bluuebunny/crossref_metadata_2025 huggingface dataset:
An ultrasensitive genetically encoded voltage indicator uncovers the electrical activity of non-excitable cells
Authors: Rühl Philipp , Nair Anagha G. , Gawande Namrata , Dehiwalage Sassrika N.C.W. , Münster Lukas , Schönherr Roland , Heinemann Stefan H.
Published: October 2023

Genetically encoded voltage indicators (GEVIs) are powerful, non-invasive tools for recording action potentials in excitable cells. However, most animal cell types are non-excitable, and yet variations in the membrane potential are biologically relevant in these cells as well. Resolving such small voltage signals demands GEVIs with exceptionally high sensitivity. In this study, we applied structure-guided engineering to the GEVI ASAP3 to generate rEstus, a sensor with optimized brightness, voltage sensitivity, and voltage range. rEstus is most sensitive in the resting voltage range of non-excitable cells, exhibits a 3.6-fold improvement in fast voltage spike detection, and allows for absolute voltage calibration at the single-cell level. Using rEstus, we resolved endogenous voltage fluctuations in several non-excitable cell types and demonstrate that correlation analysis of these optically recorded fluctuations provides an easy, non-invasive, real-time readout of electrical gap-junction coupling. Our work provides greatly enhanced tools and methods for the non-invasive study of electrical signaling in excitable and non-excitable cells.
Geometric constraint of mechanosensing in bone marrow stromal cell cultures prevents stiffness-induced differentiation
Authors: Hernandez-Miranda M , Xu D , Johnston DA , Browne M , Cook RB , Sengers BG , Evans ND
Published: October 2023

Extracellular matrix (ECM) stiffness is fundamental in cell division, movement and differentiation. The stiffness that cells sense is determined not only by the elastic modulus of the ECM material, but also by ECM geometry and cell density. We hypothesised that these factors would influence cell-traction-induced matrix deformations and cellular differentiation in bone marrow stromal cells (BMSCs). To achieve this, we cultivated BMSCs on polyacrylamide hydrogels that varied in elastic modulus and geometry and measured cell spreading, cell-imparted matrix-deformations and differentiation. At low cell density BMSCs spread to a greater extent on stiff compared to soft hydrogels, or on thin compared to thick hydrogels. Cell-imparted matrix deformations were greater on soft compared to stiff hydrogels or thick compared to thin hydrogels. There were no significant differences in osteogenic differentiation relative to hydrogel elastic modulus and thickness. However, increased cell density and/or prolonged culture significantly reduced matrix deformations on soft hydrogels to levels similar to those on stiff substrates. This suggests that at high cell densities cell traction-induced matrix displacements are reduced by both neighbouring cells and the constraint imposed by an underlying stiff support. This may explain observations of the lack of difference in osteogenic differentiation as a function of stiffness.
Deciphering Cell-types and Gene Signatures Associated with Disease Activity in Rheumatoid Arthritis using Single Cell RNA-sequencing
Authors: Binvignat M. , Miao B. Y. , Wibrand C. , Yang M.M. , Rychkov D. , Flynn E. , Nititham J. , Tamaki W. , Khan U. , Carvidi A. , Krueger M. , Niemi E. , Sun Y. , Fragiadakis G. , Sellam J. , Mariotti-Ferrandiz E. , Klatzmann D. , Gross A. , Ye J. , Butte A. J. , Criswell L.A , Nakamura M. , Sirota M.
Published: October 2023

Objective

Single cell profiling of synovial tissue has previously identified gene signatures associated with rheumatoid arthritis (RA) pathophysiology, but synovial tissue is difficult to obtain. This study leverages single cell sequencing of peripheral blood mononuclear cells (PBMCs) from patients with RA and matched healthy controls to identify disease relevant cell subsets and cell type specific signatures of disease.

Methods

Single-cell RNA sequencing (scRNAseq) was performed on peripheral blood mononuclear cells (PBMCs) from 18 RA patients and 18 matched controls, accounting for age, gender, race, and ethnicity). Samples were processed using standard CellRanger and Scanpy pipelines, pseudobulk differential gene expression analysis was performed using DESeq2, and cell-cell communication analysis using CellChat.

Results

We identified 18 distinct PBMC subsets, including a novel IFITM3+ monocyte subset. CD4+ T effector memory cells were increased in patients with moderate to high disease activity (DAS28-CRP ≥ 3.2), while non-classical monocytes were decreased in patients with low disease activity or remission (DAS28-CRP < 3.2). Differential gene expression analysis identified RA-associated genes in IFITM3+ and non-classical monocyte subsets, and downregulation of pro-inflammatory genes in the Vδ subset. Additionally, we identified gene signatures associated with disease activity, characterized by upregulation of pro-inflammatory genesTNF, JUN, EGR1, IFIT2, MAFB, G0S2, and downregulation ofHLA-DQB1, HLA-DRB5, TNFSF13B. Notably, cell-cell communication analysis revealed upregulation of immune-associated signaling pathways, including VISTA, in patients with RA.

