bluuebunny/crossref_metadata_2025 huggingface dataset:
The pattern of genetic variability in a core collection of 2,021 cowpea accessions
Authors: Fiscus Christopher J. , Herniter Ira A. , Tchamba Marimagne , Paliwal Rajneesh , Muñoz-Amatriaín María , Roberts Philip A. , Abberton Michael , Alaba Oluwafemi , Close Timothy J. , Oyatomi Olaniyi , Koenig Daniel
Published: December 2023

Cowpea is a highly drought-adapted leguminous crop with great promise for improving agricultural sustainability and food security. Here, we report analyses derived from array-based genotyping of 2,021 accessions constituting a core subset of the world’s largest cowpea collection, held at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria. We used this dataset to examine genetic variation and population structure in worldwide cowpea. We confirm that the primary pattern of population structure is two geographically defined subpopulations origining in West and East Africa, respectively, and that population structure is associated with shifts in phenotypic distribution. Furthermore, we establish the cowpea core collection as a resource for genome-wide association studies by mapping the genetic basis of several phenotypes, with a focus on seed coat pigmentation patterning and color. We anticipate that the genotyped IITA cowpea core collection will serve as a powerful tool for mapping complex traits, facilitating the acceleration of breeding programs to enhance the resilience of this crop in the face of rapid global climate change.
Assessing tissue-specific gene expression of essential genes from human and mouse
Authors: Zheng Huiwen , Fard Atefeh Taherian , Mar Jessica C
Published: December 2023

A gene satisfies the definition of essentiality when the loss of its function compromises an organism’s viability. Identifying essential genes is useful for understanding the core components that regulate a biological system and ensure its survival. Advances in gene editing techniques like CRISPR-Cas9 generate the capacity to comprehensively interrogate a genome to elucidate what genes are essential. However, these techniques are often applied in the context of a single cell line and even when studies have collated essential gene sets across multiple cell lines, this information is rarely probed at a level that incorporates multiple cell and tissue types. The recent availability of large-scale single-cell RNA-sequencing (scRNA-seq) atlases provides an unprecedented opportunity to investigate the distribution of essential gene expression in cell and tissue types.

Here, we leverage information contained in benchmarking datasets, single cell tissue atlases, and databases of essential genes, and develop a computational method, scEssentials, which uses a statistical framework to report on the robustness and specificity of essential genes in human and mouse across multiple cell types. Using scEssentials, both mouse and human models showed consistently high in expression and exhibit limited variably across more than 60 cell types. We also demonstrate a substantial number of significantly correlated gene pairs within scEssentials, which produce densely connected co-expression networks with functional annotation. Furthermore, we showed high frequencies of scEssentials across 200 pathways. Finally, we develop a score to quantify the relative essentiality of genes within scEssentials, which further validates with significant association with gene mutation frequency and chromatin accessibility.

Using the heterogeneous ageing process, we demonstrate the application of scEssentials and their robust gene expression profile. Only one-fifth of scEssentials showed significant ageing-related differential expression among three age groups, occurring primarily in muscle satellite cells of varying tissue origins and highly interacting brain cells. Collectively, the robustness of scEssentials serves as a reference for analysing scRNA-seq data and provides insight into the heterogeneous nature of biological processes such as ageing.
Resource-efficient nitrogen removal from source separated urine with partial nitritation/anammox in a membrane aerated biofilm reactor
Authors: Timmer Marijn J. , De Paepe Jolien , Van Winckel Tim , Spiller Marc , Spacova Irina , De Paepe Kim , Pintelon Isabel , Lebeer Sarah , De Vos Winnok H. , Lasseur Christophe , Ganigué Ramon , Udert Kai M. , Vlaeminck Siegfried E.
Published: December 2023

