bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination Use of Compressed Sensing and Deep Learning for Shoulder Magnetic Resonance Imaging With Various Sequences
Authors: Shiraishi Kaori , Nakaura Takeshi , Uetani Hiroyuki , Nagayama Yasunori , Kidoh Masafumi , Kobayashi Naoki , Morita Kosuke , Yamahita Yuichi , Miyamoto Takeshi , Hirai Toshinori
Published: March 2023

Objective

For compressed sensing (CS) to become widely used in routine magnetic resonance imaging (MRI), it is essential to improve image quality. This study aimed to evaluate the usefulness of combining CS and deep learning–based reconstruction (DLR) for various sequences in shoulder MRI.

Methods

This retrospective study included 37 consecutive patients who underwent undersampled shoulder MRIs, including T1-weighted (T1WI), T2-weighted (T2WI), and fat-saturation T2-weighted (FS-T2WI) images. Images were reconstructed using the conventional wavelet-based denoising method (wavelet method) and a combination of wavelet and DLR-based denoising methods (hybrid-DLR method) for each sequence. The signal-to-noise ratio and contrast-to-noise ratio of the bone, muscle, and fat and the full width at half maximum of the shoulder joint were compared between the 2 image types. In addition, 2 board-certified radiologists scored the image noise, contrast, sharpness, artifacts, and overall image quality of the 2 image types on a 4-point scale.

Results

The signal-to-noise ratios and contrast-to-noise ratios of the bone, muscle, and fat in T1WI, T2WI, and FS-T2WI obtained from the hybrid-DLR method were significantly higher than those of the conventional wavelet method (P < 0.001). However, there were no significant differences in the full width at half maximum of the shoulder joint in any of the sequences (P > 0.05). Furthermore, in all sequences, the mean scores of the image noise, sharpness, artifacts, and overall image quality were significantly higher in the hybrid-DLR method than in the wavelet method (P < 0.001), but there were no significant differences in contrast among the sequences (P > 0.05).

Conclusions

The DLR denoising method can improve the image quality of CS in T1-weighted images, T2-weighted images, and fat-saturation T2-weighted images of the shoulder compared with the wavelet denoising method alone.
Factors Contributing to Computed Tomography Trauma Scan Times at a Tertiary Center: Improving Emergency Department Trauma Imaging Workflow Through Targeted Interventions
Authors: Wood Erika J. , Stabo Nicholas , Garrett John W. , Rose Sean , Bartels Carrie , Szczykutowicz Timothy P. , Avey Greg , Mao Lu , Lubner Meghan G.
Published: March 2023

Purposes

The aims of the study are to identify factors contributing to computed tomography (CT) trauma scan turnaround time variation and to evaluate the effects of an automated intervention on time metrics.

Methods

Throughput metrics were captured via picture archiving and communication system from January 1, 2018, to December 16, 2019, and included 17,709 CT trauma scans from our institution. Initial data showed that imaging technologist variation played a significant role in trauma imaging turnaround time. In December 2019, we implemented a 2-pronged intervention: (1) educational intervention to techs and (2) modified trauma CT abdomen/pelvis to autogenerate and autosend reformats to picture archiving and communication system. A total of 13,169 trauma CT scans were evaluated from the postintervention period taking place from January 2020 to March 2021. Throughput metrics such as last image to first report interval and emergency department length of stay were captured and compared with performing technologist, time of day, and weekday versus weekend scans.

Results

Substantial variability among trauma CT scans was observed. For CT trauma abdomen/pelvis, the interval from last image to initial report decreased from 26.4 to 24.0 minutes (P = 0.001) while the interval between first and last image time decreased from 11.4 to 4.2 minutes (P < 0.001). Emergency department length of stay also decreased from 3.9 to 3.7 hours (P < 0.0001) in the postintervention period. Variation among imaging technologist was statistically significant and became less significant after intervention (P = 0.09, P = 0.54)

Conclusions

Factors such as imaging technologist variability, time of day, and day of the week of trauma scans played a significant role in CT trauma turnaround time variability. Automation interventions can help with efficiency in image turnaround time.
Rapid Response Team Activation Triggers in Adults and Children: An Integrative Review
Authors: Feroz Ali Naheed , Amir Asma , Punjwani Aleena , Bhimani Rozina
Published: March 2023

Purpose

This integrative review aims to identify the triggers for rapid response team (RRT) activation and their outcomes in pediatric patients and to compare them with those of adult patients. In addition, this integrative review synthesizes the outcomes of cardiopulmonary resuscitation (CPR), intensive care unit (ICU) admission, length of hospital stay, and mortality following RRT activation.

Method

An integrative review using the Whittemore and Knafl methodology was undertaken with a search of three large databases (PubMed, Ovid MEDLINE, and CINAHL) and found 25 relevant studies published in the years 2017 through 2022.

Results

Tachypnea, decreased oxygen saturation, tachycardia, changes in blood pressure, and level of consciousness were the most common triggers in both populations. However, specific activation triggers differed between children and adults.

