bluuebunny/crossref_metadata_2025 huggingface dataset:
Repolarisation-relaxation dyscoupling and TRPA1 activation permit systolic mechano-arrhythmogenesis
Authors: Cameron Breanne A. , Stoyek Matthew R. , Greiner Joachim , Peyronnet Rémi , Kohl Peter , Quinn T Alexander
Published: February 2024

Background

The heart’s mechanical state feeds back to its electrical activity, potentially contributing to arrhythmias (mechano-arrhythmogenesis, MAR). MAR has been mechanistically explained during electrical diastole, when cardiomyocytes are at their resting membrane potential. Conversely, during electrical systole, cardiomyocytes appear to be protected from MAR, even as membrane potential and cytosolic calcium concentration ([Ca2+]i) are simultaneously restored to resting levels during repolarisation (repolarisation-relaxation coupling, RRC). Yet, systolic MAR has been reported in ischaemic myocardium, with unclear underlying mechanisms.

Methods

Rabbit left ventricular cardiomyocytes were electrically paced and exposed to a simulated ischaemia solution (including hyperkalaemia, acidosis, and block of oxidative phosphorylation) or pinacidil (to simulate ischaemia-induced opening of ATP-sensitive potassium [KATP] channels), with or without glibenclamide (to block KATPchannels). RRC was assessed by simultaneous measurement of membrane voltage and [Ca2+]idynamics with fluorescence imaging. Acute stretch at increasing magnitudes was applied using carbon fibres, with stretch timed to diastole or late systole. Stretch mechanics and the incidence of MAR was assessed by video-based measurement of sarcomere length. Mechanisms contributing to MAR were assessed by buffering [Ca2+]i(BAPTA-AM), stabilising ryanodine receptors (dantrolene), non-specifically blocking mechano-sensitive channels (streptomycin), activating (AITC) or blocking (HC-030031) transient receptor potential kinase ankyrin 1 channels (TRPA1), or chelating (NAC) or blocking production of (DPI) reactive oxygen species (ROS).

Results

It was reconfirmed that MAR during physiological RRC is rare, while ischaemia- or pharmacologically-induced RRC dyscoupling generates a vulnerable period for systolic MAR. This systolic MAR depends on TRPA1, [Ca2+]i, and ROS, which contribute to stretch-induced excitation and arrhythmia sustenance. An increase in systolic MAR can be prevented by mitigating RRC dyscoupling with KATPchannel block, or by blocking TRPA1, buffering [Ca2+]i, or reducing ROS.

Conclusion

RRC dyscoupling may be arrhythmogenic in ischaemia and other pathologies associated with systolic MAR, and TRPA1 may be a novel anti-arrhythmic target.
Macropinocytosis mediates resistance to loss of glutamine transport in triple-negative breast cancer
Authors: Wahi Kanu , Freidman Natasha , Wang Qian , Devadason Michelle , Quek Lake-Ee , Pang Angel , Lloyd Larissa , Larance Mark , Zanini Fabio , Harvey Kate , O’Toole Sandra , Guan Yi Fang , Holst Jeff
Published: February 2024

Triple-negative breast cancer (TNBC) relies on glutamine uptake by the transporter ASCT2 to sustain their unique glutamine metabolism and growth. Despite previous data showing cell growth inhibition after ASCT2 knockdown, ASCT2 CRISPR knockout was well-tolerated by breast cancer cell lines. Despite the loss of a glutamine transporter and low rate of glutamine uptake, intracellular glutamine steady state levels were higher in ASCT2 knockout compared to control TNBC cells. Proteomics data revealed upregulation of macropinocytosis, reduction in glutamine efflux and glutamine synthesis in ASCT2 knockout cells. Loss of ASCT2 in TNBC cell line HCC1806 induced a 5-10-fold increase in macropinocytosis across 5 separate ASCT2 knockout clones, compared to a modest 2-fold increase in the shRNA ASCT2 knockdown. By comparison, ASCT2 knockout impaired cell proliferation in a non-macropinocytic breast cancer cell line, HCC1569. These data suggest that macropinocytosis provides a novel resistance mechanism to strategies targeting glutamine uptake alone. Despite this adaptation, TNBC cells continue to rely on glutamine metabolism for their growth, which suggests therapeutic targeting may need to focus on downstream glutamine metabolism pathways.
Monkeys engage in visual simulation to solve complex problems
Authors: Ahuja Aarit , Yusif Rodriguez Nadira , Ashok Alekh Karkada , Serre Thomas , Desrochers Theresa , Sheinberg David
Published: February 2024