Source separation and decentralized urine treatment can cut costs in centralized wastewater treatment by diverting 80% of the nitrogen load in sewage. One promising approach for nitrogen removal in this context is partial nitritation/anammox (PN/A), reducing the aeration demand by 67% and organics dosage by 100% compared to nitrification/denitrification. Whilst previous studies with suspended biomass have encountered stability issues during PN/A treatment of urine, a PN/A biofilm was hypothesized to be more resilient. Its use for urine treatment was pioneered here for maximum rates and efficiencies in the energy efficient membrane-aerated biofilm reactor (MABR). Nitrogen removal rates of 1.0 g N L-1d-1and removal efficiencies of 80-95% were achieved during a 335-day stable operation at 28°C on stabilized (pH>11), diluted urine (10%). A balance between N2and NO3-formation was observed whilst optimizing the supply of O2and was rate limiting for the conversion towards N2. Short-term operation on less- and undiluted urine yielded N removal rates of 0.6-0.8 g N L-1d-1and removal efficiencies of 93% on 66% urine and 85% on undiluted urine. Metataxonomic analysis and fluorescencein-situhybridization confirmed the presence of biofilms consisting of nitrifiers (Nitrosomonas, Nitrospira) at the membrane side and anammox bacteria (“CandidatusBrocadia”) at the anoxic bulk side. The findings suggest that a biofilm approach to PN/A treatment of urine overcomes stability issues, and a PN/A-MABR has significant potential for resource efficient decentralized treatment. In human long-duration deep-space missions, this gravity-independent technology could produce N2to compensate artificial atmosphere losses whilst facilitating water recovery from urine.

[GRAPHICAL ABSTRACT, COLOR]
Connecting Hippo pathway and cytoophidia inDrosophilaposterior follicle cells
Authors: Weng Rui-Yu , Zhang Lei , Liu Ji-Long
Published: December 2023

CTP synthase (CTPS), the rate-limiting enzyme inde novosynthesis of CTP, assembles into filamentous structures termed cytoophidia. Here we study the impact of Hippo pathway on the posterior follicle cells (PFCs) inDrosophilaegg chambers. We find that the inactivation of Hippo pathway correlates with a reduction in cytoophidium length and number within PFCs. During the overexpression of CTPS, the presence of Hippo mutations also reduces the length of cytoophidia in PFCs. In addition, we observe that knocking down CTPS mitigateshpo(Hippo)-associated over-proliferation. In summary, our results suggest a connection between the Hippo pathway and the nucleotide biosynthesis enzyme CTPS in PFCs.
Systems genetics uncover new loci containing functional gene candidates inMycobacterium tuberculosis-infected Diversity Outbred mice
Authors: Gatti DM , Tyler AL , Mahoney JM , Churchill GA , Yener B , Koyuncu D , Gurcan MN , Niazi MKK , Tavolara T , Gower AC , Dayao D , McGlone E , Ginese ML , Specht A , Alsharaydeh A , Tessier PA , Kurtz SL , Elkins K , Kramnik I , Beamer G
Published: December 2023

Mycobacterium tuberculosis,the bacillus that causes tuberculosis (TB), infects 2 billion people across the globe, and results in 8-9 million new TB cases and 1-1.5 million deaths each year. Most patients have no known genetic basis that predisposes them to disease. We investigated the complex genetic basis of pulmonary TB by modelling human genetic diversity with the Diversity Outbred mouse population. When infected withM. tuberculosis, one-third develop early onset, rapidly progressive, necrotizing granulomas and succumb within 60 days. The remaining develop non-necrotizing granulomas and survive longer than 60 days. Genetic mapping using clinical indicators of disease, granuloma histopathological features, and immune response traits identified five new loci on mouse chromosomes 1, 2, 4, 16 and three previously identified loci on chromosomes 3 and 17. Quantitative trait loci (QTLs) on chromosomes 1, 16, and 17, associated with multiple correlated traits and had similar patterns of allele effects, suggesting these QTLs contain important genetic regulators of responses toM. tuberculosis. To narrow the list of candidate genes in QTLs, we used a machine learning strategy that integrated gene expression signatures from lungs ofM. tuberculosis-infected Diversity Outbred mice with gene interaction networks, generating functional scores. The scores were then used to rank candidates for each mapped trait in each locus, resulting in 11 candidates:Ncf2, Fam20b, S100a8, S100a9, Itgb5, Fstl1, Zbtb20, Ddr1, Ier3, Vegfa,andZfp318. Importantly, all 11 candidates have roles in infection, inflammation, cell migration, extracellular matrix remodeling, or intracellular signaling. Further, all candidates contain single nucleotide polymorphisms (SNPs), and some but not all SNPs were predicted to have deleterious consequences on protein functions. Multiple methods were used for validation including (i) a statistical method that showed Diversity Outbred mice carrying PWH/PhJ alleles on chromosome 17 QTL have shorter survival; (ii) quantification of S100A8 protein levels, confirming predicted allele effects; and (iii) infection of C57BL/6 mice deficient for theS100a8gene. Overall, this work demonstrates that systems genetics using Diversity Outbred mice can identify new (and known) QTLs and new functionally relevant gene candidates that may be major regulators of granuloma necrosis and acute inflammation in pulmonary TB.
Post-synaptic competition between calcineurin and PKA regulates mammalian sleep-wake cycles
Authors: Wang Yimeng , Cao Siyu , Tone Daisuke , Fujishima Hiroshi , Yamada Rikuhiro G. , Ohno Rei-ichiro , Shi Shoi , Matsuzawa Kyoko , Kaneko Mari , Ukai-Tadenuma Maki , Ukai Hideki , Hanashima Carina , Kiyonari Hiroshi , Sumiyama Kenta , Ode Koji L. , Ueda Hiroki R.
Published: December 2023