Conclusions

The most common triggers for RRT are detectable through vital signs monitoring; therefore, vigilant tracking of patients’ vital signs is critical and can provide early clues to clinical deterioration.
Comparison of Magnetic Resonance-Based Elastography and Ultrasound Shear Wave Elastography in Patients With Suspicion of Nonalcoholic Fatty Liver Disease
Authors: Tahmasebi Aylin , Wessner Corinne E. , Guglielmo Flavius F. , Wang Shuo , Vu Trang , Liu Ji-Bin , Civan Jesse , Lyshchik Andrej , Forsberg Flemming , Li Hongbo , Qu Enze , Eisenbrey John R.
Published: March 2023

This study investigated the correlation between magnetic resonance elastography (MRE) and shear wave ultrasound elastography (SWE) in patients with clinically diagnosed or suspected nonalcoholic fatty liver disease (NAFLD). Subjects with or at risk of NAFLD identified by magnetic resonance imaging (MRI) proton density fat fraction (PDFF) were prospectively enrolled. For each patient, 6 valid 2-dimensional SWE measurements were acquired using a Logiq E10 scanner (GE HealthCare, Waukesha, WI). A reliability criterion of an interquartile range to median ratio of ≤15% was used for SWE to indicate quality dataset. Magnetic resonance elastography, and MR-fat quantification data were collected the same day as part of the patient's clinical standard of care. Magnetic resonance imaging PDFF was used as a reference to quantify fat with >6.4% indicating NAFLD. Pearson correlation and t-test were performed for statistical analyses. A total of 140 patients were enrolled, 112 of which met SWE reliability measurement criteria. Magnetic resonance elastography and 2-dimensional SWE showed a positive correlation across all study subjects (r = 0.27; P = 0.004). When patients were grouped according to steatosis and fibrosis state, a positive correlation was observed between MRE and SWE in patients with fibrosis (r = 0.30; P = 0.03), without fibrosis (r = 0.27; P = 0.03), and with NAFLD (r = 0.28; P = 0.02). No elastography technique correlated with liver fat quantification (P > 0.52). Magnetic resonance elastography was significantly different between patients with and without fibrosis (P < 0.0001). However, this difference was not apparent with SWE (P = 0.09). In patients with suspected or known NAFLD, MRE, and SWE demonstrated a positive correlation. In addition, these noninvasive imaging modalities may be useful adjunct techniques for monitoring NAFLD.
Evaluation of vascular repair by tissue-engineered human acellular vessels or expanded polytetrafluoroethylene grafts in a porcine model of limb ischemia and reperfusion
Authors: Kirkton Robert D. , Watson J. Devin B. , Houston Robert , Prichard Heather L. , Niklason Laura E. , Rasmussen Todd E.
Published: March 2023

BACKGROUND

This study evaluated performance of a tissue-engineered human acellular vessel (HAV) in a porcine model of acute vascular injury and ischemia. The HAV is an engineered blood vessel consisted of human vascular extracellular matrix proteins. Limb reperfusion and vascular outcomes of the HAV were compared with those from synthetic expanded polytetrafluoroethylene (ePTFE) grafts.

METHODS

Thirty-six pigs were randomly assigned to four treatment groups, receiving either the HAV or a PTFE graft following a hind limb ischemia period of either 0 or 6 hours. All grafts were 3-cm-long interposition 6-mm diameter grafts implanted within the right iliac artery. Animals were not immunosuppressed and followed for up to 28 days after surgery. Assessments performed preoperatively and postoperatively included evaluation of graft patency, hind limb function, and biochemical markers of tissue ischemia or reperfusion injury. Histological analysis was performed on explants to assess host cell responses.

RESULTS

Postoperative gait assessment and biochemical analysis confirmed that ischemia and reperfusion injury were caused by 6-hour ischemia, regardless of vascular graft type. Hind limb function and tissue damage biomarkers improved in all groups postoperatively. Final patency rates at postoperative day 28 were higher for HAV than for ePTFE graft in both the 0-hour (HAV, 85.7%; ePTFE, 66.7%) and 6-hour (HAV, 100%; ePTFE, 75%) ischemia groups, but these differences were not statistically significant. Histological analyses identified some intimal hyperplasia and host reactivity to the xenogeneic HAV and also to the synthetic ePTFE graft. Positive host integration and vascular cell infiltration were identified in HAV but not ePTFE explants.

CONCLUSION

Based on the functional performance and the histologic profile of explanted HAVs, this study supports further investigation to evaluate long-term performance of the HAV when used to repair traumatic vascular injuries.
Gordonia sputi -associated bloodstream infection in a renal transplant patient with chronic indwelling central venous catheter: a case report and literature review
Authors: Lo Calvin Ka-Fung , Broderick Conor , Stefanovic Aleksandra , Connors William , Murray Melanie
Published: March 2023