Visual simulation — i.e., using internal reconstructions of the world to experience potential future versions of events that are not currently happening — is among the most sophisticated capacities of the human mind. But is this ability in fact uniquely human? To answer this question, we tested monkeys on a series of experiments involving the ‘Planko’ game, which we have previously used to evoke visual simulation in human participants. We found that monkeys were able to successfully play the game using a simulation strategy, predicting the trajectory of a ball through a field of planks while demonstrating a level of accuracy and behavioral signatures comparable to humans. Computational analyses further revealed that the monkeys’ strategy while playing Planko aligned with a recurrent neural network (RNN) that approached the task using a spontaneously learned simulation strategy. Finally, we carried out awake functional magnetic resonance imaging while monkeys played Planko. We found activity in motion-sensitive regions of the monkey brain during hypothesized simulation periods, even without any perceived visual motion cues. This neural result closely mirrors previous findings from human research, suggesting a shared mechanism of visual simulation across species. In all, these findings challenge traditional views of animal cognition, proposing that nonhuman primates possess a complex cognitive landscape, capable of invoking imaginative and predictive mental experiences to solve complex everyday problems.
A highly scalable approach to topic modelling in single-cell data by approximate pseudobulk projection
Authors: Subedi Sishir , Sumida Tomokazu S , Park Yongjin P
Published: February 2024

Probabilistic topic modelling has become essential in many types of single-cell data analysis. Based on probabilistic topic assignments in each cell, we identify the latent representation of cellular states, and topic-specific gene frequency vectors provide interpretable bases to be compared with known cell-type-specific marker genes. However, fitting a topic model on a large number of cells would require heavy computational resources–specialized computing units, computing time and memory. Here, we present a scalable approximation method customized for single-cell RNA-seq data analysis, termed ASAP, short for Annotating Single-cell data by Approximate Pseudobulk estimation. Our approach is more accurate than existing methods but requires orders of magnitude less computing time, leaving much lower memory consumption. We also show that our approach is widely applicable for atlas-scale data analysis; our method seamlessly integrates single-cell and bulk data in joint analysis, not requiring additional preprocessing or feature selection steps.
Spiral ganglion neuron degeneration in deafened rats involves innate and adaptive immune responses not requiring complement
Authors: Gansemer Benjamin M. , Rahman Muhammad T. , Zhang Zhenshen , Green Steven H.
Published: February 2024

Spiral ganglion neurons (SGNs) transmit auditory information from cochlear hair cells to the brain. SGNs are thus not only important for normal hearing, but also for effective functioning of cochlear implants, which stimulate SGNs when hair cells are missing. SGNs slowly degenerate following aminoglycoside-induced hair cell loss, a process thought to involve an immune response. However, the specific immune response pathways involved remain unknown. We used RNAseq to gain a deeper understanding immune-related and other transcriptomic changes that occur in the rat spiral ganglion after kanamycin-induced deafening. Among the immune and inflammatory genes that were selectively upregulated in deafened spiral ganglia, the complement cascade genes were prominent. We then assessed SGN survival, as well as immune cell infiltration and activation, in the spiral ganglia of rats with a CRISPR-Cas9-mediated knockout of complement component 3 (C3). Similar to previous findings in our lab and other deafened rodent models, we observed infiltration of macrophages and increased expression of CD68, a marker of phagocytic activity and cell activation, in the deafened ganglia. Moreover, we found that the immune response also includes MHCII+ macrophages and CD45+ and lymphocytes, indicative of an adaptive response. However, C3 knockout did affect SGN survival or macrophage infiltration/activation, indicating that complement activation does not play a role in SGN death after deafening. Together, these data suggest that both innate and adaptive immune responses are activated in the deafened spiral ganglion, with the adaptive response directly contributing to cochlear neurodegeneration.
Thermostablein vitrotranscription-translation for enzyme screening in microdroplets
Authors: Ribeiro Ana L. J. L. , Pérez-Arnaiz Patricia , Sánchez-Costa Mercedes , Pérez Lara , Almendros Marcos , van Vliet Liisa , Gielen Fabrice , Lim Jesmine , Charnock Simon , Hollfelder Florian , González-Pastor J. Eduardo , Berenguer José , Hidalgo Aurelio
Published: February 2024

Background

In vitroexpression involves the utilization of the transcription and translation machinery derived from the cell to produce one or more proteins of interest and has found widespread application in the optimization of gene circuits or metabolic pathways in synthetic biology but also in pharmaceutical manufacturing. Mostin vitroexpression systems available are active at moderate temperatures but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Moreover, given the fact that the main barrier toward the general use ofin vitroexpression is its high price compared with host-based recombinant expression, there is a need to develop alternativein vitroexpression systems operating at high temperatures and compatible with technologies that enable ultrahigh-throughput screening in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.

Results

To this end, we produced high-expression cell-free extracts fromThermus thermophilusforin vitrotranslation and supplemented them with thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable to other thermostablein vitroexpression systems, while the preparation procedure is simpler and can be suited to differentThermus thermophilusstrains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Although the composition of these extracts showed a high background in carboxyl esterase assays, β-glucosidase and cellobiose hydrolase activities could be measured with minimal background.

Conclusions

Cell-free extracts fromThermus thermophilusrepresent a simpler alternative to heavily optimized or pure component thermostablein vitroexpression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.
Unravelling the roadblocks to 1,2-propanediol biosynthesis in select solventogenicClostridiumspecies
Authors: Agyeman-Duah Eric , Kumar Santosh , Ujor Victor C.
Published: February 2024

Background

The compound 1,2-propanediol is an important industrial bulk chemical that has proven particularly recalcitrant to bio-production. Solvent-producingClostridiumspecies represent promising candidates for engineering 1,2-propaediol production. Co-production of 1,2-popanediol and butanol has the potential to improve the economics of the acetone-butanol-ethanol (ABE) fermentation.

