bluuebunny/crossref_metadata_2025 huggingface dataset:
Can lateral tenodesis improve the rotational stability of the ACL reconstruction? A finite element analysis
Authors: Risvas Konstantinos , Stanev Dimitar , Moustakas Konstantinos
Published: October 2023

One of the most common knee injuries is the Anterior Cruciate Ligament (ACL) rupture with severe implications on knee stability. The usual treatment is the ACL Reconstruction (ACLR) surgery where the surgeon replaces the torn ligament with a graft in an effort to restore knee kinematics. In case of excessive rotatory instability, Lateral Extra - Articular Tenodesis (LET) can be performed in combination with ACLR. Additionally, LET appears to reduce ACLR graft forces reducing graft failure chances. However, there are concerns about overconstraining physiological rotation. To gain insight in this controversial topic, we developed an automatic, open-source tool to create a series of Finite Element (FE) models attempting to investigate the interactions of ACLR and LET through simulation. We started by creating a validated model of the healthy knee joint that served as reference for subsequent FE simulations. Then, we created FE models of standalone ACLR and combined ACLR - LET. Each model was assessed by applying a loading profile that resembles the Pivot - Shift clinical exam. We measured the External Tibia Rotation (ETR), the Anterior Tibia Translation (ATT) of the lateral tibial compartment, and the ACLR graft stress developed around the femoral tunnel insertion site. We observed the following: a) LET reduces ETR and ATT compared to isolated ACLR, b) combined ACLR - LET is more sensitive to LET graft pretension with lower values showcasing performance closer to the healthy joint, c) LET reduces ACLR graft forces for the same pretension values, d) LET exhibits significant overconstraint for higher pretension values. In general, these findings are in agreement with relevant clinical studies and accentuate the potential of the developed framework as a tool that can assist orthopaedists during surgery planning. We provide open access for the FE models of this study to enhance research transparency, reproducibility and extensibility.
The Rho GTPase Rac1 mediates exercise training adaptations
Authors: Raun Steffen H. , Henriquez-Olguín Carlos , Frank Emma , Knudsen Jonas Roland , Ali Mona S. , Andersen Nicoline R. , Møller Lisbeth L. V. , Davey Jonathan , Qian Hongwei , Coelho Ana , Carl Christian S. , Voldstedlund Christian T. , Kiens Bente , Holmdahl Rikard , Gregorevic Paul , Jensen Thomas E. , Richter Erik A. , Sylow Lykke
Published: October 2023

Exercise training elicits tremendous health benefits; however, the molecular underpinnings are poorly understood. As one of the most regulated groups of proteins following acute exercise in human muscle, Rho GTPases are unexplored candidates for mediating the beneficial effects of exercise. The Rho GTPase Rac1 was activated during multiple exercise modalities and remained elevated hours after resistance exercise in human muscle. Inducible muscle-specific Rac1 knockout (Rac1 imKO) mice, displayed attenuated muscle protein synthesis, glycogen resynthesis and p38 MAPK signaling in recovery from contractions. Exercise training upregulated Rac1 protein content in human and mouse muscle. Overexpression of hyperactive Rac1 elevated reactive oxidant species production during exercise yet did not induce a trained muscle phenotype. In Rac1 imKO mice, the improvements in running capacity and muscle mass after exercise training were diminished. Using gain- and loss-of-function mouse models and human muscle biopsies, we identify Rac1 as a regulator of exercise training adaptions.

Highlights

Various exercise modalities activate Rac1 signaling in human skeletal muscle.

HSP27, MNK1, and CREB are Rac1-dependent contraction-responsive targets in muscle.

Post-contraction protein synthesis requires Rac1 but not NOX2.

Rac1-NOX2 signaling is necessary for post-contraction glycogen resynthesis.

Exercise training increases Rac1 protein content in human and mouse muscles.

Rac1 mediates critical adaptations to exercise training.
To touch or to be touched? Comparing pleasantness of vicarious execution and reception of interpersonal touch
Authors: Butti Niccolò , Urgesi Cosimo , McGlone Francis P. , Oldrati Viola , Montirosso Rosario , Cazzato Valentina
Published: October 2023

