these factors also increase the risk of chronic kidney disease and diabetes. We estimated mortality from cardiovascular diseases, chronic kidney disease, and diabetes that was attributable to these four cardiometabolic risk factors for all countries and regions from 1980 to 2010. Methods We used data for exposure to risk factors by country, age group, and sex from pooled analyses of populationbased health surveys. We obtained relative risks for the eff ects of risk factors on cause-specifi c mortality from metaanalyses of large prospective studies. We calculated the population attributable fractions for- each risk factor alone, and for the combination of all risk factors, accounting for multicausality and for mediation of the eff ects of BMI by the other three risks. We calculated attributable deaths by multiplying the cause-specifi c population attributable fractions by the number of disease-specifi c deaths. We obtained cause-specifi c mortality from the Global Burden of Diseases, Injuries, and Risk Factors 2010 Study. We propagated the uncertainties of all the inputs to the fi nal estimates. Findings In 2010, high blood pressure was the leading risk factor for deaths due to cardiovascular diseases, chronic kidney disease, and diabetes in every region, causing more than 40% of worldwide deaths from these diseases; high BMI and glucose were each responsible for about 15% of deaths, and high cholesterol for more than 10%. After accounting for multicausality, 63% (10\ub78 million deaths, 95% CI 10\ub71\u201311\ub75) of deaths from these diseases in 2010 were attributable to the combined eff ect of these four metabolic risk factors, compared with 67% (7\ub71 million deaths, 6\ub76\u20137\ub76) in 1980. The mortality burden of high BMI and glucose nearly doubled from 1980 to 2010. At the country level, age-standardised death rates from these diseases attributable to the combined eff ects of these four risk factors surpassed 925 deaths per 100 000 for men in Belarus, Kazakhstan, and Mongolia, but were less than 130 deaths per 100 000 for women and less than 200 for men in some high-income countries including Australia, Canada, France, Japan, the Netherlands, Singapore, South Korea, and Spain. Interpretation The salient features of the cardiometabolic disease and risk factor epidemic at the beginning of the 21st century are high blood pressure and an increasing eff ect of obesity and diabetes. The mortality burden of cardiometabolic risk factors has shifted from high-income to low-income and middle-income countries. Lowering cardiometabolic risks through dietary, behavioural, and pharmacological interventions should be a part of the globalresponse to non-communicable diseases

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

Intraperitoneal drain placement and outcomes after elective colorectal surgery: international matched, prospective, cohort study

Despite current guidelines, intraperitoneal drain placement after elective colorectal surgery remains widespread. Drains were not associated with earlier detection of intraperitoneal collections, but were associated with prolonged hospital stay and increased risk of surgical-site infections.Background Many surgeons routinely place intraperitoneal drains after elective colorectal surgery. However, enhanced recovery after surgery guidelines recommend against their routine use owing to a lack of clear clinical benefit. This study aimed to describe international variation in intraperitoneal drain placement and the safety of this practice. Methods COMPASS (COMPlicAted intra-abdominal collectionS after colorectal Surgery) was a prospective, international, cohort study which enrolled consecutive adults undergoing elective colorectal surgery (February to March 2020). The primary outcome was the rate of intraperitoneal drain placement. Secondary outcomes included: rate and time to diagnosis of postoperative intraperitoneal collections; rate of surgical site infections (SSIs); time to discharge; and 30-day major postoperative complications (Clavien-Dindo grade at least III). After propensity score matching, multivariable logistic regression and Cox proportional hazards regression were used to estimate the independent association of the secondary outcomes with drain placement. Results Overall, 1805 patients from 22 countries were included (798 women, 44.2 per cent; median age 67.0 years). The drain insertion rate was 51.9 per cent (937 patients). After matching, drains were not associated with reduced rates (odds ratio (OR) 1.33, 95 per cent c.i. 0.79 to 2.23; P = 0.287) or earlier detection (hazard ratio (HR) 0.87, 0.33 to 2.31; P = 0.780) of collections. Although not associated with worse major postoperative complications (OR 1.09, 0.68 to 1.75; P = 0.709), drains were associated with delayed hospital discharge (HR 0.58, 0.52 to 0.66; P &lt; 0.001) and an increased risk of SSIs (OR 2.47, 1.50 to 4.05; P &lt; 0.001). Conclusion Intraperitoneal drain placement after elective colorectal surgery is not associated with earlier detection of postoperative collections, but prolongs hospital stay and increases SSI risk