Results

In this study, the methylglyoxal synthase gene (mgsA) fromClostridium beijerinckiiNCIMB 8052 was homologously expressed in this organism. Additionally, a separate strain ofClostridium beijerinckiiNCIMB 8052 was engineered by cloning and expressingmgsAand methylglyoxal/glyoxal reductase (mgR) fromClostridium pasteurianumATCC 6013 as a fused protein linked by polyglycine linker in the former. Both strains ofC. beijerinckiiNCIMB 8052 failed to produce 1,2-propaneol. Instead, traces of acetol—the precursor of 1,2-propanediol—were detected in cultures of both strains. When the recombinant strains were exposed to acetol, both strains exhibited ∼100% acetol-to-1,2-propanediol conversion efficiency. Conversely, methylglyoxal supplementation led to the production of traces of acetol but not lactaldehyde or 1,2-propanediol. When wildtypeC. beijerinckiiNCIMB 8052,C. pasteurianumATCC 6013 andClostridium tyrobutyricumATCC 25755 were challenged with methylglyoxal,C. beijerinckiiproduced ∼0.1 g/L (S)-(+)-1,2-Propanediol, whileC. tyrobutyricumproduced traces of lactate.C. pasteurianumproduced neither 1,2-propanediol nor lactate. The wild types of all three species above exhibited ∼100% acetol-to-1,2-propanediol conversion efficiency. The recombinant strain ofC. beijerinckiiexpressing fused MgsA and MgR fromC. pasteurianumATCC 6013 showed enhanced growth and solvent production, producing as high as 88% more butanol on both glucose and lactose than the control strain and the recombinant strain of the same organism expressing the native MgsA.

Conclusions

Recombinant and native strains ofC. beijerinckii,C. pasteurianumandC. tyrobutyricumstudied in this work exhibit extremely poor capacity to catalyze the conversion of the intermediates of the methylglyoxal bypass to 1,2-propanediol. This is indicative of lack of appropriate enzymes to catalyze the reactions from methylglyoxal to acetol or lactaldehyde. Inability to detect methylglyoxal in the recombinant strains harboringmgsA(both homologous and heterologous)— whereas the strain expressing bothmgsAandmgRfromC. pasteurianum, under the same promoter (Padc) produced higher concentrations of butanol—suggests thatC. beijerinckiimight possess a regulatory mechanism that limits the activity of methylglyoxal-producing MgsA. The protein product ofmgRfromC. pasteurianumrepresents a promising metabolic engineering candidate towards increasing butanol production.
Synaptic vesicle characterization of iPSC-derived dopaminergic neurons provides insight into distinct secretory vesicle pools
Authors: Fujise Kenshiro , Rosenfeld Martin Shaun , Rafiq Nisha Mohd
Published: February 2024

The impairment of dopaminergic (DA) neurons plays a central role in the development of Parkinson’s disease. Evidence for distinct populations of synaptic vesicles (SVs) differing in neurotransmitter content (glutamate versus dopamine) has been attributed to differences in trafficking pathways and their exocytosis kinetics. However, the molecular and ultrastructural organization of the two types of vesicles remains poorly understood. Here we examined the development of axonal varicosities in human iPSC-derived DA neurons and glutamatergic neurons (i3Neurons). While i3Neurons are comprised of 40-50 nm small clear SVs, DA neurons are predominantly comprised of large pleiomorphic vesicles including empty and dense core vesicles, in addition to the classical SVs. The large vesicles were positive for VMAT2, the monoamine vesicular transporter responsible for loading dopamine, and are distinctly larger in size and spatially segregated from the VGLUT1/2-positive vesicles when expressed in an ectopic SV-like organelle reconstitution system. Moreover, these VMAT2-positive vesicles were also colocalized to known SV markers such as Rab3, SCAMP5, VAMP2, SV2C and can be clustered by the matrix protein synapsin. Our results show that DA neurons display inherent differences in their populations of neurotransmitter-containing secretory vesicles, and iPSC-derived neurons are powerful models for the study of presynaptic structures.
A multi-symptomatic model of heroin use disorder in rats reveals distinct behavioral profiles and neuronal correlates of heroin vulnerability versus resiliency
Authors: Kuhn Brittany N. , Cannella Nazzareno , Crow Ayteria D. , Lunerti Veronica , Gupta Arkobrato , Walterhouse Stephen J. , Allen Carter , Chalhoub Reda M. , Dereschewitz Eric , Roberts Analyse T. , Cockerham Mackenzie , Beeson Angela , Nall Rusty W. , Palmer Abraham A. , Hardiman Gary , Solberg Woods Leah C. , Chung Dongjun , Ciccocioppo Roberto , Kalivas Peter W.
Published: February 2024

Objective

The behavioral and diagnostic heterogeneity within human opioid use disorder (OUD) diagnosis is not readily captured in current animal models, limiting translational relevance of the mechanistic research that is conducted in experimental animals. We hypothesize that a non-linear clustering of OUD-like behavioral traits will capture population heterogeneity and yield subpopulations of OUD vulnerable rats with distinct behavioral and neurocircuit profiles.