Conclusions

We provide a novel single-cell transcriptomics dataset of PBMCs from patients with RA, and identify insights into the systemic cellular and molecular mechanisms underlying RA disease activity.
RNA structure modulates Cas13 activity and enables mismatch detection
Authors: Kimchi Ofer , Larsen Benjamin B. , Dunkley Owen R. S. , te Velthuis Aartjan J.W. , Myhrvold Cameron
Published: October 2023

The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging1–6. Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA1,7. Though Cas13 is not activated by double-stranded RNA (dsRNA)in vitro, it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured2,8. Understanding Cas13’s mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown9. Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3’ to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection, enhancing mismatch discrimination by up to 50-fold and enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.
Epigenetic field defect discriminates normal tissues from healthy and tumor-bearing kidneys
Authors: Serth Jurgen , Reese Christel , Hubscher Tatyana , Hill Bastian , Klintschar Michael , Hennenlotter Jorg , Kuczyk Markus Antonius
Published: October 2023

Background

The detection of alterations in DNA methylation (DNAm) in normal human tissues may provide molecular insights for cancer development, the estimation of personal cancer risk, and a basis for early diagnosis of malignancies to reduce cancer lethality. Thus, whether DNAm allows discrimination between healthy normal and normal tumor-adjacent renal tissues is an important question.

Methods

DNAm of loci in the LINE1 repetitive sequence andANKRD34B, NHLH2, TBR1, andZIC1was measured in a total of 493 tissue samples representing 342 normal autopsy and 151 histopathological normal tumor-adjacent renal tissues by pyro-sequencing a total of 22 CpG sites.

Results

Unsupervised k-means clustering demonstrated a significant imbalance of tissue samples in three stable tissue clusters. Random forest classification demonstrated discrimination of normal and normal tumor-adjacent renal tissues with a median area under the ROC curve of 0.81 (p=2.7×10−9, diagnostic odds ratio 10.4). Variable importance analysis revealed CpG sites in LINE1 andANKRD34Bas the most important model contributors.

Conclusions

DNAm alterations in normal tissues allow detection of renal cancer with high diagnostic efficiency, defining a DNAm field effect in the kidney. The methylation signature may serve as a basis for an epigenetic DNAm clock permitting estimation of individual renal cancer risk.
The developmental trajectory of1H-MRS brain metabolites from childhood to adulthood
Authors: Thomson Alice R. , Hwa Hannah , Pasanta Duanghathai , Hopwood Benjamin , Powell Helen J. , Lawrence Ross , Garcia Tabuenca Zeus , Arichi Tomoki , Edden Richard A. E. , Chai Xiaoqian , Puts Nicolaas A.
Published: October 2023

Human brain development is ongoing throughout childhood, with for example myelination of nerve fibres and refinement of synaptic connections continuing until early adulthood.1H-Magnetic Resonance Spectroscopy (1H-MRS)can be used to quantify the concentrations of endogenous metabolites (e.g., glutamate and γ-aminobutyric acid (GABA)) in the human brain in vivo and so can provide valuable, tractable insight into the biochemical processes that support postnatal neurodevelopment. This can feasibly provide new insight into and aid management of neurodevelopmental disorders by providing chemical markers of atypical development. This study aims to characterize the normative developmental trajectory of various brain metabolites, as measured by1H-MRS from a midline posterior parietal voxel. We find significant non-linear trajectories for GABA+, Glx, tNAA and tCr concentrations. Glx and GABA+ concentrations steeply decrease across childhood. tNAA concentrations are relatively stable in childhood but gradually decrease from early adulthood, while tCr concentrations increase from childhood to early adulthood. tCho was the only metabolite to have a strictly linear association with age. Trajectories likely reflect fundamental neurodevelopmental processes (including local circuit refinement) which occur from childhood to early adulthood and can be associated with cognitive development; we find GABA+ concentrations significantly positively correlate with recognition memory scores across post-natal development.
Rapid threat assessment in theDrosophilathermosensory system
Authors: Jouandet Genevieve C. , Alpert Michael H. , Simões José Miguel , Suhendra Richard , Frank Dominic D. , Levy Joshua I. , Para Alessia , Kath William L. , Gallio Marco
Published: October 2023

Neurons that participate in sensory processing often display “ON” responses, i.e. fire transiently at the onset of a stimulus. ON transients are widespread, perhaps universal to sensory coding, yet their function is not always well-understood. Here, we show that ON responses in theDrosophilathermosensory system extrapolate the trajectory of temperature change, priming escape behavior if unsafe thermal conditions are imminent. First, we show that second-order thermosensory projection neurons (TPN-IIIs) and their Lateral Horn targets (TLHONs), display ON responses to thermal stimuli, independent of direction of change (heating or cooling) and of absolute temperature. Instead, they track the rate of temperature change, with TLHONs firing exclusively to rapid changes (>0.2°C/sec). Next, we use connectomics to track TLHON’s output to descending neurons that control walking and escape, and modeling and genetic silencing to demonstrate how ON transients can flexibly amplify aversive responses to small thermal change. Our results suggest that, across sensory systems, ON transients may represent a general mechanism to systematically anticipate and respond to salient or dangerous conditions.
HERV activation segregates ME/CFS from fibromyalgia while defining a novel nosologic entity
Authors: Giménez-Orenga Karen , Martín-Martínez Eva , Nathanson Lubov , Oltra Elisa
Published: October 2023