Unmyelinated C-Tactile (CT) fibres are activated by caress-like touch, eliciting a pleasant feeling that decreases for static and faster stroking. Previous studies documented this effect also for vicarious touch, hypothesising ‘mirror-neurons’ mechanisms driving the perception and appreciation of observed interpersonal touch. Notably, less is known about appreciation of vicarious execution of touch, that is as referred to the one giving gentle touch. To address this issue, 53 healthy participants were asked to view and rate a series of videoclips displaying an individual being touched by another on hairy (i.e., hand dorsum) or glabrous (i.e., palm) skin sites, with touch being delivered at CT-optimal (5 cm/s) or non-CT optimal velocities (0 cm/s or 30 cm/s). Following the observation of each clip, participants were asked to complete self- and other-directed ratings of the pleasantness of touch for both executer (toucher-referred) and receiver (touchee-referred). Consistent with the CT fibres properties, for both self- and other-directed judgements of touch execution and reception, participants provided higher ratings for touch delivered at CT-optimal than other velocities, and when CT-optimal touch was delivered to the hand-dorsum compared to the palm. However, higher ratings were attributed to reception compared to execution of CT-optimal touch. Notably, individual differences in interoceptive trusting and attitude to interpersonal touch were positively correlated with, respectively, toucher- and touchee-related overall pleasantness of touch. These findings suggest that the appreciation of both toucher- and touchee-referred vicarious touch is specifically attuned to CT-optimal touch, even though they might rely on different neurocognitive mechanisms to understand affective information conveyed by interpersonal tactile interactions.
In silico MS/MS prediction for peptidoglycan profiling uncovers novel anti-inflammatory peptidoglycan fragments of the gut microbiota
Authors: Kwan Jeric Mun Chung , Liang Yaquan , Ng Evan Wei Long , Sviriaeva Ekaterina , Li Chenyu , Zhao Yilin , Zhang Xiao-Lin , Liu Xue-Wei , Wong Sunny H. , Qiao Yuan
Published: October 2023

Peptidoglycan is an essential exoskeletal polymer present across all bacteria. The gut microbiota-derived peptidoglycan fragments (PGNs) are increasingly recognized as key effector molecules that impact host biology, offering attractive yet untapped potential to combat microbiome-associated diseases in humans. Unfortunately, comprehensive peptidoglycan profiling of gut bacteria has been hampered by the lack of a robust and automated analysis workflow. Currently, PGN identification still relies on manual deconvolutions of acquired tandem mass spectrometry (MS/MS) data, which are highly laborious and inconsistent. Recognizing the unique sugar and amino acid makeup of bacterial peptidoglycan and guided by the experimental MS/MS fragmentation patterns of known PGNs, we developed a computational tool PGN_MS2 that reliably simulates MS/MS spectra of PGNs. Integrating PGN_MS2 into the customizablein silicoPGN database, we built an open-access PGN MS library of predicted MS/MS spectra for all molecules in the user-definedin silicoPGN search space. With this library, automated searching and spectral matching can be used to identify PGN. We then performed comprehensive peptidoglycan profiling for several gut bacteria species, revealing distinct PGN structural features that may be implicated in microbiota-host crosstalk. Strikingly, the probioticBifidobacteriumspp. has an exceedingly high proportion of anhydro-PGNs, which exhibit anti-inflammatory effectsin vitro. We further identified MltG and RfpB homologs inBifidobacteriumas lytic transglycosylases (LTs), which demonstrate distinct substrate preferences to produce anhydro-PGNs. Overall, our novel PGN_MS2 prediction tool contributes to the robust and automated peptidoglycan analysis workflow, advancing efforts to elucidate the structures and functions of gut microbiota-derived PGNs in the host.
Variability in radiocesium activity concentration in growing hardwood shoots in Fukushima, Japan
Authors: Itô Hiroki , Miura Satoru , Komatsu Masabumi , Kanasashi Tsutomu , Nagakura Junko , Hirai Keizo
Published: October 2023

The radiocesium contamination caused by the Fukushima Daiichi Nuclear Power Plant accident has made it difficult to use coppice woods as bed logs for mushroom cultivation. Evaluating the variability in the radiocesium activity concentration of logs is necessary in order to predict how many coppice woodlands are available for producing mushroom bed logs. To clarify the variability in radiocesium activity concentrations and to estimate the sample size required to estimate these concentrations with sufficient accuracy, we modeled the log-transformed radiocesium activity concentrations in growing shoots of hardwoods. We designed two models: (1) a model with mean concentrations that varied among stands with a standard deviation that was the same among stands, and (2) a model with varying means and standard deviations. We fit the data pertaining to onlyQuercus serratato both models and calculated the widely applicable information criterion values. Consequently, we adopted the simpler model (1). Applying the selected model to data for all species, we examined the relationship between the number of measurement individuals and the predictive distribution of the expected concentration. Based on previous recommendations and measurement costs, we proposed that five individuals would be appropriate for estimating radiocesium activity concentration in a stand.
Population genomic analysis reveals genetic structure and thermal-tolerant genotypes in remnant Tasmanian giant kelp populations
Authors: Iha Cintia , Layton Cayne , Amancio Carlos E. , Flentje Warren , Lenton Andrew , Fraser Ceridwen I. , Johnson Craig , Willis Anusuya
Published: October 2023