Methods

Over 900 male and female heterogeneous stock rats, a line capturing genetic and behavioral heterogeneity present in humans, were assessed for several measures of heroin use and rewarded and non-rewarded seeking behaviors. Using a non-linear stochastic block model clustering analysis, rats were assigned to OUD vulnerable, intermediate and resilient clusters. Additional behavioral tests and circuit analyses using c-fos protein activation were conducted on the vulnerable and resilient subpopulations.

Results

OUD vulnerable rats exhibited greater heroin taking and seeking behaviors relative to those in the intermediate and resilient clusters. Akin to human OUD diagnosis, further vulnerable rat sub-clustering revealed subpopulations with different combinations of behavioral traits, including sex differences. Lastly, heroin cue-induced neuronal patterns of circuit activation differed between resilient and vulnerable phenotypes. Behavioral sex differences were recapitulated in patterns of circuitry activation, including males preferentially engaging extended amygdala stress circuitry, and females cortico-striatal drug cue-seeking circuitry.

Conclusion

Using a non-linear clustering approach in rats, we captured behavioral diagnostic heterogeneity reflective of human OUD diagnosis. OUD vulnerability and resiliency were associated with distinct neuronal activation patterns, posing this approach as a translational tool in assessing neurobiological mechanisms underpinning OUD.
Transcription factor-mediated generation of dopaminergic neurons from human iPSCs – a comparison of methods
Authors: McDonald Kirstin O. , Lyons Nikita M.A. , Schoderboeck Lucia , Hughes Stephanie M. , Basak Indranil
Published: February 2024

Dopaminergic neurons are the predominant brain cells affected in Parkinson’s disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson’s disease, dopaminergic neurons have been generated from human skin cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, transcription factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compare and contrast three protocols to generate induced pluripotent stem cell-derived dopaminergic neurons using transcription factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate transcription factors into induced pluripotent stem cells, followed by differentiation using different media compositions. We introduced three transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells, and in combination with small molecules, we generated more than 80% neurons in the culture, out of which more than 80% neurons were dopaminergic neurons. Therefore, a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons, a prerequisite for cell replacement therapies.
A novel truncated CHAP modular endolysin, CHAPSAP26-161, that lysesStaphylococcus aureus,Acinetobacter baumannii,andClostridioides difficile
Authors: Choi Yoon-Jung , Kim Shukho , Dahal Ram Hari , Kim Jungmin
Published: February 2024

Background

Development of novel antimicrobial agents is imperative due to the increasing threat of antibiotic-resistant pathogens. This study aimed to validate the enhanced antibacterial activity andin vivoefficacy of a novel truncated endolysin, CHAPSAP26-161, derived from the CHAP domain of LysSAP26, against multidrug-resistant bacteria.

Methods

Two deletion mutants, CHAPSAP26-139 and CHAPSAP26-161, were constructed by deleting the C-terminal portion of LysSAP26. These were cloned and expressed, and their antibacterial activities, together with protein purification efficiency, were evaluated against 12 bacterial species under various environmental conditions. To test the temperature and pH stability of the three recombinant proteins, the antibacterial effects of the proteins at various temperatures (4°C–60°C) and pH values (3–10) were measured. Time-kill assay measured the optical density (600 nm) and colony-forming units after incubation for 0, 2, 4, 6, 8, and 24 h. We verified this throughin vivoexperiments using mouse models to evaluate the therapeutic potential of CHAPSAP26-161 againstAcinetobacter baumannii.

Results

CHAPSAP26-161 exhibited higher protein purification efficiency and antibacterial activity than LysSAP26. Moreover, CHAPSAP26-161 showed the highest lytic activity againstA. baumanniiwith a minimal bactericidal concentration (MBC) of 5–10 µg/mL, followed byStaphylococcus aureuswith an MBC of 10–25 µg/mL. Interestingly, CHAPSAP26-161 could lyse anaerobic bacteria, such asC. difficile, with an MBC of 25–50 µg/mL. At pH 4–8 and temperatures of 4°C–45°C, CHAPSAP26-161 exhibited optimal hydrolase activity. The lytic activity of CHAPSAP26-161 was dependent on divalent metal ions, especially Zn2+, and increased in the presence of ethylenediamine tetraacetic acid. CHAPSAP26-161 demonstrated superior protein purification efficiency and antibacterial activity than LysSAP26. It showed high lytic activity against gram-positive, gram-negative, and anaerobic bacteria, includingS. aureusandClostridioides difficile.Enhanced stability under varied temperatures and pH conditions.In vivo,tests demonstrated promising therapeutic effects of CHAPSAP26-161 in murine systemicA. baumanniiinfection models.

