Ewing's Sarcoma (EWS) has been reported in seven children with Down Syndrome (DS). To date, a detailed assessment of this solid tumor in DS patients is still missing. Here, we characterized a chemo-resistant mediastinal EWS in a 2-year-old DS child, the youngest ever reported case, by exploiting sequencing approaches. The tumor showed a neuroectodermal development driven by the EWSR1-FLI1 fusion. The inherited myeloperoxidase deficiency of the patient caused failure of neutrophil-mediated cell death and promoted genomic instability. In this context, the tumor underwent nearly genome-wide haploidization resulting in a massive overexpression of pro-inflammatory cytokines. Recruitment of defective neutrophils fostered the fast evolution of this EWS. | Make paid
Background Multivessel coronary artery disease (MVD) is highly prevalent in patients presenting with non-ST-segment elevation myocardial infarction (NSTE-ACS) and is associated with worse clinical outcomes compared with single vessel disease patients. Complete revascularization of the culprit and all significant non-culprit lesions reduces the incidence of major adverse cardiac events, but the optimal timing of non-culprit artery revascularization remains unclear. Methods This prespecified substudy of the randomized BIOVASC trial included patients who presented with NSTE-ACS and MVD, defined as [≥] 1 non-culprit related coronary artery with a diameter of [≥] 2.5 mm and [≥] 70% stenosis as per visual estimation or positive coronary physiology testing. Risk differences of the composite of all-cause mortality, myocardial infarction, unplanned ischemia driven revascularization or cerebrovascular events and its individual components were compared between the patients who were randomized to immediate and staged complete revascularization at 30 days and 1 year. Results The BIOVASC trial enrolled 1525 patients, 917 patients presented with NSTE-ACS, of whom 459 were allocated to the immediate complete and 458 to the staged complete revascularization group. The incidences of the primary composite outcome were similar in the two groups (7.9% vs. 10.1%, risk difference 2.2%, 95%CI -1.5 to 6.0, p = 0.24). Immediate complete revascularization was associated with a significant reduction in the incidence of myocardial infarction (2.0% vs. 5.3%, risk difference 3.3%, 95% confidence interval [CI] 0.9 to 5.7, p = 0.008), which was maintained after exclusion of procedure related myocardial infarctions occurring at the index or staged procedure (2.0% vs. 4.4%, risk difference 2.4%, 95%CI 0.1 to 4.7, p = 0.039). Unplanned ischemia driven revascularizations were also reduced in the immediate complete revascularization group (4.2% vs. 7.8%, risk difference 3.5%, 95%CI 0.4 to 6.6, p = 0.025). Conclusions Immediate complete revascularization is safe in NSTE-ACS and was associated with a reduction in myocardial infarctions and unplanned ischemia driven revascularizations in patients presenting with NSTE-ACS and MVD at 1 year. | Make paid
Background: The worldwide increase in obesity triggers the increase in the incidence of obesity during pregnancy. In this study, it was aimed to examine the effects of maternal obesity on some obstetric features. Methods: This study was conducted in a tertiary hospital with a comparative group, cross-sectional and descriptive design. The data of the study were collected with a Data Collection Form created by the researchers. Descriptive statistics, Pearsons chi-squared test, independent-samples t-test, and multinomial regression analysis were used to analyze the data. Results: In this study, women were divided into two groups, namely the obese group (BMI>29.9 kg/m2) and the normal weight group (BMI 18.5-24.9 kg/m2). There was no significant difference between the groups in terms of their height, education level, employment status, income level, and place of residence (p>0.05). It was determined that the obese group had higher rates of multiparity, postmature deliveries, and fetal distress developing in their babies on labor (p<0.05). There was no significant difference between the groups in terms of their modes of delivery, onset of labor, and the requirement of interventions during the second stage of the labor (p>0.05). In the logistic regression analysis, maternal obesity was found to increase the rates of multiparity by 1.758 times (1.038-2.978 CI, p=0.036), post-maturity by 5.902 times (1.283-27.164 CI, p=0.023), and moderate postpartum hemorrhage by 2.286 times (1.433-3.646 CI, p=0.001). Conclusion: It is important that women who have obesity problems in the preconception period are counseled to help them reach a normal BMI. Health care professionals should advise women on healthy nutrition and weight control during both the pregnancy and postpartum periods. | Make paid