Introduction: Although rare, human infections caused by Gordonia spp. have been reported especially within the immunocompromised population and those with long-term indwelling devices. We report a case of Gordonia spp. bacteremia in a renal transplant patient and present a literature review on microbiologic identification methods of this organism.
Case Presentation: A 62-year-old female renal transplant recipient admitted to hospital with a 2-month history of dry cough and fevers occurring weekly when receiving electrolyte replacement infusions via Groshong line. Over two weeks, blood cultures on multiple occasions repeatedly isolated a Gram-positive bacillus in aerobic bottle only, initially reported as Rhodococcus spp. by local microbiology laboratory. Chest computed tomography (CT) showed multiple ground-glass lung opacities suggestive of septic pulmonary emboli. As central line-associated blood stream infection was suspected, empiric antibiotics were initiated and the Groshong line was removed. The Gram-positive bacillus was later confirmed by reference laboratory as Gordonia sputi via 16S-rRNA sequencing. Vancomycin and ciprofloxacin for duration of six weeks was completed as targeted antimicrobial therapy. After treatment, she remained symptom-free with marked improvement on repeat CT chest imaging.
Conclusion: This case illustrates the challenges surrounding identification of Gordonia spp. and other aerobic actinomycetes.16S-rRNA gene sequencing may be a preferred identification method, especially when initial workup of a weakly acid-fast organism fails to identify or shows discrepant results using traditional diagnostic modalities.
OASIS: An interpretable, finite-sample valid alternative to Pearson’sX2for scientific discovery
Authors: Baharav Tavor Z. , Tse David , Salzman Julia
Published: March 2023

Contingency tables, data represented as counts matrices, are ubiquitous across quantitative research and data-science applications. Existing statistical tests are insufficient however, as none are simultaneously computationally efficient and statistically valid for a finite number of observations. In this work, motivated by a recent application in reference-free genomic inference (1), we develop OASIS (Optimized Adaptive Statistic for Inferring Structure), a family of statistical tests for contingency tables. OASIS constructs a test-statistic which is linear in the normalized data matrix, providing closed form p-value bounds through classical concentration inequalities. In the process, OASIS provides a decomposition of the table, lending interpretability to its rejection of the null. We derive the asymptotic distribution of the OASIS test statistic, showing that these finitesample bounds correctly characterize the test statistic’s p-value up to a variance term. Experiments on genomic sequencing data highlight the power and interpretability of OASIS. The same method based on OASIS significance calls detects SARS-CoV-2 and Mycobacterium Tuberculosis strains de novo, which cannot be achieved with current approaches. We demonstrate in simulations that OASIS is robust to overdispersion, a common feature in genomic data like single cell RNA-sequencing, where under accepted noise models OASIS still provides good control of the false discovery rate, while Pearson’sX2test consistently rejects the null. Additionally, we show on synthetic data that OASIS is more powerful than Pearson’sX2test in certain regimes, including for some important two group alternatives, which we corroborate with approximate power calculations.

Significance Statement

Contingency tables are pervasive across quantitative research and data-science applications. Existing statistical tests fall short, however; none provide robust, computationally efficient inference and control Type I error. In this work, motivated by a recent advance in reference-free inference for genomics, we propose a family of tests on contingency tables called OASIS. OASIS utilizes a linear test-statistic, enabling the computation of closed form p-value bounds, as well as a standard asymptotic normality result. OASIS provides a partitioning of the table for rejected hypotheses, lending interpretability to its rejection of the null. In genomic applications, OASIS performs reference-free and metadata-free variant detection in SARS-CoV-2 and M. Tuberculosis, and demonstrates robust performance for single cell RNA-sequencing, all tasks without existing solutions.
Mitigation of membrane morphology defects explain stability and orientational specificity of CLC dimers
Authors: Ozturk Tugba N. , Bernhardt Nathan , Schwartz Noah , Chadda Rahul , Robertson Janice L. , Faraldo-Gómez José D.
Published: March 2023

Most membrane proteins are oligomers, but the physical forces explaining the stable association of these complexes inside the lipid bilayer are not well understood. The homodimeric antiporter CLC-ec1 highlights the puzzling nature of this reaction. This complex is thermodynamically stable even though it associates via a large hydrophobic protein-protein interface that appears well adapted to interact with the membrane interior. In a previous study, however, we discovered that when CLC-ec1 is dissociated, this interface introduces a morphological defect in the surrounding membrane, leading us to hypothesize association is driven by the elimination of this defect upon dimerization. This study tests this hypothetical mechanism directly and shows it is supported by molecular and physical models. First, using coarse-grained umbrella-sampling molecular simulations, we calculated the membrane contribution to the potential-of-mean-force for dimerization in a POPC bilayer. This shows the stable association of CLC subunits prior to formation of direct protein-protein contacts, but only via the native interface that presents the membrane defect, and not others. Single-molecule photobleaching experiments show that addition of short-chain DLPC lipids, known to alleviate the membrane defect, also shifts the association equilibrium from dimers to monomers. We explain this destabilizing effect through additional umbrella-sampling and alchemical free-energy simulations, which show DLPC enrichment of the defect diminishes the membrane contribution to the association free energy, as it improves the lipid-solvation energetics of the monomer but not the dimer. In summary, this study establishes a physical model that explains the stability and orientational specificity of CLC dimers in terms of membrane-mediated forces, rather than protein-protein interactions. We posit that cells might ubiquitously leverage morphological defects in the bilayer to drive organization of membrane proteins into functional complexes, and that cellular regulation of lipid composition can modulate this organizing effect.
Trends in mosquito species distribution modeling: insights for vector surveillance and disease control
Authors: Lippi Catherine A. , Mundis Stephanie J. , Sippy Rachel , Flenniken J. Matthew , Chaudhary Anusha , Hecht Gavriella , Carlson Colin J. , Ryan Sadie J.
Published: March 2023