Conclusions

CHAPSAP26-161, a truncated modular endolysin containing only the CHAP domain of LysSAP26, demonstrated higher protein purification efficiency and antibacterial activity than LysSAP26. It also exhibited extended-spectrum antibacterial activity against gram-positive, gram-negative, and anaerobic bacteria, such asS. aureus,A. baumannii, andC. difficile. Its successfulin vivoapplication in murine models highlights its potential as an alternative therapeutic agent in combating antibiotic resistance.
stMMR: accurate and robust spatial domain identification from spatially resolved transcriptomics with multi-modal feature representation
Authors: Zhang Daoliang , Yu Na , Li Wenrui , Sun Xue , Zou Qi , Li Xiangyu , Liu Zhiping , Yuan Zhiyuan , Zhang Wei , Gao Rui
Published: February 2024

Deciphering spatial domains using spatially resolved transcriptomics (SRT) is of great value for the characterizing and understanding of tissue architecture. However, the inherent heterogeneity and varying spatial resolutions present challenges in the joint analysis of multi-modal SRT data. We introduce a multi-modal geometric deep learning method, named stMMR, to effectively integrate gene expression, spatial location and histological information for accurate identifying spatial domains from SRT data. stMMR uses graph convolutional networks (GCN) and self-attention module for deep embedding of features within unimodal and incorporates similarity contrastive learning for integrating features across modalities. Comprehensive benchmark analysis on various types of spatial data shows superior performance of stMMR in multiple analyses, including spatial domain identification, pseudo-spatiotemporal analysis, and domain-specific gene discovery. In chicken heart development, stMMR reconstruct the spatiotemporal lineage structures indicating accurate developmental sequence. In breast cancer and lung cancer, stMMR clearly delineated the tumor microenvironment and identified marker genes associated with diagnosis and prognosis. Overall, stMMR is capable of effectively utilizing the multi-modal information of various SRT data to explore and characterize tissue architectures of homeostasis, development and tumor.
Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing
Authors: De Rijk Peter , Watzeels Tijs , Küçükali Fahri , Van Dongen Jasper , Faura Júlia , Willems Patrick , De Deyn Lara , Duchateau Lena , Grones Carolin , Eekhout Thomas , De Pooter Tim , Joris Geert , Rombauts Stephane , De Rybel Bert , Rademakers Rosa , Van Breusegem Frank , Strazisar Mojca , Sleegers Kristel , De Coster Wouter
Published: February 2024

We introducescywalker, an innovative and scalable package developed to comprehensively analyze long-read nanopore sequencing data of full-length single-cell or single-nuclei cDNA. Existing nanopore single-cell data analysis tools showed severe limitations in handling current data sizes. We developed novel scalable methods for cell barcode demultiplexing and single-cell isoform calling and quantification and incorporated these in an easily deployable package. Scywalker streamlines the entire analysis process, from sequenced fragments in FASTQ format to demultiplexed pseudobulk isoform counts, into a single command suitable for execution on either server or cluster. Scywalker includes data quality control, cell type identification, and an interactive report. Assessment of datasets from the human brain, Arabidopsis leaves, and previously benchmarked data from mixed cell lines, demonstrate excellent correlation with short-read analyses at both the cell-barcoding and gene quantification levels. At the isoform level, we show that scywalker facilitates the direct identification of cell-type-specific expression of novel isoforms.
Centriolar satellites are sites of translation of centrosomal and ciliary proteins
Authors: Pachinger Claudia , Dobbelaere Jeroen , Rumpf-Kienzl Cornelia , Raina Shiviya , Garcia-Baucells Júlia , Sarantseva Marina , Brauneis Andrea , Dammermann Alexander
Published: February 2024

Centriolar satellites are cytoplasmic particles found in the vicinity of centrosomes and cilia whose functional contribution to the formation of these cellular structures has long been unclear. By characterizing the main scaffolding component of satellites, PCM1 or Combover inDrosophila, we show that satellites are not involved in cellular trafficking as previously thought but rather act as sites for the coordinate translation of centrosomal and ciliary proteins through the interaction with a set of RNA binding proteins and proteins involved in quality control. Strikingly, the concentration of satellites near centrosomes and cilia in vertebrates is not a conserved feature and therefore dispensable for satellite function. Such coordinate synthesis may be a general feature in eukaryotic cells to facilitate protein complex formation and cellular compartmentalization.

One-Sentence Summary

Centriolar satellites facilitate the coordinate synthesis of centrosomal and ciliary proteins.
Metabolic reprogramming of hypoxic tumor-associated macrophages through CSF-1R targeting favours treatment efficiency in colorectal cancers
Authors: Gharzeddine Khaldoun , Gonzalez Pietro Cristina , Malier Marie , Hennot Clara , Grespan Renata , Yamaryo-Botté Yoshiki , Botté Cyrille Y. , Thomas Fabienne , Laverriere Marie-Hélène , Girard Edouard , Roth Gael , Millet Arnaud
Published: February 2024