BACKGROUND Monitoring stroke patients in critical-care units for 24 hours after thrombolysis or endovascular thrombectomy is considered standard of care but is not evidence-based. Due to the Covid-19 pandemic, our center modified its protocol in April 2021 with 24-hour critical-care monitoring no longer being guaranteed for stroke patients. We aim to compare the incidence and timing of complications over the first 24 hours post-reperfusion therapies and their association to hospital unit in 2019, 2020 and 2021. METHODS We conducted a single-center retrospective cohort study. We analyzed data from stroke patients treated with thrombolysis and/or endovascular thrombectomy at our center in 2019 (pre-Covid-19, standard of care), 2020 (during Covid-19, standard of care) and 2021 (during Covid-19, new protocol). Data extracted included demographics, the nature and timing of complications within the first 24 hours, and the unit at the time of any complication. Major complications included neurologic deterioration, symptomatic intracranial hemorrhage, recurrent stroke, myocardial infarction, systemic bleeding, rapid assessment of critical events call, and death. RESULTS Three hundred forty-nine patients were included in our study: 78 patients in 2019, 115 patients in 2020, and 156 patients in 2021. In 2021, 32% of patients experienced at least one complication within the first 24 hours compared to 34% in 2020 and 27% in 2019. In 2021, 33% of patients admitted to critical-care units had a complication compared to 31% in 2020 and 26% in 2019. In 2021, 70% of complications had occurred by hour eight compared to 49% in 2020 and 29% in 2019. CONCLUSIONS Despite the change of protocol in April 2021, the incidence and timing of complications did not significantly worsen compared to prior years and were not associated with hospital location. Further research is required to evaluate the necessity of critical care monitoring for 24 hours in this population. | Make paid
Compared to the general population, science trainees experience significant challenges and stressors that increase negative mental health outcomes. With COVID-19, the stressors of social distancing, isolation, truncated lab time, and uncertainty about the future have all likely exacerbated the effect. Now, more than ever, practical and effective interventions are vitally needed to address the core causes of science trainee stress and to increase resilience amongst trainee populations. This paper discusses a new resilience program targeted to biomedical trainees and scientists - Becoming a Resilient Scientist Series (BRS), a 5-part workshop coupled with facilitated group discussions dedicated to increasing resilience, specifically focusing on academic and research environments. Results show that BRS increases trainee resilience (primary outcome), with reductions in perceived stress, anxiety, and work presenteeism, and increases in ability to shift and persist, self-awareness, and self-efficacy (secondary outcomes). Furthermore, participants in the program reported high level of satisfaction, would highly recommend the program to others, and perceived positive changes in their resilience skills. To our knowledge, this is the first resilience program explicitly targeted for biomedical trainees and scientists, catering to the unique professional culture and environment in which these individuals work. | Make paid
Background: It has been suggested that glycoprotein acetyls (GlycA) better reflects chronic inflammation than high sensitivity C-reactive protein (hsCRP), but paediatric/life-course data are sparse. Method: Using data from the Avon Longitudinal Study of Parents and Children and UK Biobank, we compared short- (over weeks) and long-term (over years) correlations of GlycA and hsCRP, cross-sectional correlations between GlycA and hsCRP, and associations of pro-inflammatory risk factors with GlycA and hsCRP across the life-course. Results: GlycA showed high short-term (weeks) stability at 15y (r=0.75; 95% CI=0.56, 0.94), 18y (r=0.74; 0.64, 0.85), 24y (r=0.74; 0.51, 0.98) and 48y (r=0.82 0.76, 0.86) and this was comparable to the short-term stability of hsCRP at 24y. GlycA stability was moderate over the long-term, for example between 15y and 18y r=0.52; 0.47, 0.56 and between 15y and 24y r=0.37; 0.31, 0.44. These were larger than equivalent correlations of hsCRP. GlycA and concurrently measured hsCRP were moderately correlated at all ages, for example at 15y (r=0.44; 0.40, 0.48) and at 18y (r=0.55; 0.51, 0.59). We found similar associations of known proinflammatory factors and inflammatory diseases with GlycA and hsCRP. For example, BMI was positively associated with GlycA (mean difference in GlycA per standard deviation change in BMI=0.08; 95% CI=0.07, 0.10) and hsCRP (0.10; 0.08, 0.11). Conclusion: This study showed that GlycA has greater long-term stability than hsCRP, however associations of proinflammatory factors with GlycA and hsCRP were broadly similar. | Make paid