Research of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM), two acquired chronic illnesses affecting mainly females, has failed to ascertain their frequent co-appearance and etiology. Despite prior detection of human endogenous retrovirus (HERV) activation in these diseases, the potential biomarker value of HERV expression profiles for their diagnosis, and the relationship of HERV expression profiles with patient immune systems and symptoms had remained unexplored. By using HERV-V3 high-density microarrays (including over 350k HERV elements and more than 1500 immune-related genes) to interrogate the transcriptomes of peripheral blood mononuclear cells from female patients diagnosed with ME/CFS, FM or both, and matched healthy controls (n=43), this study fills this gap of knowledge. Hierarchical clustering of HERV expression profiles strikingly allowed perfect participant assignment into four distinct groups: ME/CFS, FM, co-diagnosed, or healthy, pointing at a potent biomarker value of HERV expression profiles to differentiate between these hard-to-diagnose chronic syndromes. Differentially expressed HERV-immune-gene modules revealed unique profiles for each of the four study groups and highlighting decreased γδ T cells, and increased plasma and resting CD4 memory T cells, correlating with patient symptom severity in ME/CFS. Moreover, activation of HERV sequences coincided with enrichment of binding sequences targeted by transcription factors which recruit SETDB1 and TRIM28, two known epigenetic silencers of HERV, in ME/CFS, offering a mechanistic explanation for the findings. Unexpectedly, HERV expression profiles appeared minimally affected in co-diagnosed patients denoting a new nosological entity with low epigenetic impact, a seemingly relevant aspect for the diagnosis and treatment of this prevalent group of patients.
Humans and a subterranean rodent have contrasting biological rhythms
Authors: Appenzeller Otto , Qualls Clifford , Bromage Tim
Published: October 2023

Here we show that naked mole rats (NMR’s) have an extraordinary survival advantage. We base this statement on a spectral analysis of the time series of measured intervals in teeth of 3 species (NMR’s, Killer Whale, and Modern Humans). We used Fourier decomposition to analyze the variability of these intervals. We find these animals go through their long life without any of the age-associated diseases seen in humans such as osteoporosis or cancer and they show no signs of muscle atrophy or slowing of mobility. Global warming will delay its effects on these animals whereas for humans it is a clear and present danger.
Microfluidics-free single-cell genomics reveals complex central-peripheral immune crosstalk in the mouse brain during peripheral inflammation
Authors: Boles Jake Sondag , Uriarte Huarte Oihane , Tansey Malú Gámez
Published: October 2023

Inflammation is a realized detriment to brain health in a growing number of neurological diseases, but querying neuroinflammation in its cellular complexity remains a challenge. This manuscript aims to provide a reliable and accessible strategy for examining the brain’s immune system. We compare the efficacy of cell isolation methods in producing ample and pure immune samples from mouse brains. Then, with the high-input single-cell genomics platform PIPseq, we generate a rich neuroimmune dataset containing microglia and many peripheral immune populations. To demonstrate this strategy’s utility, we interrogate the well-established model of LPS-induced neuroinflammation with single-cell resolution. We demonstrate the activation of crosstalk between microglia and peripheral phagocytes and highlight the unique contributions of microglia and peripheral immune cells to neuroinflammation. Our approach enables the high-depth evaluation of inflammation in longstanding rodent models of neurological disease to reveal novel insight into the contributions of the immune system to brain health.
FMNL2 regulates actin for ER and mitochondria distribution in oocyte meiosis
Authors: Pan Meng-Hao , Pan Zhen-Nan , Sun Ming-Hong , Li Xiao-Han , Ju Jia-Qian , Luo Shi-Ming , Ou Xiang-Hong , Sun Shao-Chen
Published: October 2023

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study we reported that an actin nucleating factor formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and endoplasmic reticulum, showing with decreased mitochondrial membrane potential and the occurrence of endoplasmic reticulum stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mouse oocytes.
A Novel Interaction Between Aquaporin 1 and Caspase-3 in Pulmonary Arterial Smooth Muscle Cells
Authors: Niedermeyer Shannon , Yun Xin , Trujillo Marielena , Andrade Manuella Ribas , Kolb Todd , Suresh Karthik , Damarla Mahendra , Shimoda Larissa A.
Published: October 2023