Giant kelp,Macrocystis pyrifera, is a foundation species that forms dense forests of complex physical habitat and supports coastal biodiversity, productivity, and other essential ecosystem services. Tasmanian coasts have suffered a massive decline in giant kelp forests due to changes in regional oceanography and environmental conditions, but efforts are being made to restore these disappearing populations using identification and selective breeding for lineages that are more tolerant to warmer temperatures. Here, we used gametophytes that originated from remnant populations collected at three sites in northeastern and three sites in southeastern Tasmania to determine the genetic structure of the giant kelp population via genotyping-by-sequencing (GBS) and assembled a draft genome from a Tasmanian giant kelp sporophyte individual. Previous research identified seven strain lines that were tolerant to warm temperatures, and we used the GBS data to test whether genotypes were associated with warm tolerance. Kelps from the north were genetically distinct from the southern ones, with much lower gene flow between regions than within regions. These results revealed that giant kelp populations from Tasmania are more genetically structured than previously thought. Two loci were significantly associated with warm temperature tolerance. They are population-specific: the alternative allele from one of the loci was found only in the northern populations, while the other was found in the southern populations. This could suggest that thermal tolerance is adapting locally or drifting given large changes in population demography, but further research is needed to confirm this hypothesis. Our research sheds light on genetic patterns in this critical habitat-forming kelp and will help inform conservation management, including selective breeding.
Volumetric Compression Shifts Rho GTPase Balance and Induces Mechanobiological Cell State Transition
Authors: Gong Xiangyu , Nguyen Ryan , Chen Zehua , Wen Zhang , Zhang Xingjian , Mak Michael
Published: October 2023

During development and disease progression, cells are subject to osmotic and mechanical stresses that modulate cell volume, which fundamentally influences cell homeostasis and has been linked to a variety of cellular functions. It is not well understood how the mechanobiological state of cells is programmed by the interplay of intracellular organization and complex extracellular mechanics when stimulated by cell volume modulation. Here, by controlling cell volume via osmotic pressure, we evaluate physical phenotypes (including cell shape, morphodynamics, traction force, and extracellular matrix (ECM) remodeling) and molecular signaling (YAP), and we uncover fundamental transitions in active biophysical states. We demonstrate that volumetric compression shifts the ratiometric balance of Rho GTPase activities, thereby altering mechanosensing and cytoskeletal organization in a reversible manner. Specifically, volumetric compression controls cell spreading, adhesion formation, and YAP nuclear translocation, while maintaining cell contractile activity. Furthermore, we show that on physiologically relevant fibrillar collagen I matrices, which are highly non-elastic, cells exhibit additional modes of cell volume-dependent mechanosensing that are not observable on elastic substrates. Notably, volumetric compression regulates the dynamics of cell-ECM interactions and irreversible ECM remodeling via Rac-directed protrusion dynamics, at both the single-cell level and the multicellular level. Our findings support that cell volume is a master biophysical regulator and reveal its roles in cell mechanical state transition, cell-ECM interactions, and biophysical tissue programming.
Adaptation to volumetric compression drives hepatoblastoma cells to an apoptosis-resistant and invasive phenotype
Authors: Gong Xiangyu , Ogino Noriyoshi , Leite M. Fatima , Chen Zehua , Nguyen Ryan , Liu Raymond , Kruglov Emma , Flores Kaitlin , Cabral Aiden , Mendes Gabriel M. M. , Ehrlich Barbara E. , Mak Michael
Published: October 2023

Liver cancer involves tumor cells rapidly growing within a packed tissue environment. Patient tumor tissues reveal densely packed and deformed cells, especially at tumor boundaries, indicative of physical crowding and compression. It is not well understood how these physical signals modulate tumor evolution and therapeutic susceptibility. Here we investigate the impact of volumetric compression on liver cancer (HepG2) behavior. We find that conditioning cells under a highly compressed state leads to major transcriptional reprogramming, notably the loss of hepatic markers, the epithelial-to-mesenchymal transition (EMT)-like changes, and altered calcium signaling-related gene expression, over the course of several days. Biophysically, compressed cells exhibit increased Rac1-mediated cell spreading and cell-extracellular matrix interactions, cytoskeletal reorganization, increased YAP and β-catenin nuclear translocation, and dysfunction in cytoplasmic and mitochondrial calcium signaling. Furthermore, compressed cells are resistant to chemotherapeutics and desensitized to apoptosis signaling. Apoptosis sensitivity can be rescued by stimulated calcium signaling. Our study demonstrates that volumetric compression is a key microenvironmental factor that drives tumor evolution in multiple pathological directions and highlights potential countermeasures to re-sensitize therapy-resistant cells.

Significance statement

Compression can arise as cancer cells grow and navigate within the dense solid tumor microenvironment. It is unclear how compression mediates critical programs that drive tumor progression and therapeutic complications. Here, we take an integrative approach in investigating the impact of compression on liver cancer. We identify and characterize compressed subdomains within patient tumor tissues. Furthermore, using in vitro systems, we induce volumetric compression (primarily via osmotic pressure but also via mechanical force) on liver cancer cells and demonstrate significant molecular and biophysical changes in cell states, including in function, cytoskeletal signaling, proliferation, invasion, and chemoresistance. Importantly, our results show that compressed cells have impaired calcium signaling and acquire resistance to apoptosis, which can be countered via calcium mobilization.
The impact of short-lived controls on the interpretation of lifespan experiments and progress in geroscience
Authors: Pabis Kamil , Barardo Diogo , Gruber Jan , Sirbu Olga , Selvarajoo Kumar , Kaeberlein Matt , Kennedy Brian K.
Published: October 2023

Although lifespan extension remains the gold standard for assessing interventions hypothesized to impact the biology of aging, there are important limitations to this approach. Our reanalysis of lifespan studies from multiple sources suggests that the use of short-lived control cohorts tends to exaggerate the relative efficacy of putative longevity interventions. Moreover, due to the high cost and long timeframes of mouse studies, it is rare that a particular longevity intervention will be independently replicated by multiple groups.