Bi-stable stimuli evoke two distinct perceptual interpretations that alternate and compete for dominance. Bi-stable perception is thought to be driven at least in part by mutual suppression between distinct neural populations that represent each percept. Abnormal visual perception is observed among people with psychotic psychopathology (PwPP), and there is evidence to suggest that these visual deficits may depend on impaired neural suppression in visual cortex. However, it is not yet clear whether bi-stable visual perception is abnormal among PwPP. Here, we examined bi-stable perception in a visual structure-from-motion task using a rotating cylinder illusion in a group of 65 PwPP, 44 first-degree biological relatives, and 43 healthy controls. Data from a 'real switch' task, in which physical depth cues signaled real switches in rotation direction were used to exclude individuals who did not show adequate task performance. In addition, we measured concentrations of neurochemicals, including glutamate, glutamine, and {gamma}-amino butyric acid (GABA), involved in excitatory and inhibitory neurotransmission. These neurochemicals were measured non-invasively in visual cortex using 7 tesla MR spectroscopy. We found that PwPP and their relatives showed faster bi-stable switch rates than healthy controls. Faster switch rates also correlated with significantly higher psychiatric symptom levels across all participants. However, we did not observe any significant relationships across individuals between neurochemical concentrations and SFM switch rates. Our results are consistent with a reduction in suppressive neural processes during structure-from-motion perception in PwPP, and suggest that genetic liability for psychosis is associated with disrupted bi-stable perception. | Make paid
BackgroundOur ethnographic team has conducted longitudinal research focused on illicit drug markets in Northern Mexico since 2018. In 2021-2022, study participants described the arrival of new, unusually potent tablets sold as ostensibly controlled substances, without a prescription, directly from pharmacies that cater to US tourists. Concurrently, fentanyl- and methamphetamine-based counterfeit prescription drugs have driven escalating overdose death rates in the US, however their presence in Mexico has not been assessed. AimsTo characterize the availability of counterfeit and authentic controlled substances at pharmacies in Northern Mexico available to English-speaking tourists without a prescription. MethodsWe employed an iterative, exploratory, mixed methods design. Longitudinal ethnographic data was used to characterize tourist-oriented micro-neighborhoods and guide the selection of n=40 pharmacies in n=4 cities in Northern Mexico. In each pharmacy, samples of "oxycodone", "Xanax", and "Adderall" were sought as single pills, during English-language encounters, after which detailed ethnographic accounts were recorded. We employed immunoassay-based testing strips to check each pill for the presence of fentanyls, benzodiazepines, amphetamines, and methamphetamines. We used Fourier-Transform Infrared Spectroscopy to further characterize drug contents. ResultsOf 40 pharmacies, these controlled substances could be obtained in any form with no prescription at 68.3% and as single pills at 46.3%. Counterfeit pills were obtained at n=11 (26.8%) of pharmacies. Of n=45 samples sold as one-off controlled substances, n=20 were counterfeit including 9 of 11 (81.8%) of samples sold as "Adderall" that contained methamphetamine, and 8 of 27 (29.6%) of samples sold as "Oxycodone" that contained fentanyl, and n=3 Oxycodone samples containing heroin. Pharmacies providing counterfeit drugs were uniformly located in tourist-serving micro-neighborhoods, and generally featured English-language advertisements for erectile dysfunction medications and painkillers. Pharmacy employees occasionally expressed concern about overdose risk and provided harm reduction guidance. DiscussionThe availability of fentanyl-, heroin-, and methamphetamine-based counterfeit medications in Northern Mexico represents a public health risk, and occurs in the context of 1) the normalization of medical tourism as a response to rising unaffordability of healthcare in the US, 2) plummeting rates of opioid prescription in the US, affecting both chronic pain patients and the availability of legitimate pharmaceuticals on the unregulated market, 3) the rise of fentanyl-based counterfeit opioids as a key driver of the fourth, and deadliest-to-date, wave of the opioid crisis. It is not possible to distinguish counterfeit medications based on appearance, because identically-appearing authentic and counterfeit versions are often sold in close geographic proximity. Nevertheless, US tourist drug consumers may be more trusting of controlled substances purchased directly from pharmacies. Due to Mexicos limited opioid overdose surveillance infrastructure, the current death rate from these substances remains unknown. | Make paid
Background In a pilot study using both cannabidiol (CBD) and tetrahydrocannabinol (THC) as single agents in advanced cancer patients undergoing palliative care in Thailand, the doses were generally well tolerated, and the outcome measure of total symptom distress scores showed overall symptom benefit. The current study aims to determine the intensity of the symptoms experienced by breast cancer patients receiving chemotherapy, to explore the microbiome profile, cytokines, and bacterial metabolites before and after the treatment with cannabis oil or no cannabis oil, and to study the pharmacokinetics parameters and pharmacogenetics profile of the doses. Methods A randomized, double-blinded, placebo-controlled trial will be conducted on the metastatic breast cancer cases receiving chemotherapy at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand. Block randomization will be used to allocate the patients into three groups: Ganja Oil (THC 2 mg/ml; THC 0.08 mg/drop, and CBD 0.02 mg/drop), Metta Osot (THC 81 mg/ml; THC 3 mg/drop), and placebo oil. The Edmonton Symptom Assessment System (ESAS), microbiome profile, cytokines, and bacterial metabolites will be assessed before and after the interventions. | Make paid