Species distribution modeling (SDM) has become an increasingly common approach to explore questions about ecology, geography, outbreak risk, and global change as they relate to infectious disease vectors. Here, we conducted a systematic review of the scientific literature, screening 563 abstracts and identifying 204 studies that used SDMs to produce distribution estimates for mosquito species. While the number of studies employing SDM methods has increased markedly over the past decade, the overwhelming majority used a single method (MaxEnt) and focused on human infectious disease vectors or their close relatives. The majority of regional models were developed for areas in Africa and Asia, while more localized modeling efforts were most common for North America and Europe. Findings from this study highlight gaps in taxonomic, geographic, and methodological foci of current SDM literature for mosquitoes that can guide future efforts to study the geography of mosquito-borne disease risk.
Coupling enzymatic activity and gating in an ancient TRPM chanzyme and its molecular evolution
Authors: Huang Yihe , Lü Wei , Du Juan
Published: March 2023

The canonical ion channels gated by chemical ligands use the free energy of agonist binding to open the channel pore, returning to a closed state upon agonist departure. A unique class of ion channels, known as channel-enzymes (chanzymes), possess additional enzymatic activity that is directly or indirectly linked to their channel function. Here we investigated a TRPM2 chanzyme from choanoflagellates, an evolutionary ancestor of all metazoan TRPM channels, which integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP ribose (ADPR) with high open probability and an enzyme module (NUDT9-H domain) consuming ADPR at a remarkably slow rate. Using time-resolved cryo- electron microscopy (cryo-EM), we captured a complete series of structural snapshots of the gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. Our results showed that the slow kinetics of the NUDT9-H enzyme module confers a novel self-regulatory mechanism, whereby the enzyme module modulates channel gating in a binary manner. Binding of ADPR to NUDT9-H first triggers tetramerization of the enzyme modules, promoting channel opening, while the subsequent hydrolysis reaction reduces local ADPR availability, inducing channel closure. This coupling enables the ion-conducting pore to alternate rapidly between open and closed states, avoiding Mg2+and Ca2+overload. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADPR hydrolase module in early species TRPM2 to a fully integrated component of a gating ring essential for channel activation in advanced species TRPM2. Our study demonstrated an example of how organisms can adapt to their environments at the molecular level.
ITNR: Inversion Transformer-based Neural Ranking for Cancer Drug Recommendations
Authors: Sotudian Shahabeddin , Paschalidis Ioannis Ch.
Published: March 2023

Personalized drug response prediction is an approach for tailoring effective therapeutic strategies for patients based on their tumors’ genomic characterization. The current study introduces a new listwise Learning-to-rank (LTR) model called Inversion Transformer-based Neural Ranking (ITNR). ITNR utilizes genomic features and a transformer architecture to decipher functional relationships and construct models that can predict patient-specific drug responses. Our experiments were conducted on three major drug response data sets, showing that ITNR reliably and consistently outperforms state-of-the-art LTR models.

Highlights

The proposed framework is a transformer-based model to predict drug responses using RNAseq gene expression profile, drug descriptors and drug fingerprints.

ITNR utilizes a Context-Aware-Transformer architecture as its scoring function that ensures the modeling of inter-item dependencies.

We introduced a novel loss function using the concept of Inversion and Approximate Permutation matrices.

Our computational results indicated that our method leads to substantially improved performance when compared to the baseline methods across all performance metrics, which can lead to selecting highly effective personalized treatment.
Pulmonary maternal immune activation does not extend through the placenta but leads to fetal metabolic adaptation
Authors: Hansen Signe Schmidt Kjølner , Krautz Robert , Rago Daria , Havelund Jesper , Færgeman Nils J. , Prézelin Audrey , Rivière Julie , Couturier-Tarrade Anne , Akimov Vyacheslav , Blagoev Blagoy , Elfving Betina , Stigliani Arnaud , Vogel Ulla Birgitte , Khodosevich Konstantin , Hougaard Karin Sørig , Sandelin Albin
Published: March 2023

Maternal immune system activation (MIA) during pregnancy can disrupt the fetal environment, causing postnatal susceptibility to disorders. How the placenta and the fetus respond to acute MIA over time is unknown. Here, we characterized the response to acute maternal pulmonary inflammation across time in maternal and fetal organs using multi-omics. Unlike maternal organs which mounted strong innate immune responses, the placenta upregulated tissue-integrity genes, likely to prevent fetal exposure to infections, and downregulated growth-associated genes. Subsequently, the placenta upregulated biosynthesis and endoplasmic reticulum stress genes in order to return to homeostasis. These responses likely protected the fetus, since we observed no immune response in fetal liver. Instead, likely due to nutrient depletion, the fetal liver displayed metabolic adaptations, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. Our study shows, for the first time, the integrated temporal response to pulmonary MIA across maternal and fetal organs.
funspace: an R package to build, analyze and plot functional trait spaces
Authors: Carmona Carlos P. , Pavanetto Nicola , Puglielli Giacomo
Published: March 2023

Functional trait space analyses are pivotal to define species’ ecological strategies across the tree of life. Yet, there is no single application that streamlines the many sometimes-troublesome steps needed to build and analyze functional trait spaces.