Pulmonary arterial hypertension (PAH) is a disease in which remodeling of the precapillary pulmonary vasculature leads to hyperplasia and hypertrophy of the muscular vascular wall, and the formation of vaso-occlusive lesions. These pathologic changes are predominantly due to abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), enhanced cellular functions that have been linked to increases in the cell membrane protein aquaporin-1 (AQP1). However, the mechanisms underlying increased AQP1 abundance have not been fully elucidated. Here we present data that establishes a novel interaction between AQP1 and the proteolytic enzyme caspase-3.In silicoanalysis of the AQP1 protein reveals two caspase-3 cleavage sites on its c-terminal tail, proximal to known ubiquitin sites. Using biotin proximity ligase techniques, we establish that AQP1 and caspase-3 interact in both HEK293A cells and rat PASMCs. Furthermore, we demonstrate that AQP1 levels increase and decrease with enhanced caspase-3 activity and inhibition respectively. Ultimately, further work characterizing this interaction could provide the foundation for novel PAH therapeutics.
Beta-Validation of a Non-Invasive Method for Simultaneous Detection of Early-Stage Female-Specific Cancers
Authors: Ramamoorthy Sudhakar , Sundaramoorthy Saranya , Gupta Ankur , Siddiqui Zaved , Sagar Ganga , Rao Kanury V.S. , Saquib Najmuddin , Mohan Surapeni Krishna
Published: October 2023

Objectives

In this report, we assessed the accuracy of our previously developed method for simultaneous diagnosis of the four female cancers of the Breast, Endometrium, Cervix, and Ovary, in a clinical set-up with blinded protocol.

Materials and Methods

Our test protocol combined global serum metabolome profiling wherein data was analyzed with machine learning algorithms to extract metabolite signatures that correlated with early-stage cancers. High-resolution mass spectrometry was employed to profile the serum metabolome and the resulting data were subjected to a pre-processing pipeline to obtain the data set. The data was then analyzed using artificial intelligence algorithms to identify early-stage cancer metabolic signatures.

Results

Overall, a total of 1000 blinded samples were analyzed by generating the serum metabolome profiles, followed by sequential algorithms for cancer detection and multiclass cancer type identification. Of these 1000 samples, 797 were identified as cancer positive, while, 203 samples were identified as cancer-negative. The multiclass algorithm was then applied to the 797 cancer-positive samples, to distinguish between samples that were from patients with either endometrial, breast, cervical, or ovarian cancer. After completion of the analysis, the sample code was broken to estimate the accuracy of the results. Concerning the identification of samples that were cancer-positive, the sensitivity obtained was 99.6% whereas the specificity was 100%. For the second stage of analysis which involves ‘tissue of origin’, all 107 breast cancer samples were correctly identified without any false calls. The accuracy for identification of cervical and ovarian cancers was between 95-96% for each, whereas 91% for endometrial cancer.

Conclusions

Our present study validates the performance of our method for the early-stage detection of female-specific cancers in a clinical setting. Importantly, the algorithms for cancer detection and ‘tissue of origin’ prediction, which were initially trained using samples from Caucasian patients, retained the accuracy on samples from Indian women patients. This suggests that the performance of these algorithms was minimally influenced by variables such as ethnicity and race. Present results, therefore, also underscore the potential clinical utility of our method for early-stage diagnosis of cancers that are specific to females.
Hs-CRP is associated with Heart Failure Hospitalization in Patients with MAFLD and Normal LVEF Undergoing Coronary Angiography
Authors: Zhou Xiao-Dong , Chen Qin-Fen , Targher Giovanni , Byrne Christopher D. , Shapiro Michael D. , Tian Na , Xiao Tie , Sung Ki-Chul , Lip Gregory Y. H. , Zheng Ming-Hua
Published: October 2023

Background

Systemic chronic inflammation plays a role in the pathophysiology of both heart failure with preserved ejection fraction (HFpEF) and metabolic dysfunction-associated fatty liver disease (MAFLD).

Aim

This study aimed to investigate whether serum high-sensitivity C-reactive protein (hs-CRP) levels were associated with the future risk of heart failure (HF) hospitalization in patients with MAFLD and a normal left ventricular ejection fraction (LVEF).

Methods

The study enrolled consecutive individuals with MAFLD and normal LVEF who underwent coronary angiography for suspected coronary heart disease. The study population was subdivided into non-HF, pre-HFpEF, and HFpEF groups at baseline. The study outcome was the first hospitalization for HF.

Results

In 10,019 middle-aged individuals (mean age 63.3±10.6 years; 38.5% female), the prevalence rates of HFpEF and pre-HFpEF were 34.2% and 34.5%, with a median serum hs-CRP level of 4.5 mg/L (IQR: 1.9-10 mg/L) and 5.0 mg/L (IQR: 2.1-10.1 mg/L), respectively. Serum hs-CRP levels were significantly higher in the pre-HFpEF and HFpEF groups than in the non-HF group. HF hospitalizations occurred in 1942 (19.4%) patients over a median of 3.2 years, with rates of 3.7% in non-HF, 20.8% in pre-HFpEF, and 32.1% in HFpEF, respectively. Cox regression analyses showed that patients in the highest hs-CRP level quartile had a ∼4.5-fold increased risk of being hospitalized for HF compared to those in the lowest hs-CRP level quartile (adjusted-Hazard Ratio 4.42, 95% CI 3.72-5.25).