To facilitate identification of successful interventions, we propose an alternative approach. The level of confidence we can have in an intervention is proportional to the degree of lifespan extension above the strain- and species-specific upper limit of lifespan, which we can estimate from comparison to historical controls. In the absence of independent replication, a putative mouse longevity intervention should only be considered with high confidence when control lifespans are close to 900 days or if the final lifespan of the treated group is considerably above 900 days. Using this “900-day rule” we identified several candidate interventions from the literature that merit follow-up studies.
Glycolytic oscillations under periodic drivings
Authors: Kim Pureun , Hyeon Changbong
Published: October 2023

In many living organisms displaying circadian rhythms, the intake of energy often occurs in a periodic manner. Glycolysis is a prototypical biochemical reaction that exhibits a self-sustained oscillation under continuous injection of glucose. Here we study the effect of periodic injection of glucose on the glycolytic oscillation from a dynamical systems perspective. In particular, we employ the Goldbeter’s allosteric model of phosphofructokinase (PFK) as a model system for glycolytic oscillations, and explore the effect of periodic substrate influx of varying frequencies and amplitudes by building the phase diagrams of Lyapunov exponents and oscillatory periods. When the frequency of driving is tuned around the harmonic and sub/super-harmonic conditions of the natural frequency, the system is entrained to a frequency-locked state, forming an entrainment band that broadens with an increasing amplitude of driving. On the other hand, if the amplitude is substantial, the system may transition, albeit infrequent, to a chaotic state which defies prediction of dynamical behavior. Our study offers in-depth understandings into the controllability of glycolytic oscillation as well as explains physical underpinnings that enable the synchronous oscillations among a dense population of cells.
Cortical networks of parkinsonian gait: a metabolic and functional connectivity study
Authors: Pellegrini Franziska , Pozzi Nicoló G. , Palmisano Chiara , Marotta Giorgio , Buck Andreas , Haufe Stefan , Isaias Ioannis U.
Published: October 2023

Objective

Locomotion is an automated voluntary movement sustained by coordinated neural synchronization across a distributed brain network. The cerebral cortex is central for adapting the locomotion pattern to the environment and alterations of cortical network dynamics can lead to gait impairments. Gait problems are a common symptom with a still unclear pathophysiology and represent an unmet therapeutical need in Parkinson’s disease. Little is known about the cortical network dynamics of locomotor control in these patients.

Methods

We studied the cortical basis of parkinsonian gait by combining metabolic brain imaging with high-density EEG recordings and kinematic measurements performed at rest and during unperturbed overground walking.

Results

We found significant changes in functional connectivity between frontal, sensorimotor and visuomotor cortical areas during walking as compared to resting. Specifically, hypokinetic gait was associated with poor information flow from the supplementary motor area (SMA) to precuneus and from calcarine to lingual gyrus as well as high information flow from calcarine to cuneus.

Interpretation

Our findings support a role for visuomotor integration processes in PD-related hypokinetic gait and suggest that reinforcing visual information may act as a compensatory strategy to allow SMA-related feedforward locomotor control in PD.
Influence of socioeconomic status on functional outcomes after stroke: a systematic review and meta-analysis
Authors: Nguyen Mai T H , Sakamoto Yuki , Maeda Toshiki , Woodward Mark , Anderson Craig S. , Catiwa Jayson , Yazidjoglou Amelia , Carcel Cheryl , Yang Min , Wang Xia
Published: October 2023

Background

Despite advances in stroke treatment and rehabilitation, socioeconomic factors have an important impact on recovery from stroke. This review aimed to quantify the impact of socioeconomic status (SES) on functional outcomes from stroke and identify the SES indicators that exhibit the highest magnitude of association.

Methods

We performed a systematic literature search across Medline and Embase databases up to May 2022, for studies fulfilling the following criteria: observational studies with ≥100, patients aged ≥18 years with stroke diagnosis based on clinical examination or in combination with neuroimaging, reported data on the association between SES and functional outcome, assessed functional outcomes with the modified Rankin Scale (mRS) or Barthel index tools, provided estimates of association (odds ratios [OR] or equivalent), and published in English. Risk of bias was assessed using the modified Newcastle Ottawa Scale.