Tumor-associated macrophages participate in the complex network of support that favors tumor growth. Among the various strategies that have been developed to target these cells, blockade of the CSF-1R receptor is one of the most promising one. Here, we characterize the resulting state of human macrophages exposed to a CSF-1R kinase inhibitor. We find that CSF-1R receptor inhibition in human macrophages is able to impair cholesterol synthesis, fatty acid metabolism and hypoxia-driven expression of dihydropyrimidine dehydrogenase, an enzyme responsible for the 5-fluorouracil macrophage-mediated chemoresistance. We show that this inhibition of the CSF-1R receptor leads to a downregulation of the expression of SREBP2, a transcription factor that controls cholesterol and fatty acid synthesis. We also show that the inhibition of ERK1/2 phosphorylation resulting from targeting the CSF-1R receptor destabilizes the expression of HIF2α in hypoxia resulting in the downregulation of dihydropyrimidine dehydrogenase expression restoring the sensitivity to 5-fluorouracil in colorectal cancer. These results reveal the unexpected metabolic rewiring resulting from the CSF-1R receptor targeting of human macrophages in tumors.
NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis
Authors: Parolia Abhijit , Eyunni Sanjana , Verma Brijesh Kumar , Young Eleanor , Liu Lianchao , George James , Aras Shweta , Das Chandan Kanta , Mannan Rahul , ur Rasool Reyaz , Luo Jie , Carson Sandra E. , Mitchell-Velasquez Erick , Liu Yihan , Xiao Lanbo , Gajjala Prathibha R. , Jaber Mustapha , Wang Xiaoju , He Tongchen , Qiao Yuanyuan , Pang Matthew , Zhang Yuping , Alhusayan Mohammed , Cao Xuhong , Tavana Omid , Hou Caiyun , Wang Zhen , Ding Ke , Chinnaiyan Arul M. , Asangani Irfan A.
Published: February 2024

The androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here, we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 expression is abnormally gained in prostate cancer cells and its functional inhibition impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites, which is greater than 65% of the AR tumor cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and instead contain the canonical palindromic motifs. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in the physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Concordantly, a dual NSD1/2 PROTAC degrader, called LLC0150, was preferentially cytotoxic in AR-dependent prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the ARneo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 as viable paralog co-targets in advanced prostate cancer.
The evolution of antimicrobial peptide resistance inPseudomonas aeruginosais severely constrained by random peptide mixtures
Authors: Antunes B. , Zanchi C. , Johnston P.R. , Maron B. , Witzany C. , Regoes R. , Hayouka Z. , Rolff J.
Published: February 2024

The prevalence of antibiotic-resistant pathogens has become a major threat to public health, requiring swift initiatives for discovering new strategies to control bacterial infections. Hence, antibiotic stewardship and rapid diagnostics, but also the development, and prudent use, of novel effective antimicrobial agents are paramount. Ideally, these agents should be less likely to select for resistance in pathogens than currently available conventional antimicrobials. The usage of antimicrobial Peptides (AMPs), key components of the innate immune response, and combination therapies, have been proposed as strategies to diminish the emergence of resistance.

Herein, we investigated whether newly developed random antimicrobial peptide mixtures (RPMs) can significantly reduce the risk of resistance evolutionin vitroto that of single sequence AMPs, using the ESKAPE pathogenPseudomonas aeruginosa(P. aeruginosa) as a model Gram-negative bacterium. Infections of this pathogen are difficult to treat due the inherent resistance to many drug classes, enhanced by the capacity to form biofilms.P. aeruginosawas experimentally evolved in the presence of AMPs or RPMs, subsequentially assessing the extent of resistance evolution and cross-resistance/collateral sensitivity between treatments. Furthermore, the fitness costs of resistance on bacterial growth were studied, and whole-genome sequencing used to investigate which mutations could be candidates for causing resistant phenotypes. Lastly, changes in the pharmacodynamics of the evolved bacterial strains were examined.

Our findings suggest that using RPMs bears a much lower risk of resistance evolution compared to AMPs and mostly prevents cross-resistance development to other treatments, while maintaining (or even improving) drug sensitivity. This strengthens the case for using random cocktails of AMPs in favour of single AMPs, against which resistance evolvedin vitro, further providing an alternative to classic antibiotics worth pursuing.
Catabolism of germinant amino acids is required to prevent premature spore germination inBacillus subtilis
Authors: Kasu Iqra R. , Reyes-Matte Octavio , Bonive-Boscan Alejandro , Derman Alan I. , Lopez-Garrido Javier
Published: February 2024

Spores ofBacillus subtilisgerminate in response to specific germinant molecules that are recognized by receptors in the spore envelope. Germinants signal to the dormant spore that the environment can support vegetative growth, so many germinants, such as alanine and valine, are also essential metabolites. As such, they are also required to build the spore. Here we show that these germinants cause premature germination if they are still present at the latter stages of spore formation and beyond, but thatB. subtilismetabolism is configured to prevent this: alanine and valine are catabolized and cleared from wild-type cultures even when alternative carbon and nitrogen sources are present. Alanine and valine accumulate in the spent media of mutants that are unable to catabolize these amino acids, and premature germination is pervasive. Premature germination does not occur if the germinant receptor that responds to alanine and valine is eliminated, or if wild-type strains that are able to catabolize and clear alanine and valine are also present in coculture. Our findings demonstrate that spore-forming bacteria must fine-tune the concentration of any metabolite that can also function as a germinant to a level that is high enough to allow for spore development to proceed, but not so high as to promote premature germination. These results indicate that germinant selection and metabolism are tightly linked, and suggest that germinant receptors evolve in tandem with the catabolic priorities of the spore-forming bacterium.
A ribosome-associating chaperone mediates GTP-driven vectorial folding of nascent eEF1A
Authors: Sabbarini Ibrahim M. , Reif Dvir , Park Kibum , McQuown Alexander J. , Nelliat Anjali R. , Trejtnar Charlotte , Dötsch Volker , Shakhnovich Eugene I. , Murray Andrew W. , Denic Vladimir
Published: February 2024