Conclusions

There was a high prevalence of baseline pre-HFpEF and HFpEF in subjects with MAFLD. There was an increased risk of HF hospitalization in those with elevated hs-CRP levels.
Linear ubiquitination at damaged lysosomes induces local NF-κB activation and controls cell survival
Authors: Zein Laura , Dietrich Marvin , Balta Denise , Bader Verian , Scheuer Christoph , Zellner Suzanne , Weinelt Nadine , Vandrey Julia , Mari Muriel C. , Behrends Christian , Zunke Friederike , Winklhofer Konstanze F. , van Wijk Sjoerd J. L.
Published: October 2023

Lysosomes are the major cellular organelles responsible for nutrient recycling and degradation of cellular material. Maintenance of lysosomal integrity is essential for cellular homeostasis and lysosomal membrane permeabilization (LMP), induced by lysosomotrophic agents, sensitizes towards cell death. Damaged lysosomes are repaired or degraded via lysophagy, during which glycans, exposed on ruptured lysosomal membranes, are recognized by galectins leading to K48- and K63-linked poly-ubiquitination (poly-Ub) of lysosomal proteins followed by recruitment of the autophagic machinery and degradation. Linear (M1) poly-Ub, catalyzed by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN) exerts important functions in immune signaling and cell survival, but the role of M1 poly-Ub in lysosomal homeostasis remains largely unexplored. Here, we demonstrate that damaged lysosomes are decorated with M1 poly-Ub in a LUBAC-, OTULIN- and K63-dependent manner. LMP-induced M1 poly-Ub at damaged lysosomes contributes to lysosome degradation, recruits nuclear factor κ-B (NF-κB) essential modulator (NEMO) and locally activates inhibitor of NF-ĸB kinase (IKK) to trigger NF-κB activation in a K63 poly-Ub-dependent manner. Inhibition of lysosomal degradation enhances LMP- and OTULIN-dependent cell death, indicating pro-survival functions of LMP and potentially lysophagy. Finally, we demonstrate that M1 poly-Ub occurs at L-leucyl-leucine methyl ester (LLOMe)-damaged lysosomes in primary mouse neurons and induced pluripotent stem cell (iPSC)-derived primary human dopaminergic neurons. Together, our results reveal novel functions of M1 poly-Ub during lysosomal homeostasis, LMP and degradation of damaged lysosomes, with important implications for NF-κB signaling, inflammation and cell death.
Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins
Authors: Harjung Alexander , Fracassi Alessandro , Devaraj Neal
Published: October 2023

A common method of generating artificial cells is to encapsulate protein expression systems within lipid vesicles. However, to communicate with the external environment, protein translocation across lipid membranes must take place. In living cells, protein transport across membranes is achieved with the aid of complex translocase systems which are difficult to reconstitute into artificial cells. Thus, there is need for simple mechanisms by which proteins can be encoded and expressed inside synthetic compartments yet still be externally displayed. Here we present a genetically encodable membrane functionalization system based on mutants of pore-forming proteins. We show that the membrane translocating loop of α-hemolysin can be engineered to translocate functional peptides up to 52 amino acids across lipid membranes. Engineered hemolysins can be used for genetically programming artificial cells to display interacting peptide pairs, enabling their assembly into artificial tissue-like structures capable of signal transduction.
MPSeqM, a tool combining multiplex PCR and high-throughput sequencing to study the polymorphism of eightLeptosphaeria maculansavirulence genes and its application to field surveys in France
Authors: Gautier Angélique , Laval Valérie , Balesdent Marie-Hélène
Published: October 2023

Leptosphaeria maculansis a fungal pathogen causing stem canker of oilseed rape (Brassica napus). The disease is mainly controlled by the deployment of varieties with major resistance genes (Rlm).Rlmgenes can rapidly become ineffective following the selection of virulent isolates of the fungus, i.e. with deletions or mutations in the corresponding avirulence genes (AvrLm). Reasoned and durable management ofRlmgenes relies on the detection and monitoring of virulent isolates in field populations. Based on previous knowledge ofAvrLmgene polymorphism, we developed a tool combining multiplex PCR and Illumina sequencing to characterise allelic variants for eightAvrLmgenes in fieldL. maculanspopulations.

We tested the method on DNA pools of 71 characterisedL. maculansisolates and of leaf spots from 32L. maculansisolates. After multiplex-PCR and sequencing with MiSeq technology, reads were mapped on an in-houseAvrLmsequence database. Data were filtered using thresholds defined from control samples included in each run. Proportions of each allelic variant per gene, including deletions, perfectly correlated with expected ones. The method was then applied to around 1300 symptoms (42 pools of mainly 32 leaf spots) from nineB. napusfields. The proportions of virulent isolates estimated by sequencing leaf spot pools perfectly correlated with those estimated by pathotyping. In addition, the proportions of allelic variants determined at the national scale also correlated with those previously determined following individual sequencing ofAvrLmgenes in a representative collection of isolates. Finally, the method also allowed us to detect still undescribed and rare allelic variants. Despite the diversity of mechanisms generating virulent isolates and the gene-dependant diversity ofAvrLmgene polymorphism, the method proved suitable for large-scale and regular monitoring ofL. maculanspopulations, which will make it possible to choose effectiveRlmgenes and to detect resistance breakdowns at early stages.
Deciphering Cancer Genomes with GenomeSpy: A Grammar-Based Visualization Toolkit
Authors: Lavikka Kari , Oikkonen Jaana , Li Yilin , Muranen Taru , Micoli Giulia , Marchi Giovanni , Lahtinen Alexandra , Huhtinen Kaisa , Lehtonen Rainer , Hietanen Sakari , Hynninen Johanna , Virtanen Anni , Hautaniemi Sampsa
Published: October 2023