In the last few years, several models trying to calculate the biological brain age have been proposed based on structural magnetic resonance imaging scans (T1-weighted MRIs, T1w) using multivariate methods and artificial intelligence. We developed and validated a convolutional neural network (CNN)-based biological brain age prediction model that uses only one T1w MRI pre-processing step to simplify implementation and increase accessibility in research settings. Our model only requires rigid image registration to the MNI space, which is an advantage compared to previous methods that require more pre-processing steps, such as feature extraction. We used a multicohort dataset of cognitively healthy individuals (age range = 32.0 - 95.7 yrs.) comprising 17296 MRIs for training and evaluation. We compared our model using hold-out (CNN1) and cross-validation (CNN2-4) approaches. To verify generalizability, we used two external datasets with different population and MRI scan characteristics to evaluate the model. To demonstrate its usability, we included the external dataset's images in the cross-validation training (CNN3). To ensure that our model used only the brain signal on the image, we also predicted brain age using skull-stripped images (CNN4). The trained models achieved a mean absolute error of 2.99, 2.67, 2.67, and 3.08 yrs. for the CNN1-4, respectively. The model's performance in the external dataset was in the typical range of mean absolute error (MAE) found in the literature for testing sets. Adding the external dataset to the training set (CNN3), overall, MAE is unaffected, but individual cohort MAE improves (2.25 to 5.63 years). Salience maps of predictions reveal that periventricular, temporal, and insular regions are the most important for age prediction. We provide indicators for using biological (predicted) brain age as a metric for age correction in neuroimaging studies as an alternative to the traditional chronological age. In conclusion, using different approaches, our CNN-based model showed good performance using only one T1w brain MRI pre-processing step. The proposed CNN model is made publicly available for the research community to be easily implemented and used to study aging and age-related disorders. | Make paid
Background: We aimed to estimate vaccine effectiveness (VE) against COVID-19 mortality, and to explore whether an increased risk of non-COVID-19 mortality exists in the weeks following a COVID-19 vaccine dose. Methods: National registries of causes of death, COVID-19 vaccination, specialized health care and long-term care reimbursements were linked by a unique person identifier using data from 1 January 2021 to 31 January 2022. We used Cox regression with calendar time as underlying time scale to, firstly, estimate VE against COVID-19 mortality after primary and first booster vaccination, per month since vaccination and, secondly, estimate risk of non-COVID-19 mortality in the 5 or 8 weeks following a first, second or first booster dose, adjusting for birth year, sex, medical risk group and country of origin. Results: VE against COVID-19 mortality was >90% for all age groups two months after completion of the primary series. VE gradually decreased thereafter, to around 80% at 7-8 months post-primary series for most groups, and around 60% for elderly receiving a high level of long-term care and for people aged 90+ years. Following a first booster dose, the VE increased to >85% in all groups. The risk of non-COVID-19 mortality was lower or similar in the 5 or 8 weeks following a first dose compared to no vaccination, as well as following a second dose compared to one dose and a booster compared to two doses, for all age and long-term care groups. Conclusion: At the population level, COVID-19 vaccination greatly reduced the risk of COVID-19 mortality and no increased risk of death from other causes was observed. | Make paid
Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo. | Make paid
The spatiotemporal dynamics of inflammation provide vital insights into the understanding of skin inflammation. Skin inflammation primarily depends on the regulatory feedback between pro- and anti-inflammatory mediators. Healthy skin exhibits faded erythema. In contrast, diseased skin exhibits expanding erythema with diverse patterns, clinically classified into five types: circular, annular, arcuate, gyrate, and polycyclic. Inflammatory diseases with expanding erythema are speculated to result from the overproduction of pro-inflammatory mediators. However, the mechanism by which feedback selectively drives the switch from a healthy fading erythema to each of the five types of diseased expanding erythema remains unclear. This study theoretically elucidates the imbalanced production between pro- and anti-inflammatory mediators and prospective treatment strategies for each expansion pattern. Our literature survey showed that eleven diseases exhibit some of the five expanding erythema, suggesting a common spatiotemporal regulation underlying different patterns and