Findings

We identified 7,698 potentially eligible records through the search after removing duplicates. Of these, 19 studies (157,715 patients, 47.7% women) met our selection criteria and were included in the meta-analyses. Ten studies (53%) were assessed as low risk of bias. Measures of SES reported were education (11 studies), income (8), occupation (4), health insurance status (3), and neighbourhood socioeconomic deprivation (3). Random-effect meta-analyses revealed low SES was significantly associated with poor functional outcomes: incomplete education or below high school level versus high school attainment and above (OR [95% CI]: 1.66 [1.40, 1.95]), lowest income versus highest income (1.36 [1.02, 1.83], a manual job/unemployed versus a non-manual job/employed (1.62 [1.29, 2.02]), and living in the most disadvantaged socioeconomic neighbourhood versus the least disadvantaged (1.55 [1.25, 1.92]). Low health insurance status was also associated with an increased risk of poor functional outcomes (1.32 [0.95, 1.84]), although not statistically significant.

Conclusions

Socioeconomic disadvantage remains a risk factor for poor functional outcomes after an acute stroke. Further research is needed to better understand causal mechanisms and disparities.

Funding

This study is supported by an NHMRC Investigator grant (APP1195237).
Unravelling Causal Associations between Population Mobility and COVID-19 Cases in Spain: a Transfer Entropy Analysis
Authors: Ponce-de-Leon Miguel , Pontes Camila , Arenas Alex , Valencia Alfonso
Published: October 2023

Human mobility is a well-known factor in the spread of infectious diseases. During the COVID-19 pandemic, the rapid spread of the SARS-CoV-2 virus led to healthcare systems collapsing in numerous countries, such as Spain and Italy, resulting in a significant number of deaths. To avoid such disastrous outcomes in the future, it is vital to understand how population mobility is linked to the spread of infectious diseases. To assess that, we applied an information theoretic approach called transfer entropy (TE) to measure the influence of the number of infected people travelling between two localities on the future number of infected people in the destination. We first validated our approach using simulated data from a SIR epidemiological model and found that the mobility-based TE was effective in filtering out non-causal influences that could otherwise arise, thereby successfully recovering the epidemic’s spreading patterns and the mobility network topology. We then applied the mobility-based TE to analyse the COVID-19 pandemic in Spain. We identified which regions acted as the main drivers of the pandemic at different periods, both globally and locally. Our results unravelled significant epidemiological events such as the outbreak in Lleida during the Summer of 2020, caused by the influx of temporary workers. We also analysed the effects of a non-pharmaceutical intervention in Catalunya, using mobility- based TE to compare the infection dynamics with a control region. These results help clarify how human mobility influences the dynamic spread of infectious diseases and could be used to inform future non-pharmaceutical interventions.
Testing the sensitivity of diagnosis-derived patterns in functional brain networks to symptom burden in a Norwegian youth sample
Authors: Voldsbekk Irene , Kjelkenes Rikka , Frogner Erik R. , Westlye Lars T. , Alnæs Dag
Published: October 2023

Aberrant brain network development represents a putative aetiological component in mental disorders, which typically emerge during childhood and adolescence. Previous studies have identified resting-state functional connectivity (RSFC) patterns reflecting psychopathology, but the generalisability to other samples and politico-cultural contexts has not been established.

We investigated whether a previously identified cross-diagnostic case-control and autism spectrum disorder (ASD)-specific pattern of RSFC (discovery sample; aged 5-21 from New York City, USA; n=1666) could be validated in a Norwegian convenience-based youth sample (validation sample; aged 9-25 from Oslo, Norway; n=531). As a test of generalisability, we investigated if these diagnosis-derived RSFC patterns were sensitive to levels of symptom burden in both samples, based on an independent measure of symptom burden.

Both the cross-diagnostic and ASD-specific RSFC pattern were validated across samples. Connectivity patterns were significantly associated with thematically appropriate symptom dimensions in the discovery sample. In the validation sample, the ASD-specific RSFC pattern showed a weak, inverse relationship with symptoms of conduct problems, hyperactivity, and prosociality, while the cross-diagnostic pattern was not significantly linked to symptoms.

Diagnosis-derived connectivity patterns in a developmental clinical US sample were validated in a convenience sample of Norwegian youth, however, they were not associated with mental health symptoms.
The tumor multi-omic landscape of endometrial cancers developed on a germline genetic background of adiposity
Authors: Richenberg George , Francis Amy , Owen Carina N. , Gray Victoria , Robinson Timothy , AG Gabriel Aurélie , Lawrenson Kate , Crosbie Emma J. , Schildkraut Joellen M. , Mckay James D. , Gaunt Tom R. , Relton Caroline L. , Vincent Emma E. , Kar Siddhartha P.
Published: October 2023