Background

Visualization is an indispensable facet of genomic data analysis. Despite the abundance of specialized visualization tools, there remains a distinct need for tailored solutions. However, their implementation typically requires extensive programming expertise from bioinformaticians and software developers, especially when building interactive applications. Toolkits based on visualization grammars offer a more accessible, declarative way to author new visualizations. Nevertheless, current grammar-based solutions fall short in adequately supporting the interactive analysis of large data sets with extensive sample collections, a pivotal task often encountered in cancer research.

Results

We present GenomeSpy, a grammar-based toolkit for authoring tailored, interactive visualizations for genomic data analysis. Users can implement new visualization designs with little effort by using combinatorial building blocks that are put together with a declarative language. These fully customizable visualizations can be embedded in web pages or end-user-oriented applications. The toolkit also includes a fully customizable but user-friendly application for analyzing sample collections, which may comprise genomic and clinical data. Findings can be bookmarked and shared as links that incorporate provenance information. A distinctive element of GenomeSpy’s architecture is its effective use of the graphics processing unit (GPU) in all rendering. GPU usage enables a high frame rate and smoothly animated interactions, such as navigation within a genome. We demonstrate the utility of GenomeSpy by characterizing the genomic landscape of 753 ovarian cancer samples from patients in the DECIDER clinical trial. Our results expand the understanding of the genomic architecture in ovarian cancer, particularly the diversity of chromosomal instability. We also show how GenomeSpy enabled the discovery of clinically actionable genomic aberrations.

Conclusions

GenomeSpy is a visualization toolkit applicable to a wide range of tasks pertinent to genome analysis. It offers high flexibility and exceptional performance in interactive analysis. The toolkit is open source with an MIT license, implemented in JavaScript, and available athttps://genomespy.app/.
Fine-tuning FAM161A gene augmentation therapy to restore retinal function
Authors: Arsenijevic Yvan , Chang Ning , Mercey Olivier , Fersioui Younes El , Koskiniemi-Kuendig Hanna , Joubert Caroline , Bemelmans Alexis-Pierre , Rivolta Carlo , Banin Eyal , Sharon Dror , Guichard Paul , Hamel Virginie , Kostic Corinne
Published: October 2023

In 15 years, inherited retinal diseases have seen gene therapy as a springboard to hope. Many preclinical investigations focused on vectors with maximal gene expression capabilities. But despite an efficient gene transfer, little physiological improvement was noted for certain ciliopathies. FAM161A is an essential protein for the structure of photoreceptor connecting cilium (CC). In the absence of FAM161A, cilia disorganize resulting in outersegment collapses and vision impairment. Within the human retina,FAM161Aproduces two isoforms: the long with exon 4, and the short, lacking it. To restore CC inFam161a-deficient mice, we compared AAV vectors with different promoter activities, doses, and human isoforms injected subretinally in 14-daysFam161atm1b/tm1bmice, shortly after the onset of cilium disorganization. All vectors improved cell survival, but only combining both isoforms using the weak FCBR1-F0.4 promoter allowed precise FAM161A expression in the CC and enhanced retinal function.

Our study onFAM161Agene replacement for RP28, a rod-cone-related disease, underscores the critical need for precise therapeutic gene regulation, appropriate vector dosing and delivery of both isoforms. Fine tuning of therapeutic gene expression, tailored to disease traits, is crucial for restoring retinal function. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.
Co-transcriptional production of programmable RNA condensates and synthetic organelles
Authors: Fabrini Giacomo , Farag Nada , Nuccio Sabrina Pia , Li Shiyi , Stewart Jaimie M. , Tang Anli A. , McCoy Reece , Owens Róisín M. , Rothemund Paul W. K. , Franco Elisa , Di Antonio Marco , Di Michele Lorenzo
Published: October 2023

Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate guest molecules. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with controlled number, size, morphology and composition, and that display the ability to selectively capture proteins. Thein situexpression of programmable RNA condensates could underpin spatial organisation of functionalities in both biological and synthetic cells.
Synergistic, Multi-level Understanding of Psychedelics: Three Systematic Reviews and Meta-analyses of Their Pharmacology, Neuroimaging and Phenomenology
Authors: Shinozuka Kenneth , Jerotic Katarina , Mediano Pedro , Zhao Alex T. , Preller Katrin H. , Carhart-Harris Robin , Kringelbach Morten L.
Published: October 2023