To fill this gap, we proposefunspace, an R package to easily handle bivariate and multivariate (PCA-based) functional trait space analyses. The six functions that constitute the package can be grouped in three modules: ‘Building and exploring’, ‘Mapping’, and ‘Plotting’.

The building and exploring module defines the main features of a functional trait space (e.g., functional diversity metrics) by leveraging kernel density-based methods. The mapping module uses general additive models to map how a target variable distributes within a trait space. The plotting module provides many options for creating flexible and high-quality figures representing the outputs obtained from previous modules. We provide a worked example to demonstrate a completefunspaceworkflow.

funspacewill provide researchers working with functional traits across the tree of life with an indispensable asset to easily explore: (i) the main features of any functional trait space, (ii) the relationship between a functional trait space and any other biological or non-biological factor that might contribute to shaping species’ functional diversity.
Functional convergence underground? The scale-dependency of community assembly processes in European cave spiders
Authors: Mammola Stefano , Graco-Roza Caio , Ballarin Francesco , Hesselberg Thomas , Isaia Marco , Lunghi Enrico , Mouron Samuel , Pavlek Martina , Tolve Marco , Cardoso Pedro
Published: March 2023

Understanding how species assemble into communities is a central tenet in ecology. One of the most elusive questions is the relative contribution of environmental filtering versus limiting similarity. Important advances in this area have been achieved by looking at communities through a functional lens (i.e., the traits they express), so as to derive principles valid across species pools. Yet, even using traitsin lieuof taxonomy, the issue remains controversial because i) environmental filtering and limiting similarity often act simultaneously in shaping communities; and ii) their effect is scale-dependent. We exploited the experimental arena offered by caves, island-like natural laboratories characterized by largely constant environmental gradients and a limited diversity of species and interactions. Leveraging uniquely available data on distribution and traits for European cave spiders, we tested explicit hypotheses about variations in community assembly rules across ecological gradients and scales. We demonstrate that environmental filtering and limiting similarity shape cave communities acting on trait evolution in opposing directions. These effects are strongly scale dependent, varying along multiple environmental gradients. Conversely, the effect of geography on trait composition is weak, indicating that trait turnover in space happens primarily by substitution of species pursuing similar functions due to strong environmental filters. Our findings reconcile contrasted views about the relative importance of the two main mechanisms shaping patterns of biodiversity, and provide a conceptual foundation to account for scaling effects in the study of community assembly.
Optimized two-color single-molecule tracking of fast-diffusing membrane receptors
Authors: Spagnolo Chiara Schirripa , Moscardini Aldo , Amodeo Rosy , Beltram Fabio , Luin Stefano
Published: March 2023

Single particle tracking (SPT) combined with total internal reflection fluorescence (TIRF) microscopy is an outstanding approach to decipher crucial molecular mechanisms on the cell membrane at the nanometric scale. In multicolor configurations it can even be the ideal tool to investigate interactions, but this is hindered by a number of experimental challenges. We systematically and quantitatively analyze the impact of all the necessary sub-elements of any SPT-TIRF setup on signal-to-noise ratio, especially in dynamic studies using minimally-invasive dyes for biomolecule labeling. We show that the dominant limiting factor is the autofluorescence originating from the commonly-used optical glass. We identify and test a different material and show a significant improvement of signal-to-noise ratio in a multichannel TIRF configuration employing the new glass covers. We also address the problem of photobleaching of fluorescent probes by presenting effective approaches suited to a multicolor implementation that requires simultaneous stabilization of multiple dyes. We apply the developed protocol to the analysis of p75 receptors labeled by two fluorophores on the membrane of living cells. Our strategy yields reliable, simultaneous two-color SPT, even for fast-diffusing receptors, enabling this study under conditions not accessible with standard experimental configurations. We argue that the present protocol can pave the way for multicolor super-resolved localization and tracking of single molecules by TIRF microscopy, much expanding the potential of SPT.
Cell type-specific and disease-associated eQTL in the human lung
Authors: Natri Heini M , Del Azodi Christina B , Peter Lance , Taylor Chase J , Chugh Sagrika , Kendle Robert , Chung Mei-i , Flaherty David K , Matlock Brittany K , Calvi Carla L , Blackwell Timothy S , Ware Lorraine B , Bacchetta Matthew , Walia Rajat , Shaver Ciara M , Kropski Jonathan A , McCarthy Davis J , Banovich Nicholas E
Published: March 2023