High body mass index (BMI) is a causal risk factor for endometrial cancer but the tumor molecular mechanisms affected by adiposity and their therapeutic relevance remain poorly understood. Here we characterize the tumor multi-omic landscape of endometrial cancers that have developed on a background of lifelong germline genetic exposure to elevated BMI. We built a polygenic score (PGS) for BMI in women using data on independent, genome-wide significant variants associated with adult BMI in 434,794 women. We performed germline (blood) genotype quality control and imputation on data from 354 endometrial cancer cases from The Cancer Genome Atlas (TCGA). We assigned each case in this TCGA cohort their genetically predicted life-course BMI based on the BMI PGS. Multivariable generalized linear models adjusted for age, stage, microsatellite status and genetic principal components were used to test for associations between the BMI germline PGS and endometrial cancer tumor genome-wide genomic, transcriptomic, proteomic, epigenomic and immune traits in TCGA. High BMI germline PGS was associated with (i) upregulated tumor gene expression in theIL6-JAK-STAT3pathway (FDR=4.2×10−7); (ii) increased estimated intra-tumor activated mast cell infiltration (FDR=0.008); (iii) increased single base substitution (SBS) mutational signatures 1 (FDR=0.03) and 5 (FDR=0.09) and decreased SBS13 (FDR=0.09), implicating age-related and APOBEC mutagenesis, respectively; and (iv) decreased tumor EGFR protein expression (FDR=0.07). Alterations inIL6-JAK-STAT3signaling gene and EGFR protein expression were, in turn, significantly associated with both overall survival and progression-free interval. Thus, we integrated germline and somatic data using a novel study design to identify associations between genetically predicted lifelong exposure to higher BMI and potentially actionable endometrial cancer tumor molecular features. These associations inform our understanding of how high BMI may influence the development and progression of this cancer, impacting endometrial tumor biology and clinical outcomes.
Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells
Authors: Hammer Quirin , Perica Karlo , van Ooijen Hanna , Mbofung Rina , Momayyezi Pouria , Varady Erika , Martin Karen E. , Pan Yijia , Jelcic Mark , Groff Brian , Abujarour Ramzey , Krokeide Silje , Lee Tom , Williams Alan , Goodridge Jode P. , Valamehr Bahram , Önfelt Björn , Sadelain Michel , Malmberg Karl-Johan
Published: October 2023

Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection, although absence ofB2Mtriggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligandsCD54andCD58on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion ofCD54andCD58inB2M-deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cellsin vitroandin vivowithout affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.
MRP1 inhibition by lipid-derived electrophiles during ferroptosis illustrates a role for protein alkylation in ferroptotic cell death
Authors: Van Kessel Antonius T. M. , Cosa Gonzalo
Published: October 2023

Ferroptosis is a regulated form of cell death characterized by lipid peroxidation and lipid hydroperoxide (LOOH) generation that offers new therapeutic opportunities. However, the molecular mechanism through which LOOH accumulation leads to cell death remains poorly understood. Importantly, LOOH breakdown forms truncated phospholipids (PLs) and highly reactive lipid-derived electrophiles (LDEs) capable of altering protein function through cysteine alkylation. While truncated PLs have been shown to mediate ferroptotic membrane permeabilization, a functional role for LDEs in the ferroptotic cell death mechanism has not been established. Here, using multidrug resistance protein 1 (MRP1) activity as an example, we demonstrate that LDEs mediate altered protein functionduring ferroptosis. Applying live cell fluorescence imaging, we first identified that inhibition of MRP1-mediated LDE detoxification occurs across a panel of ferroptosis inducers (FINs) with differing mechanisms of ferroptosis induction (Types I-IV FINs erastin, RSL3, FIN56 and FINO2). This MRP1 inhibition was recreated by both initiation of lipid peroxidation and treatment with the LDE 4-hydroxy-2-nonenal (4-HNE). Importantly, treatment with radical-trapping antioxidants prevented impaired MRP1 activity when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of inhibited MRP1 activity during ferroptosis. Our findings, when combined with reports of widespread LDE-alkylation of key proteins during ferroptosis, sets a precedent for LDEs as critical mediators of ferroptotic cell death. LOOH breakdown to truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function during late stage ferroptosis, offering a unified explanation of the molecular ferroptotic cell death mechanism.
Nanoscale Structural Mapping of Protein Aggregates in Live Cells Modeling Huntington’s Disease
Authors: Guo Zhongyue , Chiesa Giulio , Yin Jiaze , Sanford Adam , Meier Stefan , Khalil Ahmad S. , Cheng Ji-Xin
Published: October 2023

Protein aggregation, in the form of amyloid fibrils, is intimately correlated with many neurodegenerative diseases. Despite recent advances in structural biology, it remains challenging to acquire structural information of proteins in live cells. Tagging with fluorescent proteins, like green fluorescent protein (GFP), is routinely used for protein visualization. Yet, this method alone cannot provide detailed structural information on the protein system of interest, and tagging proteins has the potential to perturb native structure and function. Here, by fluorescence-detected as well as label-free scattering-based mid-infrared photothermal (MIP) microscopy, we demonstrate nanoscale mapping of secondary structure of protein aggregates in a yeast model of Huntington’s disease. We first used GFP as a highly sensitive photothermal reporter to validate β-sheet enrichment in huntingtin (htt) protein aggregates. We then obtained label-free structural maps of protein aggregates. Our data showed that the fluorescent protein tag indeed perturbed the secondary structure of the aggregate, evident by a spectral shift. Live cell MIP spectroscopy further revealed the fine spatial distribution of structurally distinct components in protein aggregates, featuring a 246-nm diameter core highly enriched in β-sheet surrounded by a ɑ-helix-rich shell. Interestingly, this structural partition exists only in presence of the [RNQ+] prion, a prion that acts to facilitate the formation of other amyloid prions. Indeed, when htt is induced to aggregate in the absence of this prion ([rnq-] state), it forms non-toxic amyloid aggregates exclusively. These results showcase the potential of MIP for unveiling detailed and subtle structural information on protein systems in live cells.