Serotonergic psychedelics induce altered states of consciousness and have shown potential for treating a variety of neuropsychiatric disorders, including depression and addiction. Yet their modes of action are not fully understood. Here, we provide a novel, synergistic understanding of psychedelics arising from systematic reviews and meta-analyses of three hierarchical levels of analysis: 1) subjective experience (phenomenology), 2) neuroimaging and 3) molecular pharmacology. Phenomenologically, medium and high doses of LSD yield significantly higher ratings of visionary restructuralisation than psilocybin on the 5-dimensional Altered States of Consciousness Scale. Our neuroimaging results reveal that, in general, psychedelics significantly strengthen between-network functional connectivity (FC) while significantly diminishing within-network FC. Pharmacologically, LSD induces significantly more inositol phosphate formation at the 5-HT2Areceptor than DMT and psilocin, yet there are no significant between-drug differences in the selectivity of psychedelics for the 5-HT2A, 5-HT2C, or D2receptors, relative to the 5-HT1Areceptor. Our meta-analyses link DMT, LSD, and psilocybin to specific neural fingerprints at each level of analysis. The results show a highly non-linear relationship between these fingerprints. Overall, our analysis highlighted the high heterogeneity and risk of bias in the literature. This suggests an urgent need for standardising experimental procedures and analysis techniques, as well as for more research on the emergence between different levels of psychedelic effects.
Single cell expression and chromatin access of theToxoplasma gondiilytic cycle identifies AP2XII-8 as an essential pivotal controller of a ribosome regulon
Authors: Lou Jingjing , Rezvani Yasaman , Arriojas Argenis , Degras David , Keroack Caroline D. , Duraisingh Manoj T. , Zarringhalam Kourosh , Gubbels Marc-Jan
Published: October 2023

Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasiteToxoplasma gondii. This parasite’s unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle were established at the single cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles mapped transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identified putatively functionally related gene groups facilitating parasite division. Promoter DNA motif mapping revealed patterns of combinatorial regulation. Pseudo-time trajectory analysis revealed transcriptional bursts at different cell cycle points. The dominant burst in G1 was driven by transcription factor AP2XII-8, which engages TGCATGCG/A and TATAAGCCG motifs, and promoted the expression of a regulon encoding 40 ribosomal proteins. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.
“The Translesion Polymerase Pol Y1 is a Constitutive Component of theB. subtilisReplication Machinery”
Authors: Marrin McKayla E. , Foster Michael R. , Santana Chloe M. , Choi Yoonhee , Jassal Avtar S. , Rancic Sarah J. , Greenwald Carolyn R. , Drucker Madeline N. , Thrall Elizabeth S.
Published: October 2023

Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low fidelity and must be tightly regulated to avoid harmful mutagenesis. The gram-negative bacteriumEscherichia colihas served as the model organism for studies of the molecular mechanisms of bacterial TLS. However, it is poorly understood whether these same mechanisms apply to other bacteria. Here, we use in vivo single-molecule fluorescence microscopy to investigate the TLS polymerase Pol Y1 in the model gram-positive bacteriumBacillus subtilis. We find significant differences in the localization and dynamics of Pol Y1 in comparison to itsE. colihomolog, Pol IV. Notably, Pol Y1 is constitutively enriched at or near sites of replication in the absence of DNA damage through interactions with the DnaN clamp; in contrast, Pol IV has been shown to be selectively enriched only upon replication stalling. These results suggest key differences in the roles and mechanisms of regulation of TLS polymerases across different bacterial species.
A green alternative to fragrant agarwood sesquiterpenoid production
Authors: Gutiérrez Sergio , Overmans Sebastian , Wellman Gordon B. , Samaras Vasilios G. , Oviedo Claudia , Gede Martin , Szekely Gyorgy , Lauersen Kyle J.
Published: October 2023

Certain endangered Thymelaeaceous trees are major sources of the fragrant and highly valued resinous agarwood, comprised of hundreds of oxygenated sesquiterpenoids (STPs). Despite growing pressure on natural agarwood sources, the chemical complexity of STPs severely limits synthetic production. Here, we catalogued the chemical diversity in 58 agarwood samples by two-dimensional gas chromatography–mass spectrometry and partially recreated complex STP mixtures through synthetic biology. We improved STP yields in the unicellular algaChlamydomonas reinhardtiiby combinatorial engineering to biosynthesise nine macrocyclic STP backbones found in agarwood. A bioprocess following green-chemistry principles was developed that exploits ‘milking’ of STPs without cell lysis, solvent–solvent STP extraction, solvent–STP nanofiltration, and bulk STP oxy-functionalisation to obtain terpene mixtures like those of agarwood. This process occurs with total solvent recycling and enables continuous production. Our synthetic-biology approach offers a sustainable alternative to harvesting agarwood trees to obtain mixtures of complex, fragrant, oxygenated STPs.
Commentary on Pang et al. (2023) Nature
Authors: Patil Kaustubh R. , Jung Kyesam , Eickhoff Simon B.
Published: October 2023