diseases. Accordingly, a reaction-diffusion model incorporating mediator feedback reproduced the five observed types of diseased expanding and healthy fading patterns. Importantly, the fading pattern transitioned to the arcuate, gyrate, and polycyclic patterns when the productions of anti-inflammatory and pro-inflammatory mediators were lower and higher, respectively, than in the healthy condition. Further depletion of anti-inflammatory mediators caused a circular pattern, whereas further overproduction of pro-inflammatory mediators caused an annular pattern. Mechanistically, the bistability due to stabilization of the diseased state exhibits circular and annular patterns, whereas the excitability exhibits the gyrate, polycyclic, arcuate, and fading patterns as the threshold of pro-inflammatory mediator concentration relative to the healthy state increases. These dynamic regulations of diffusive mediator feedback provide effective treatment strategies for mediator production wherein skins recover from each expanding pattern toward a fading pattern. Thus, these strategies can estimate disease severity and risk based on erythema patterns, paving the way for developing noninvasive and personalized treatments for inflammatory skin diseases. | Make paid
Trypanosoma brucei is an early divergent parasitic protozoan that causes a fatal disease, African trypanosomiasis. T. brucei possesses a unique and essential translocase of the mitochondrial inner membrane, the TbTIM17 complex. TbTim17 associates with 6 small TbTims, (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction pattern of the small TbTims with each other and TbTim17 are not clear. Here, we demonstrated by yeast two-hybrid (Y2H) analysis that all six small TbTims interact with each other, but stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. Each of the small TbTims also interact directly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial to maintain the steady-state levels of the TbTIM17 complex. Co-immunoprecipitation analyses from T. brucei mitochondrial extracts also showed that TbTim10 has a stronger association with TbTim9 and TbTim8/13, but a weaker association with TbTim13, whereas TbTim13 has a stronger connection with TbTim17. Analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except TbTim13, is present in ~70 kDa complexes, which could be heterohexameric forms of the small TbTims. However, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionated with TbTim17. Altogether, our results demonstrated that TbTim13 is a part of the TbTIM complex and the smaller complexes of the small TbTims likely interact with the larger complex dynamically. Therefore, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific in T. brucei. | Make paid
Multidomain proteins with flexible linkers and disordered regions play important roles in many cellular processes, but characterizing their conformational ensembles is difficult. In simulations, the situation is complicated further in multi-component systems--such as in the presence of a membrane--since the conformational ensemble depends on subtle balances between the interactions between and within protein, membrane, and water. We have previously shown that, for intrinsically disordered proteins (IDPs) and a small set of multidomain proteins, the widely used coarse grained force field, Martini 3, produces too compact ensembles in solution, and that increasing the strength of protein-water interactions in Martini 3 (by 10%) improves the agreement between simulations and small-angle X-ray scattering (SAXS) for these proteins. Here, we examine whether, as an alternative approach, decreasing the strength of interactions between protein beads can provide equivalent or further improved agreement with the experimental data, and explore the effects of these choices on the interactions with lipid bilayers. We have expanded the set of multidomain proteins to include a wider variety of sizes and domain architectures. Consistent with our previous results, we find that Martini 3 underestimates the global dimensions of this set of multidomain proteins, and that increasing the strength of protein-water interactions (by 10%) or decreasing the strength of non-bonded interactions between protein beads (by 12%) substantially improves the agreement with experimental SAXS data. We show that the \"symmetry\" between rescaling protein-water and protein-protein interactions breaks down when studying interactions with membranes, and that rescaling protein-protein interactions better preserves the binding specificity of peripheral membrane proteins, multidomain proteins, and IDPs with lipid membranes. We conclude that decreasing the strength of protein-protein interactions improves the accuracy of Martini 3 for IDPs and multidomain proteins, both in solution and in the presence of a lipid membrane, providing a favorable alternative to rescaling protein-water interactions. | Make paid
The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP.BeF3, and GroEL-ADP.AlF3-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP.BeF3 to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP.BeF3-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, explaining how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP.AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues. | Make paid