Eukaryotic translation elongation factor 1A (eEF1A) is a highly abundant, multi-domain GTPase. Post-translational steps essential for eEF1A biogenesis are carried out by bespoke chaperones but co-translational mechanisms tailored to eEF1A folding remain unexplored. Here, we find that the N-terminal, GTP-binding domain of eEF1A is prone to co-translational misfolding and using computational approaches, yeast genetics, and microscopy analysis, we identify the conserved yet uncharacterized yeast protein Ypl225w as a chaperone dedicated to solving this problem. Proteomics and biochemical reconstitution reveal that Ypl225w’s interaction with ribosomal eEF1A nascent chains depends on additional binding of Ypl225w to the UBA domain of nascent polypeptide-associated complex (NAC). Lastly, we show by orthogonal chemical genetics that Ypl225w primes eEF1A nascent chains for their subsequent binding to GTP and release from Ypl225w. Our work establishes eEF1A as a model system for chaperone-dependent co-translational folding and unveils a novel mechanism for GTP-driven folding on the ribosome.
Laboratory evolution reveals transcriptional mechanisms underlying thermal adaptation ofEscherichia coli
Authors: Rychel Kevin , Chen Ke , Catoiu Edward A. , Olson Connor A. , Sandberg Troy E. , Gao Ye , Xu Sibei , Hefner Ying , Szubin Richard , Patel Arjun , Feist Adam M. , Palsson Bernhard O.
Published: February 2024

Adaptive laboratory evolution (ALE) is able to generate microbial strains which exhibit extreme phenotypes, revealing fundamental biological adaptation mechanisms. Here, we use ALE to evolveEscherichia colistrains that grow at temperatures as high as 45.3°C, a temperature lethal to wild type cells. The strains adopted a hypermutator phenotype and employed multiple systems-level adaptations that made global analysis of the DNA mutations difficult. Given the challenge at the genomic level, we were motivated to uncover high temperature tolerance adaptation mechanisms at the transcriptomic level. We employed independently modulated gene set (iModulon) analysis to reveal five transcriptional mechanisms underlying growth at high temperatures. These mechanisms were connected to acquired mutations, changes in transcriptome composition, sensory inputs, phenotypes, and protein structures. They are: (i) downregulation of general stress responses while upregulating the specific heat stress responses; (ii) upregulation of flagellar basal bodies without upregulating motility, and upregulation fimbriae; (iii) shift toward anaerobic metabolism, (iv) shift in regulation of iron uptake away from siderophore production, and (v) upregulation ofyjfIJKL, a novel heat tolerance operon which we characterized using AlphaFold. iModulons associated with these five mechanisms explain nearly half of all variance in the gene expression in the adapted strains. These thermotolerance strategies reveal that optimal coordination of known stress responses and metabolism can be achieved with a small number of regulatory mutations, and may suggest a new role for large protein export systems. ALE with transcriptomic characterization is a productive approach for elucidating and interpreting adaptation to otherwise lethal stresses.
Deep plasma proteomics with data-independent acquisition: A fastlane towards biomarkers identification
Authors: Ward Bradley , Pyr dit Ruys Sébastien , Balligand Jean-Luc , Belkhir Leïla , Cani Patrice D. , Collet Jean-François , De Greef Julien , Dewulf Joseph P. , Gatto Laurent , Haufroid Vincent , Jodogne Sébastien , Kabamba Benoît , Lingurski Maxime , Yombi Jean Cyr , Vertommen Didier , Elens Laure
Published: February 2024

Plasma proteomic is a precious tool in human disease research, but requires extensive sample preparation in order to perform in-depth analysis and biomarker discovery using traditional Data-Dependent Acquisition (DDA). Here, we highlight the efficacy of combining moderate plasma prefractionation and Data-Independent Acquisition (DIA) to significantly improve proteome coverage and depth, while remaining cost- and time-efficient.

Using human plasma collected from a 20-patient COVID-19 cohort, our method utilises commonly available solutions for depletion, sample preparation, and fractionation, followed by 3 LC-MS/MS injections for a 360-minutes DIA run time. DIA-NN software was then used for precursor identification, and the QFeatures R package was used for protein aggregation.

We detect 1,321 proteins on average per patient, and 2,031 unique proteins across the cohort. Filtering precursors present in under 25% of patients, we still detect 1,230 average proteins and 1,590 unique proteins, indicating robust protein identification. Differential analysis further demonstrates the applicability of this method for plasma proteomic research and clinical biomarker identification.