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis (PF). Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA-sequencing of lung tissue from 67 PF and 49 unaffected donors. Employing a pseudo-bulk approach, we mapped expression quantitative trait loci (eQTL) across 38 cell types, observing both shared and cell type-specific regulatory effects. Further, we identified disease-interaction eQTL and demonstrated that this class of associations is more likely to be cell-type specific and linked to cellular dysregulation in PF. Finally, we connected PF risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression, and implicates context-specific eQTL as key regulators of lung homeostasis and disease.
How total mRNA influences cell growth
Authors: Calabrese Ludovico , Ciandrini Luca , Lagomarsino Marco Cosentino
Published: March 2023

Experimental observations tracing back to the 1960s imply that ribosome quantities play a prominent role in determining a cell’s growth. Nevertheless, in biologically relevant scenarios, growth can also be influenced by the levels of mRNA and RNA polymerase. Here, we construct a quantitative model of biosynthesis providing testable scenarios for these situations. The model explores a theoretically-motivated regime where RNA polymerases compete for genes and ribosomes for transcripts, and gives general expressions relating growth rate, mRNA concentrations, ribosome and RNA polymerase levels. On general grounds, the model predicts how the fraction of ribosomes in the proteome depends on total mRNA concentration, and inspects an underexplored regime in which the trade-off between transcript levels and ribosome abundances sets the cellular growth rate. In particular, we show that the model predicts and clarifies three important experimental observations, in budding yeast andE. colibacteria: (i) that the growth-rate cost of unneeded protein expression can be affected by mRNA levels, (ii) that resource optimization leads to decreasing trends in mRNA levels at slow growth, and (iii) that ribosome allocation may increase, stay constant, or decrease, in response to transcription-inhibiting antibiotics. Since the data indicate that a regime of joint limitation may apply in physiological conditions and not only to perturbations, we speculate that this regime is likely self-imposed.
Tuft cell-derived acetylcholine regulates epithelial fluid secretion
Authors: Billipp Tyler E. , Fung Connie , Webeck Lily M. , Sargent Derek B. , Gologorsky Matthew B. , McDaniel Margaret M. , Kasal Darshan N. , McGinty John W. , Barrow Kaitlyn A. , Rich Lucille M. , Barilli Alessio , Sabat Mark , Debley Jason S. , Myers Richard , Howitt Michael R. , von Moltke Jakob
Published: March 2023

Tuft cells are solitary chemosensory epithelial cells that can sense lumenal stimuli at mucosal barriers and secrete effector molecules to regulate the physiology and immune state of their surrounding tissue. In the small intestine, tuft cells detect parasitic worms (helminths) and microbe-derived succinate, and signal to immune cells to trigger a Type 2 immune response that leads to extensive epithelial remodeling spanning several days. Acetylcholine (ACh) from airway tuft cells has been shown to stimulate acute changes in breathing and mucocilliary clearance, but its function in the intestine is unknown. Here we show that tuft cell chemosensing in the intestine leads to release of ACh, but that this does not contribute to immune cell activation or associated tissue remodeling. Instead, tuft cell-derived ACh triggers immediate fluid secretion from neighboring epithelial cells into the intestinal lumen. This tuft cell-regulated fluid secretion is amplified during Type 2 inflammation, and helminth clearance is delayed in mice lacking tuft cell ACh. The coupling of the chemosensory function of tuft cells with fluid secretion creates an epithelium-intrinsic response unit that effects a physiological change within seconds of activation. This response mechanism is shared by tuft cells across tissues, and serves to regulate the epithelial secretion that is both a hallmark of Type 2 immunity and an essential component of homeostatic maintenance at mucosal barriers.
The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal
Authors: Ashraf Humza M. , Fernandez Brianna , Spencer Sabrina L.
Published: March 2023

Senescence, a state of permanent cell-cycle withdrawal, is difficult to distinguish from quiescence, a transient state of cell-cycle withdrawal. This difficulty arises because quiescent and senescent cells are defined by overlapping biomarkers, raising the question of whether quiescence and senescence are truly distinct states. To address this, we used single-cell time-lapse imaging to distinguish slow-cycling quiescent cells frombona fidesenescent cells after chemotherapy treatment, followed immediately by staining for various senescence biomarkers. We found that the staining intensity of multiple senescence biomarkers is graded rather than binary and primarily reflects the duration of cell-cycle withdrawal, rather than senescence per se. Together, our data suggest that quiescence and senescence are not distinct cellular states but rather fall on a continuum of cell-cycle withdrawal, where the intensities of canonical senescence biomarkers reflect the likelihood of cell-cycle re-entry.
CMS Final Rule for Organ Donation: Unintended Consequences of the Pancreas Research Loophole
Authors: Goldberg David , Chyou Darius , Wulf Rachael , Wadsworth Matthew
Published: March 2023