Phosphorylation of synaptic proteins is a pivotal biochemical reaction that controls the sleep-wake cycle in mammals. Protein phosphorylation in vivo is reversibly regulated by kinases and phosphatases. In this study, we investigated a pair of kinases and phosphatases that reciprocally regulate sleep duration. Through comprehensive screening of Protein kinase A (PKA) and phosphoprotein phosphatase (PPP) family genes via the generation of 40 gene knockout mouse lines including post-natal CRISPR targeting, we identified a regulatory subunit of PKA (Prkar2b), a regulatory subunit of protein phosphatase (PP) 1 (Pppr1r9b), and catalytic and regulatory subunits of PP2B (calcineurin) (Ppp3caandPpp3r1) as sleep control genes. AAV-mediated stimulation of PKA and PP1/calcineurin activities confirmed PKA as a wake-promoting kinase, while PP1 and calcineurin function as sleep-promoting phosphatases. The importance of these phosphatases in sleep regulation is supported by the dramatic changes in sleep duration associated with their increased and decreased activity, ranging from approximately 17.3 hours/day (PP1 expression) to 6.7 hours/day (post-natal CRISPR targeting of calcineurin). For these phosphatases to exert their sleep-promoting effects, localization signals to the excitatory post-synapse were necessary. Furthermore, the wake-promoting effect of PKA localized to the excitatory post-synapse negated the sleep-promoting effect of calcineurin, suggesting that PKA and calcineurin construct a hierarchical phosphorylation control network for sleep regulation at excitatory post-synapses.
A direct estrogenic involvement in the expression of human hypocretin
Authors: Li Haimei , Chen Xinlu , Dong Jingyi , Liu Ripeng , Duan Jinfeng , Huang Manli , Hu Shaohua , Lu Jing
Published: December 2023

Background

Hypocretin-1 may play an important role in depression, which was increased in female depression in LHA in our previous study. Estrogen acts through its nuclear transcriptional receptors.

Methods

We studied the possibility of a direct action of estrogen receptors on the expression of human hypocretin. To explore the activation of ERs by hypocretin, we first investigated the potential presence of co-localization of the estrogen receptors (ERs) in hypocretin-immunoreactive neurons in the lateral hypothalamic area (LHA) in human and nuclear ERs in hypocretin immunoreactive neuroblastoma SK-N-SH cells. We investigated the relationship between E2 and hypocretin in female rats. After we explored the regulating role of exogenous estrogen on hypocretin gene expression via ERs through Chromatin immunoprecipitation, Electrophoretic mobility shift assay and Luciferase assay.

Results

We found that hypocretin-1 plasma level was significantly higher in female depression. Estrogen receptors (ERα and ERβ) and hypocretin are co-localized in female depression LHA, PC12 and SK-N-SH cell lines. The estrogen receptor response elements (EREs) existing in the hypocretin promoter region may directly regulate the expression of hypocretin, the synchronicity of change of hypocretin and estradiol both in hypothalamus and plasma was verified in female rats. With the presence of estradiol, there is specific binding between human ER and the Hypocretin-ERE. Expression of ER combined with estradiol repressed hypocretin promoter activity through the ERE.