Pang et al. (2023) observe that the geometric eigenmodes, derived from the shape of the cortical surface, are better at reconstructing patterns of both spontaneous and stimulus-evoked activity, when contrasted with three alternative connectome-based models including structural connectome derived eigenmodes. Based on this observation they propose that geometric eigenmodes offer a good model for explaining brain function, noting that “wave dynamics offer a more accurate and parsimonious mechanistic account of macroscale, spontaneous cortical dynamics captured by fMRI”. They then question the prevailing view that brain activity is “localized to focal, spatially isolated clusters” and it is driven by “intricate patterns of anatomical connections”. While the observation that geometric properties fit brain activity well is intriguing, we argue that accepting geometric eigenmodes as a model for brain function risks the logical fallacy of “affirming the consequent”. A representation that effectively describes the underlying geometry is inherently adept at fitting patterns within that geometric space; it does not necessarily shed light on mechanisms of the brain’s functional attributes. To this end, we provide two lines of empirical results: (a) Basic parcel-based representations, which capture localized structures, can reconstruct activity patterns as well. (b) Geometric eigenmodes demonstrate a high flexibility when fit to a range of manipulated patterns, which evokes the danger of overfitting. Based on those results, theoretical considerations, and previous data we argue that more consideration is needed regarding “parsimony, robustness and generality of geometric eigenmodes as a basis set for brain function”. While we recognize the potential role of the brain’s geometry in influencing its dynamics, assertions regarding its efficacy should be weighed against the performance of simpler models, an inherent risk of overfitting and anatomical evidence. Pang et al.1put forth harmonic modes derived from the brain’s geometry as a previously underrecognized model to explain brain-wide dynamics. Their reconstruction framework relies on multiple linear regression to fit brain patterns using a basis set and then calculating Pearson’s correlation between original data and fitted data, both parcellated using an atlas with 180 parcels in each hemisphere2. In addition to the overarching challenge of accepting any model as an actual reflection of the real world3, we here provide empirical results and theoretical arguments that highlight the need for further consideration regarding geometric eigenmodes as a model of macroscale brain activity.
Characterization of a novel mouse model for Fuchs Endothelial Corneal Dystrophy
Authors: Murugan Subashree , de Campos Viviane Souza , Ghag Sachin Anil , Ng Matthew , Shyam Rajalekshmy
Published: October 2023

Purpose

Fuchs Endothelial Corneal Dystrophy (FECD) is a progressive blinding disorder prevalent in 4% of Americans over 40. Corneal transplantation is the standard treatment. Animal models with partial FECD features exist, but a model encompassing all the major disease characteristics is desirable to improve the understanding of the pathogenesis and to identify signaling pathways involved in the disease onset and progression. Such an animal model can be helpful to develop intervention strategies. Here, we developed a mouse model that recapitulates all the features of FECD.

Method

Loss of function mutations inSlc4a11and a knock-in mutation inCol8a2(Q455K) are implicated in FECD. Mice with Slc4a11 and Col8a2 mutations in C57BL/6J background were crossed to generate double mutant mice at F2 generation. At five weeks of age, a subset of the animals were fed tamoxifen-enriched chow or standard chow for two weeks, followed by standard chow. Corneal thickness, endothelial cell density, and guttae were measured at 5 (baseline) and 16 weeks of age. Corneas collected from mice at 16 weeks were stained for tight and adherens junctions, and reactive oxygen species. A lactate assay was performed to evaluate the endothelial pump function.

Results

The double mutant tamoxifen-fed mice showed increased corneal thickness, decreased endothelial cell density, presence of guttae, and elevated stromal lactate levels. The endothelial cells showed altered morphology with disrupted adherens junctions and elevated ROS.

Conclusion

Overall, this mouse model recapitulates all the important phenotypic features associated with FECD.
IL-15 complex-induced IL-10 enhancesPlasmodium-specific CD4+Tfh differentiation and antibody production
Authors: Bravo Morgan , Dileepan Thamotharampillai , Dolan Molly , Hildebrand Jacob , Wolford Jordan , Hamilton Sara E. , Frosch Anne E. , Burrack Kristina S.
Published: October 2023

Malaria, which results from infection withPlasmodiumparasites, remains a major public health problem. While humans do not develop long-lived, sterilizing immunity, protection against symptomatic disease develops after repeated exposure toPlasmodiumparasites and correlates with the acquisition of humoral immunity. Despite the established role antibodies play in protection from malaria disease, dysregulated inflammation is thought to contribute to the sub-optimal immune response toPlasmodiuminfection.Plasmodium bergheiANKA (PbA) infection results in a fatal severe malaria disease in mice. We previously demonstrated that treatment of mice with IL-15 complex (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) induces IL-10 expression in NK cells, which protects mice from PbA-induced death. Using a novel MHC class II tetramer to identify PbA-specific CD4+T cells, herein we demonstrate that IL-15C treatment enhances Tfh differentiation. Moreover, genetic deletion of NK cell-derived IL-10 or IL-10R expression on T cells prevents IL-15C-induced Tfh differentiation. Additionally, IL-15C treatment results in increased anti-PbA IgG antibody levels and improves survival following reinfection. Overall, these data demonstrate that IL-15C treatment, via its induction of IL-10 from NK cells, modulates the dysregulated inflammation duringPlasmodiuminfection to promote Tfh differentiation and antibody generation, correlating with improved survival from reinfection. These findings will facilitate improved control of malaria infection and protection from disease by informing therapeutic strategies and vaccine design.