The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques. | Make paid
Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis is not well understood. To investigate this, we study the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical-trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether-force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by a saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes as well as transferrin-labelled pits close to the basal plasma membrane also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked but proceeded uncontrolled. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested on depleting both. The data strongly supports pit-formation to be central to the reduction in fluctuations whether in normal or ATP depleted condition. Furthermore, while in normal conditions the contribution of clathrin-mediated endocytosis is clear, under ATP-depleted conditions we propose that cholesterol-dependent pits spontaneously regulate tension. | Make paid
Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein Phosphatase 4 (PP4) plays a pivotal role in coordinating cellular response to DNA damage. Meanwhile, Centrobin (Ctb), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with Ctb in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and Ctb and shed light on the importance of two specific motifs in Ctb, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in DNA repair process. Moreover, we examine cells depleted of PP4 or Ctb and cells harboring FRVP and SQ mutations in Ctb, which result in similar abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms of PP4 and Ctb-regulated HR repair and their interrelation. | Make paid
Imaging mass spectrometry enables spatial metabolomics, yet metabolites can be assigned only to a fraction of the data generated. METASPACE-ML is a machine learning-based approach addressing this challenge. Integrating new scores and computationally-efficient False Discovery Rate estimation, trained and evaluated on 444 representative datasets from 44 groups, METASPACE-ML surpasses the rule-based predecessor in precision, coverage, and computational efficiency. Our work helps illuminate dark matter in spatial metabolomics with machine learning. | Make paid
Shape changes of epithelia during animal development, such as convergent extension, are achieved through concerted mechanical activity of individual cells. While much is known about the corresponding large scale tissue flow and its genetic drivers, the question of cell-scale coordination remains open. We propose that this coordination can be understood in terms of mechanical interactions and instantaneous force balance within the tissue. Using whole embryo imaging data for Drosophila gastrulation, we exploit the relation between balance of local cortical tension forces and cell geometry. This unveils how local positive feedback on active tension and passive global deformations account for coordinated cell rearrangements. We develop a model that bridges the cell and tissue scale dynamics and predicts the dependence of total tissue extension on initial anisotropy and hexagonal order of the cell packing. Our study provides general insight into the encoding of global tissue shape in local cell-scale activity. | Make paid
Lipid bilayer provides a two-dimensional hydrophobic solvent milieu for membrane proteins in cells. Although the native bilayer is widely recognized as an optimal environment for folding and function of membrane proteins, the underlying physical basis remains elusive. Here, employing the intramembrane protease GlpG of Escherichia coli as a model, we elucidate how the bilayer stabilizes a membrane protein and engages the residue interaction network compared to the nonnative hydrophobic medium, micelles. We find that the bilayer enhances GlpG stability by promoting residue burial in the protein interior compared to micelles. Strikingly, while the cooperative residue interactions cluster into multiple distinct regions in micelles, the whole packed regions of the protein act as a single cooperative unit in the bilayer. Molecular dynamics (MD) simulation indicates that lipids less efficiently solvate GlpG than detergents. Thus, the bilayer-induced enhancement of stability and cooperativity likely stems from the dominant intraprotein interactions outcompeting the weak lipid solvation. Our findings reveal a foundational mechanism in the folding, function, and quality control of membrane proteins. The enhanced cooperativity benefits function facilitating propagation of local structural perturbation across the membrane. However, the same phenomenon can render the conformational integrity of a protein vulnerable to missense mutations causing conformational diseases. | Make paid