Significance

Protein aggregation is a hallmark of neurodegenerative diseases, such as Huntington’s Disease. Understanding the nature of neurotoxic aggregates could lead to better therapeutic approaches. The limited progress in this direction is partly due to the lack of tools for extracting structural information in the physiological context of the aggregates. Here, we report a photothermally detected mid-infrared micro-spectroscopy technique able to dissect the secondary structure of aggregates of the huntingtin protein in live cells. We describe for the first time a nanoscale partition of secondary structures between β-rich core and ɑ-rich shell of the aggregates. This work demonstrates the potential of mid-infrared photothermal microscopy for structural and functional mapping of proteins in live cells.
A robust life-or-death selection platform for enzyme evolution
Authors: Jansen Suzanne C. , Mayer Clemens
Published: October 2023

Life-or-death selections evaluate the fitness of individual organisms on a population level. In enzyme engineering, such growth selections allow the rapid and straightforward identification of highly efficient biocatalysts from extensive libraries. However, selection-based improvement of (industrially-relevant) biocatalysts is challenging, as they require highly dependable strategies that artificially link their activities to host survival. Here, we showcase a robust and scalable life-or-death selection platform centered around the complementation of non-canonical amino acid-dependent bacteria. Specifically, we demonstrate how serial passaging of populations featuring millions of carbamoylase variants autonomously selects biocatalysts with up to 90,000-fold higher initial rates. Notably, selection of replicate populations enriched diverse biocatalysts, which feature distinct amino-acid motifs that drastically boost carbamoylase activity. As beneficial substitutions also originated from unintended copying errors during library preparation or cell division, we anticipate that our life-or-death selection platform will be applicable to the continuous, autonomous evolution of diverse biocatalysts in the future.
Discovery of Novel Replication Proteins for Large Plasmids in Cyanobacteria and Their Potential Applications in Genetic Engineering
Authors: Ohdate Kazuma , Sakata Minori , Maeda Kaisei , Sakamaki Yutaka , Nimura-Matsune Kaori , Ohbayashi Ryudo , Hess Wolfgang R. , Watanabe Satoru
Published: October 2023

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on Rep proteins necessary for plasmid replication initiation in cyanobacteria is limited.Synechocystissp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes inSynechocystis6803,slr6031andslr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 inSynechococcus elongatusPCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. Theslr6031/slr6090-containing plasmids exhibited lower copy numbers and instability inSynechococcus7942 cells compared to the expression vector pYS. While recombination occurred in the case ofslr6090, the engineered plasmid withslr6031coexisted with plasmids encoding CyRepA1 or Slr6090 inSynechococcus7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (CyanobacterialRep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with “two Reps in one plasmid”. Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps inSynechococcus7942 andSynechocystis6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.
Optimization for High-Throughput BiFC Screening
Authors: Jia Yunlong , Reboulet Jonathan , Bleicher Françoise , Dumont Agnès , Ruscio Sylvie Di , Gillet Benjamin , Hughes Sandrine , Merabet Samir
Published: October 2023

The Cell-PCA screen, since its inception, has provided an efficient method for analyzing cellular interactomes and has been used in various biological studies involving proteins like MYC, PER2, and ERK. With rapid advancements in biotechnology, including tools for protein function investigation, the Cell-PCA screen remains relevant. However, despite its successful application in recent studies, there are areas for optimization to ensure its continued relevance in the face of evolving technological advancements.
An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal stem cells
Authors: Cotton Michael J , Ariel Pablo , Chen Kaiwen , Walcott Vanessa A , Dixit Michelle , Breau Keith A , Hinesley Caroline M , Kedziora Kasia , Tang Cynthia Y , Zheng Anna , Magness Scott T , Burclaff Joseph
Published: October 2023

Background and Aims

The intestinal epithelium exhibits dynamic control of cell cycle phase lengths, yet no experimental platform exists for directly analyzing cell cycle phases in living human intestinal stem cells (ISCs). Here, we develop primary human ISC lines with two different reporter constructs to provide fluorescent readouts to analyze cell cycle phases in cycling ISCs.

Methods

3D printing was used to construct a collagen press for making chamber slides that support primary human ISC growth and maintenance within the working distance of a confocal microscope objective. The PIP-FUCCI fluorescent cell cycle reporter and a variant with H2A-mScarlet that allows for automated tracking of cell cycle phases (PIP-H2A) were used in human ISCs along with live imaging and EdU pulsing. An analysis pipeline combining free-to-use programs and publicly available code was compiled to analyze live imaging results.

Results

Chamber slides with soft collagen pressed to a thickness of 0.3 mm concurrently support ISC cycling and confocal imaging. PIP-FUCCI ISCs were found to be optimal for snapshot analysis wherein all nuclei are assigned to a cell cycle phase from a single image. PIP-H2A ISCs were better suited for live imaging since constant nuclear signal allowed for more automated analysis. CellPose2 and TrackMate were used together to track cycling cells.