CMS updated the ‘Final Rule’ for OPO Conditions for Coverage on 11/20/2020 to include new OPO metrics, tiers of classification, and a new definition of a donor that included an individual with: a) ≥1 organ transplanted; or b) pancreas procured for research or islet cell transplantation. We conducted a retrospective cohort study using data from the OPTN/ UNOS. The number of pancreata procured for research increased four-fold from 2020 to 2022, despite stable numbers of other organs procured for research. Of the 57 OPOs, 8 (14.0%) procured >100 pancreata for research in 2022, accounting for 1,548 (58.2%) pancreata research procurements. Of those 8 OPOs, seven would be at risk for possible (Tier 2; n=3) or definite (Tier 3; n=4) decertification based on CMS’s 2022 interim report. Based on these data, two Tier 3 OPOs would be reclassified as Tier 2 and one as Tier 1; three Tier 2 OPOs would be reclassified as Tier 1; and three Tier 3s would be reclassified as borderline Tier 2. The pancreas research carveout may detrimentally impact the system of organ donation by failing to accurately measure OPO performance and flag (and decertify) underpeforming OPOs unless CMS revises its final rule.
Usefulness of Current sgRNA Design Guidelines andin vitroCleavage Assays for Plant CRISPR/Cas Genome Editing: A Case in Eggplant (Solanum melongenaL.)
Authors: Sagarbarria Mark Gabriel S. , Caraan John Albert M.
Published: March 2023

The advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed on the development of new crop varieties that can withstand agricultural challenges of the 21stcentury. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to a multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether anin vitroassay is needed to validate the targeting efficiency of a particular sgRNA prior toin vivodelivery of the CRISPR/Cas9 system. Here, we demonstrate thein vitroandin vivoactivity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have highin vitrocleavage activity did not produce editsin vivo, suggesting that anin vitroassay may not be a reliable basis to predict sgRNAs with highly efficientin vivocleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observedin vitroandin vivocleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensurein vivocleavage of chosen sgRNAs.

Key Message

in vitrocleavage assay of sgRNAs was able to identify low activity sgRNAs but did not 13 reliably predictin vivomutagenesis.

Using multiple sgRNAs that meet the plant-validated parameters and have high activityin vitroin plant genome editing is critical to ensure success.
Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer
Authors: Maurer H. Carlo , Garcia-Curiel Alvaro , Holmstrom Sam R. , Castillo Cristina , Palermo Carmine F. , Sastra Steven A. , Andren Anthony , Zhang Li , Le Large Tessa Y.S. , Sagalovskiy Irina , Ross Daniel R. , Wong Winston , Shaw Kaitlin , Genkinger Jeanine , Manji Gulam A. , Iuga Alina C. , Schmid Roland M. , Johnson Kristen , Badgley Michael A. , Lyssiotis Costas A. , Shah Yatrik , Califano Andrea , Olive Kenneth P.
Published: March 2023

To identify drivers of malignancy in human pancreatic ductal adenocarcinoma (PDAC), we performed regulatory network analysis on a large collection of expression profiles from laser capture microdissected samples of PDAC and benign precursors. We discovered that BMAL2 plays a role in the initiation, progression, post resection survival, and KRAS activity in PDAC. Functional analysis of BMAL2 target genes led us to hypothe-size that it plays a role in regulating the response to hypoxia, a critical but poorly understood feature of PDAC physiology. Knockout of BMAL2 in multiple human PDAC cell lines revealed effects on viability and invasion, particularly under hypoxic conditions. Loss of BMAL2 also affected glycolysis and other metabolic processes. We found that BMAL2 directly regulates hypoxia-responsive target genes. We also found that BMAL2 is necessary for the stabilization of HIF1A upon exposure to hypoxia, but destabilizes HIF2A under hypoxia. These data demonstrate that BMAL2 is a master transcriptional regulator of hypoxia responses in PDAC and may serve as a long-sought molecular switch that distinguishes HIF1A- and HIF2A-dependent modes of hypoxic metabolism.

Statement of significance

We provide a comprehensive portrait of the molecular drivers behind pancreatic cancer initiation, progression and outcome. We discover a new genetic regulator of metabolic responses to low oxygen environments, a key feature of pancreatic cancer biology. This protein, BMAL2, is a new driver of pancreatic cancer malignancy.
The Sde Phosphoribosyl-Linked Ubiquitin Transferases protect theLegionella pneumophilavacuole from degradation by the host
Authors: Kim Seongok , Isberg Ralph R.
Published: March 2023

Legionella pneumophilagrows intracellularly within a host membrane-bound vacuole that is formed in response to a bacterial type IV secretion system (T4SS). T4SS translocated Sde proteins promote phosphoribosyl-linked ubiquitination of endoplasmic reticulum protein Rtn4, but the role played by this modification is obscure due to lack of clear growth defects of mutants. To identify the steps in vacuole biogenesis promoted by these proteins, mutations were identified that unmasked growth defects in Δsdestrains. Mutations in thesdhA,ridLandlegA3genes aggravated the Δsdefitness defect, resulting in disruption of theLegionella-containing vacuole (LCV) membrane within 2 hrs of bacterial contact with host cells. Depletion of Rab5B and sorting nexin 1 partially bypassed loss of Sde proteins, consistent with Sde blocking early endosome and retrograde trafficking, similar to roles previously demonstrated for SdhA and RidL proteins. Sde protein protection of LCV lysis was only observed shortly after infection, presumably because Sde proteins are inactivated by the metaeffector SidJ during the course of infection. Deletion of SidJ extended the time that Sde proteins could prevent vacuole disruption, indicating that Sde proteins are negatively regulated at the posttranslational level and are limited to protecting membrane integrity at the earliest stages of replication. Transcriptional analysis was consistent with this timing model for an early point of execution of Sde protein. Therefore, Sde proteins act as temporally-regulated vacuole guards during establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments early during biogenesis of the LCV.