In summary, this study introduces a streamlined, cost- and time-effective approach to deep plasma proteome analysis, expanding its utility beyond classical research environments and enabling larger-scale multi-omics investigations in clinical settings.
Hyperbaric Oxygen-Induced acute lung injury: A Mouse Model Study on Pathogenesis and Recovery Dynamics
Authors: Wang Shu , Li Zhi , Xu Guangxu , Bao Xiaochen
Published: February 2024

Hyperbaric oxygen (HBO) therapy is extensively used to treat a number of ailments. Although oxygen is crucial for survival, too much oxygen can be harmful. Excessive oxygen inhalation in a short period of time can lead to injury, and the lung is one of the main target organs. Lung injury induced by hyperbaric oxygen is notably more severe than that caused by normobaric oxygen, yet systematic research on such injury and its regression is scarce. In this study, mice were exposed to 2 atmospheres absolute (ATA), ⩾ 95% oxygen for 2, 4, 6, 8 hours. Changes in lung histopathology, inflammation and expression of chemokines, vascular endothelial permeability, 8-OHdG and apoptotic cells were detected before and after the exposure. These parameters were also measured immediately, 12 hours, and 24 hours following 6 hours of exposure to 2 ATA of ⩾95% oxygen. The study indicates that lung damage from HBO is histologically characterized by bronchiolar and alveolar dilation, atelectasis, inflammatory cell infiltration, and hemorrhage. At 2 ATA with over 95% oxygen for 4 hours, there is a significant increase in pulmonary vascular permeability, as evidenced by elevated Evans blue scores (p= 0.02). After 6 hours of HBO exposure, there is a significant rise in pulmonary tissue pathology scores, 8-OHdG levels, and inflammatory and chemotactic factors (such as IL-6, CCL2, CCL3, CXCL5, and CXCL10), along with intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1(VCAM1). Moreover, it was observed that these markers continued to progress even after leaving the hyperoxic environment, peaking at 12 hours and starting to recover after 24 hours, suggesting that the peak of inflammatory lung damage occurs within 12 hours post-exposure, with recovery beginning at 24 hours. However, the content of Evans Blue, reflecting vascular endothelial damage, and ICAM1, VCAM1 remain significantly elevated 24 hours after leaving the hyperoxic environment, indicating that the pulmonary vasculature endothelium is the most sensitive to damage and the slowest to recover in HBO-induced lung injury. These findings provide evidence for the prevention and treatment of acute lung injury complications caused by HBO.

NEW & NOTEWORTHY

This study systematically observed the development and outcome changes of ALI induced by HBO. In lung injuries caused by high partial pressure of oxygen, the pulmonary vascular endothelial cells are the first to be damaged and the slowest to recover. A 6-hour exposure to 2 ATA, ⩾95% oxygen of hyperbaric oxygen primarily causes oxidative DNA damage and inflammatory responses without significant apoptosis. The lung injury progresses even after leaving the high-oxygen setting, with inflammation peaking at 12 hours post-exposure and beginning to subside after 24 hours.
Molecular and Cellular Mechanisms of Teneurin Signaling in Synaptic Partner Matching
Authors: Xu Chuanyun , Li Zhuoran , Lyu Cheng , Hu Yixin , McLaughlin Colleen N. , Wong Kenneth Kin Lam , Xie Qijing , Luginbuhl David J. , Li Hongjie , Udeshi Namrata D. , Svinkina Tanya , Mani D. R. , Han Shuo , Li Tongchao , Li Yang , Guajardo Ricardo , Ting Alice Y. , Carr Steven A. , Li Jiefu , Luo Liqun
Published: February 2024

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionally conserved teneurins were the first identified transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurin’s functions is unclear. Here, we usein situproximity labeling to obtain the intracellular interactome of teneurin (Ten-m) in theDrosophilabrain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.

HIGHLIGHTS

In situspatial proteomics reveal the first intracellular interactome of teneurins

Ten-m signals via a RhoGAP and Rac1 GTPase to regulate synaptic partner matching

Single-axon analyses reveal a stabilization-upon-contact model for partner matching

Ten-m signaling promotes F-actin in axon branches contacting partner dendrites
Development and validation of a gene expression score to account for tumour purity and improve prognostication in breast cancer
Authors: Barreca Marco , Dugo Matteo , Galbardi Barbara , Győrffy Balázs ,   NA-PHER2 consortium,   NeoTRIP consortium, Valagussa Pinuccia , Besozzi Daniela , Viale Giuseppe , Bianchini Giampaolo , Gianni Luca , Callari Maurizio
Published: February 2024

The prevalence of malignant cells in clinical specimens, or tumour purity, is affected by both intrinsic biological factors and extrinsic sampling bias. Molecular characterization of large clinical cohorts is typically performed on bulk samples; data analysis and interpretation can be biased by tumour purity variability. Transcription-based strategies to estimate tumour purity have been proposed, but no breast cancer specific method is available yet.

We interrogated over 4400 expression profiles from 9 breast cancer datasets to develop and validate a 9-gene Breast Cancer Purity Score (BCPS). BCPS outperformed existing methods for estimating tumour content. Adjusting transcriptomic profiles using the BCPS reduce sampling bias and aid data interpretation. BCPS-estimated tumour purity improved prognostication in luminal breast cancer, correlated with pathologic complete response in on-treatment biopsies from triple-negative breast cancer patients undergoing neoadjuvant treatment and effectively stratified the risk of relapse in HER2+ residual disease post-neoadjuvant treatment.