Fluorescence microscopy is a fundamental tool in the life sciences, but the availability of sophisticated equipment required to yield high-quality, quantitative data is a major bottleneck in data production in many laboratories worldwide. This problem has long been recognized and the abundancy of low-cost electronics and the simplification of fabrication through 3D-printing have led to the emergence of open-source scientific hardware as a research field. Cost effective fluorescence microscopes can be assembled from cheaply mass-produced components, but lag behind commercial solutions in image quality. On the other hand, blueprints of sophisticated microscopes such as light-sheet or super-resolution systems, custom-assembled from high quality parts are available, but require a high level of expertise from the user. Here we combine the UC2 microscopy toolbox with high-quality components and integrated electronics and software to assemble an automated high-resolution fluorescence microscope. Using this microscope, we demonstrate high resolution fluorescence imaging for fixed and live samples. When operated inside an incubator, long-term live-cell imaging over several days was possible. Our microscope reaches single molecule sensitivity, and we performed single particle tracking and SMLM super-resolution microscopy experiments in cells. Our setup costs a fraction of its commercially available counterparts but still provides a maximum of capabilities and image quality. We thus provide a proof of concept that high quality scientific data can be generated by lay users with a low-budget system and open-source software. Our system can be used for routine imaging in laboratories that do not have the means to acquire commercial systems and through its affordability can serve as teaching material to students. | Make paid
Facial aging is the most visible manifestation of aging. People desire to look younger than others of the same chronological age. Hence, perceived age is often used as a visible marker of aging, while biological age, often estimated by methylation markers, is used as an objective measure of age. Multiple epigenetics-based clocks have been developed for accurate estimation of general biological age and the age of specific organs, including the skin. However, it is not clear whether the epigenetic biomarkers (CpGs) used in these clocks are drivers of aging processes or consequences of aging. In this proof-of-concept study, we integrate data from GWAS on perceived facial aging, and EWAS on CpGs measured in blood. By running EW Mendelian randomization, we identify hundreds of putative CpGs that are potentially causal to perceived facial aging with similar numbers of damaging markers that causally drive or accelerate facial aging and protective methylation markers that causally slow down or protect from aging. We further demonstrate that while candidate causal CpGs have little overlap with known epigenetics-based clocks, they affect genes or proteins with known functions in skin aging such as skin pigmentation, elastin, and collagen levels. Overall, our results suggest that blood methylation markers reflect facial aging processes, and thus can be used to quantify skin aging and develop anti-aging solutions that target the root causes of aging. | Make paid
Fluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single molecule or single particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during FRET analysis in the presence of a significant donor-only fraction. To address this problem, we combined particle localization with phasor-based FLIM analysis. Using simulations, we first showed that an average of ~300 photons, spread over the different pixels encompassing single fluorescing particles and without background, is enough to determine a correct phasor signature (standard deviation <5% for a 4 ns lifetime). For immobilized single- or double-labeled dsDNA molecules, we next validated that particle-based phasor-FLIM-FRET readily allows estimating fluorescence lifetimes and FRET from single molecules. Thirdly, we applied particle-based phasor-FLIM-FRET to investigate protein-protein interactions in sub diffraction HIV-1 viral particles. To do this, we first quantitatively compared the fluorescence brightness, lifetime and photostability of different popular fluorescent protein-based FRET probes when genetically fused to the HIV-1 integrase enzyme (IN) in viral particles, and conclude that eGFP, mTurquoise2 and mScarlet perform best. Finally, for viral particles co-expressing FRET-donor/acceptor labeled IN, we determined the absolute FRET efficiency of IN oligomers. Available in a convenient open-source graphical user interface, we believe that particle-based phasor-FLIM-FRET is a promising tool to provide detailed insights in samples suffering from low overall signal intensities. | Make paid
A critical step in how malaria parasites invade red blood cells (RBCs) is the wrapping of the membrane around the egg-shaped merozoites. Recent experiments have revealed that RBCs can be protected from malaria invasion by high membrane tension. While cellular and biochemical aspects of parasite actomyosin motor forces during the malaria invasion have been well studied, the important role of the biophysical forces induced by the RBC membrane cytoskeleton composite has not yet been fully understood. In this study, we use a theoretical model for lipid bilayer mechanics, cytoskeleton deformation, and membrane-merozoite interactions to systematically investigate the influence of effective RBC membrane tension, which includes contributions from the lipid bilayer tension, spontaneous tension, interfacial tension, and the resistance of cytoskeleton