Significance statement

Maintaining replication compartment integrity is critical for growth of intravacuolar pathogens within host cells. By identifying genetically redundant pathways,Legionella pneumophilaSde proteins that promote phosphoribosyl-linked ubiquitination of target eukaryotic proteins are shown to be temporally-regulated vacuole guards, preventing replication vacuole dissolution during early stages of infection. As targeting of reticulon 4 by these proteins leads to tubular endoplasmic reticulum aggregation, Sde proteins are likely to construct a barrier that blocks access of disruptive early endosomal compartments to the replication vacuole. Our study provides a new framework for how vacuole guards function to support biogenesis of theL. pneumophilareplicative niche.
Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling
Authors: Chaiamarit Tai , Verhelle Adriaan , Chassefeyre Romain , Shukla Nandini , Novak Sammy Weiser , Andrade Leonardo R. , Manor Uri , Encalada Sandra E.
Published: March 2023

Dystrophic axons comprising misfolded mutant prion protein (PrP) aggregates are a characteristic pathological feature in the prionopathies. These aggregates form inside endolysosomes -called endoggresomes-, within swellings that line up the length of axons of degenerating neurons. The pathways impaired by endoggresomes that result in failed axonal and consequently neuronal health, remain undefined. Here, we dissect the local subcellular impairments that occur within individual mutant PrP endoggresome swelling sites in axons. Quantitative high-resolution light and electron microscopy revealed the selective impairment of the acetylated vs tyrosinated microtubule cytoskeleton, while micro-domain image analysis of live organelle dynamics within swelling sites revealed deficits uniquely to the MT-based active transport system that translocates mitochondria and endosomes toward the synapse. Cytoskeletal and defective transport results in the retention of mitochondria, endosomes, and molecular motors at swelling sites, enhancing mitochondria-Rab7 late endosome contacts that induce mitochondrial fission via the activity of Rab7, and render mitochondria dysfunctional. Our findings point to mutant Pr Pendoggresome swelling sites as selective hubs of cytoskeletal deficits and organelle retention that drive the remodeling of organelles along axons. We propose that the dysfunction imparted locally within these axonal micro-domains spreads throughout the axon over time, leading to axonal dysfunction in prionopathies.
Expansion microscopy with trypsin digestion and tyramide signal amplification (TT-ExM) for protein and lipid staining
Authors: Wang Ueh-Ting Tim , Tian Xuejiao , Liou Yae-Huei , Lee Sue-Ping , Lu Chieh-Han , Chen Peilin , Cheb Bi-Chang
Published: March 2023

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers two major drawbacks, namely proteolysis and swelling effects that, respectively, induce protein loss and dilute fluorescence signals. Here, we report two improvements to expansion microscopy that overcome these two challenges, i.e., deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. Notably, we demonstrate better protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures following 2-h trypsin treatment. Subsequent lipid staining revealed the complex 3D membrane structures in entire cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. Thus, TT-ExM significantly enhances fluorescent signals and generates high-quality and ultrafine-resolution images under confocal microscopy.
Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal
Authors: Kandola Tej , Venkatesan Shriram , Zhang Jiahui , Lerbakken Brooklyn , Schulze Alex Von , Blanck Jillian F , Wu Jianzheng , Unruh Jay , Berry Paula , Lange Jeffrey J. , Box Andrew , Cook Malcolm , Sagui Celeste , Halfmann Randal
Published: March 2023

A long-standing goal of amyloid research has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of nucleation has made this goal unachievable with existing biochemistry, structural biology, and computational approaches. Here, we addressed that limitation for polyglutamine (polyQ), a polypeptide sequence that causes Huntington’s and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. To identify essential features of the polyQ amyloid nucleus, we used a direct intracellular reporter of self-association to quantify frequencies of amyloid appearance as a function of concentration, conformational templates, and rational polyQ sequence permutations. We found that nucleation of pathologically expanded polyQ involves segments of three glutamine (Q) residues at every other position. We demonstrate using molecular simulations that this pattern encodes a four-stranded steric zipper with interdigitated Q side chains. Once formed, the zipper poisoned its own growth by engaging naive polypeptides on orthogonal faces, in a fashion characteristic of polymer crystals with intramolecular nuclei. We further show that self-poisoning can be exploited to block amyloid formation, by genetically oligomerizing polyQ prior to nucleation. By uncovering the physical nature of the rate-limiting event for polyQ aggregation in cells, our findings elucidate the molecular etiology of polyQ diseases.