Conclusions

We present two complete platforms for analyzing cell cycle phases in living primary human ISCs. The PIP-FUCCI construct allows for cell cycle phase assignment from one image of living cells, the PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths in human ISCs using our computational pipeline. These platforms hold great promise for future studies on how pharmaceutical agents affect the intestinal epithelium, how cell cycle is regulated in human ISCs, and more.
A Suite of Designed Protein Cages Using Machine Learning Algorithms and Protein Fragment-Based Protocols
Authors: Meador Kyle , Castells-Graells Roger , Aguirre Roman , Sawaya Michael R. , Arbing Mark A. , Sherman Trent , Senarathne Chethaka , Yeates Todd O.
Published: October 2023

Designed protein cages and related materials provide unique opportunities for applications in biotechnology and medicine, while methods for their creation remain challenging and unpredictable. In the present study, we apply new computational approaches to design a suite of new tetrahedrally symmetric, self-assembling protein cages. For the generation of docked poses, we emphasize a protein fragment-based approach, while forde novointerface design, a comparison of computational protocols highlights the power and increased experimental success achieved using the machine learning program ProteinMPNN. In relating information from docking and design, we observe that agreement between fragment-based sequence preferences and ProteinMPNN sequence inference correlates with experimental success. Additional insights for designing polar interactions are highlighted by experimentally testing larger and more polar interfaces. In all, using X-ray crystallography and cryo-EM, we report five structures for seven protein cages, with atomic resolution in the best case reaching 2.0 Å. We also report structures of two incompletely assembled protein cages, providing unique insights into one type of assembly failure. The new set of designed cages and their structures add substantially to the body of available protein nanoparticles, and to methodologies for their creation.
Studentsourcing - aggregating and re-using data from a practical cell biology course
Authors: Goedhart Joachim
Published: October 2023

Practical courses mimic experimental research and may generate valuable data. Yet, data that is generated by students during a course is often lost as there is no centrally organized collection and storage of the data. The loss of data prevents its re-use. To provide access to these data, I present an approach that I call studentsourcing. It collects, aggregates and reuses data that is generated by students in a practical course on cell biology. The course runs annually, and I have recorded the data that was generated by >100 students over 3 years. Two use cases illustrate how the data can be aggregated and re-used either for the scientific record or for teaching. As the data is obtained by different students, in different groups, over different years, it is an excellent opportunity to discuss experimental design and modern data visualization methods such as the superplot. The first use case demonstrates how the data can be presented as an online, interactive dashboard, providing real-time data of the measurements. The second use case shows how central data storage provides a unique opportunity to get precise quantitative data due to the large sample size. Both use cases illustrate how data can be effectively aggregated and re-used.

Abstract Figure
HSQC2STRUC: A Machine Learning Model for Protein Secondary Structure Prediction using Unassigned NMR Spectra
Authors: Dietrich Jonas , Bellstedt Peter
Published: October 2023

Dynamic changes in the secondary structure content of proteins can provide valuable insights into protein function or dysfunction. Predicting these dynamic changes is still a significant challenge but is of paramount importance for basic research as well as drug development. Here, we present a machine learning-based model that predicts the secondary structure content of proteins based on their un assigned1H,15N-HSQC NMR spectra with an RMSE of 0.11 forα-helix, 0.08 forβ-sheet and 0.12 for random coil content. Our model has been implemented into an easy-to-use and publicly available web service that estimates secondary structure content based on a provided peak list. Furthermore, a Python version is provided, ready to be integrated into Bruker’s TopSpin software or own scripts.
ReMASTER: Improved phylodynamic simulation for BEAST 2.7
Authors: Vaughan Timothy G.
Published: October 2023

Phylodynamic models link phylogenetic trees to biologically-relevant parameters such as speciation and extinction rates (macroevolution), effective population sizes and migration rates (ecology and phylogeography), and transmission and removal/recovery rates (epidemiology) to name a few. Being able to simulate phylogenetic trees and population dynamics under these models is the basis for (a) developing and testing of phylodynamic inference algorithms, (b) performing simulation studies which quantify the biases stemming from model-misspecification, and (c) performing so-called model adequacy assessments by simulating samples from the posterior predictive distribution. Here I introduceReMASTER, a package for the BEAST 2 phylogenetic inference platform which provides a simple and efficient approach to specifying and simulating the phylogenetic trees and population dynamics arising from phylodynamic models. ReMASTER is a complete rewrite of an earlier package, MASTER, and boasts improved efficiency, ease of use, flexibility of model specification, and integration with BEAST 2.

Availability and Implementation

ReMASTER can be installed directly from the BEAST 2 package manager, and its documentation is available online athttps://tgvaughan.github.io/remaster. ReMASTER is free software, and is distributed under version 3 of the GNU General Public License. The Java source code for ReMASTER is available fromhttps://github.com/tgvaughan/remaster.