against shear deformation on the progression of membrane wrapping during the process of malaria invasion. Our model reveals that this effective membrane tension creates a wrapping energy barrier for a complete merozoite entry. We calculate the tension threshold required to impede the malaria invasion. We find that the tension threshold is a nonmonotonic function of spontaneous tension and undergoes a sharp transition from large to small values as the magnitude of interfacial tension increases. We also predict that the physical properties of the RBC cytoskeleton layer, particularly the resting length of the cytoskeleton, play key roles in specifying the degree of the membrane wrapping. We also found that the shear energy of cytoskeleton deformation diverges at the full wrapping state, suggesting the local disassembly of the cytoskeleton is required to complete the merozoite entry. Additionally, using our theoretical framework, we predict the landscape of myosin-mediated forces and the physical properties of the RBC membrane in regulating successful malaria invasion. Our findings on the crucial role of RBC membrane tension in inhibiting malaria invasion can have implications for developing novel antimalarial therapeutic or vaccine-based strategies. | Make paid
Biological systems are regulated by molecular interactions which are tuned by the concentrations of each of the molecules involved. Cells exploit this feature by regulating protein expression, to adapt their responses to overstimulation. Correlating events in single cells to the concentrations of proteins involved can therefore provide important mechanistic insight into cell behavior. Unfortunately, quantification of molecular densities by fluorescence imaging becomes non-trivial due to the diffraction limited resolution of the imaged volume. We show here an alternative approach to overcome this limitation in optical quantification of protein concentrations which is based on calibrating protein volume and surface densities in a model membrane system. We exploit the ability of fluorescently labeled annexin V to bind membranes in presence of calcium. By encapsulating known concentrations of annexin V, we can directly infer the membrane density of annexin V after addition of Ca2+ and correlate the density with the measured fluorescence signal. Our method, named Calmet, enables quantitative determination of the concentration of cytosolic and membrane associated proteins. The applicability of Calmet is demonstrated by quantification of a transmembrane protein receptor (beta 1 adrenergic receptor) labeled by SNAP tagged fluorophores and expressed in HEK293 cells. Calmet is a generic method suitable for the determination of a broad range of concentrations and densities and can be used on regular fluorescence images captured by confocal laser scanning microscopy. | Make paid
Large-scale pretrained models have become foundation models, leading to breakthroughs in natural language processing and related fields. Developing foundation models in life science, aimed at deciphering the "languages" of cells and facilitating biomedical research, is challenging yet promising. We developed a large-scale pretrained model, scFoundation, for this purpose. scFoundation was trained on over 50 million human single-cell transcriptomics data, which contain high-throughput observations on the complex molecular features in all known types of cells. scFoundation is currently the largest model in terms of the size of trainable parameters, dimensionality of genes and the number of cells used in the pre-training. Experiments showed that scFoundation can serve as a foundation model for single-cell transcriptomics and can achieve state-of-the-art performances in a diverse array of downstream tasks, such as gene expression enhancement, tissue drug response prediction, single-cell drug response classification, and single-cell perturbation prediction. | Make paid
The emerging field of living technologies aims to create new functional hybrid materials in which living systems interface and interact with inanimate ones. Combining research into living technologies with emerging developments in computing architecture has enabled the generation of organic electronics from plants and slime mould. Here, we expand on this work by studying capacitive properties of a substrate colonised by mycelium of grey oyster fungi, Pleurotus ostreatus. Capacitors play a fundamental role in traditional analogue and digital electronic systems and have a range of uses including sensing, energy storage and filter circuits. Mycelium has the potential to be used as an organic replacement for traditional capacitor technology. Here, were show that the capacitance of mycelium is in the order of hundreds of picofarads and at the same time a voltage-dependent pseudocapacitance of the order of hundreds of microfarads. We also demonstrate that the charge density of the mycelium 'dielectric' decays rapidly with increasing distance from the source probes. This is important as it indicates that small cells of mycelium could be used as a charge carrier or storage medium. | Make paid
CDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA Pol II, and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation. | Make paid