Conclusions

we defined that estradiol may affect hypocretin neurons in the human hypothalamus via ER binding to the Hypocretin-ERE directly, which may thus lead to the sex-specific pathogenesis of depression.
On the mechanism for winter stem pressure build-up in walnut trees
Authors: Bozonnet Cyril , Saudreau Marc , Badel Eric , Charrier Guillaume , Améglio Thierry
Published: December 2023

Xylem embolism is a significant factor in tree mortality. Restoration of hydraulic conductivity after massive embolisation of the vascular system requires the application of positive pressure to the vessels and/or the creation of new conductive elements. Some species generate positive pressure from the root system to propagate pressure in distal, aboveground, organs in spring, whereas other species generate positive pressure locally at the stem level during winter. We provide a mechanistic explanation for winter stem pressure build-up in the walnut tree. We have developed a physical model that accounts for temperature fluctuations and phase transitions. This model is based on the exchange of water and sugars between living cells and vessels. Our computations demonstrate that vessel pressurization can be attributed to the transfer of water between vessels across the parenchyma rays, which is facilitated by a radial imbalance in sugar concentration. The ability to dispose of soluble sugars in living cells, and to transport them between living cells and up to the vessels, are identified as the main drivers of stem pressure build-up in the walnut tree.
Cryo-EM structure of monomeric CXCL12-bound CXCR4 in active state
Authors: Liu Yezhou , Liu Aijun , Li Xinyu , Liao Qiwen , Zhang Weijia , Zhu Lizhe , Ye Richard D.
Published: December 2023

CXC chemokine receptor 4 (CXCR4) and its chemokine ligand CXCL12 are crucial to embryonic development, bone marrow retention of hematopoietic progenitor cells, cancer metastasis, angiogenesis and HIV-1 infection. Yet the structural basis for CXCR4 recognition of full-length CXCL12 remains unknown despite available structures of CXCR4 in complex with small molecule antagonists and a viral chemokine. Here we present a cryo-EM structure of monomeric CXCL12-CXCR4 in complex with heterotrimeric Gi proteins at a resolution of 2.65 Å. The CXCL12-CXCR4 interaction at their N termini is stabilized by a polar interaction between the PC motif (C28NTin CXCR4) and CXC motif (the unique P10 in CXCL12). The N terminal 8 amino acids in CXCL12 insert into the transmembrane binding pocket (chemokine recognition site 2, CRS2) of CXCR4 with S4 defining an upward turn of V3, P2 and K1. Polar interactions involving C186ECL2and D187ECL2, along with hydrogen bonding with E2887,39and D2626,58stabilize the N terminus of CXCL12 in CRS2. Hydrophobic interactions between side chains aligning CRS2 and P2, L5 and S6 of CXCL12 further strengthen its binding to the receptor. CXCL12 inserts deep into the binding pocket, and the 3.2Å distance measured between V3 and the ‘toggle switch’ W6.48for G protein activation is among the shortest of all chemokine-receptor pairs. Our findings provide structural insights into the recognition mechanism of CXCR4 for its chemokine ligand CXCL12.
A system-wide analysis of lipid transfer proteins delineates lipid mobility in human cells
Authors: Titeca Kevin , Chiapparino Antonella , Türei Dénes , Zukowska Joanna , van Ek Larissa , Moqadam Mahmoud , Triana Sergio , Nielsen Inger Ødum , Foged Mads Møller , Gehin Charlotte , Maeda Kenji , Alexandrov Theodore , Saez-Rodriguez Julio , Reuter Nathalie , Hennrich Marco L. , Gavin Anne-Claude
Published: December 2023

Lipid transfer proteins (LTPs) maintain the specialised lipid compositions of biological membranes, and many are associated with disease. In eukaryotes, they support organellar functions by transporting lipids between compartmentalised metabolic pathways. However, for the majority of the hundreds of human LTPs, the cargoes remain unknown. We combined biochemical, lipidomic and computational methods to characterize LTP-lipid complexes assembledin celluloand in anin vitrobiochemical assay. We identified bound lipids for about half of the LTPs analysed, and confirmed known cargoes, while discovering new ones for most LTP families. The data represents a systematic resource that captures the general principles of non-vesicular lipid transport in humans. The specificity of LTPs for lipids involves not only the recognition of specific head groups, but also of specific acyl chains. This selectivity defines lipid species within a lipid class with different metabolic or functional fates. The generalised ability of LTPs to form complexes with more than one class of lipids delineates new relationships between lipids and regulatory mechanisms that may contribute to the coordination of metabolism between different organelles. This work represents a resource and a framework for further analyses in different cell types, in pathological states or following various cellular perturbations.
Kappa Opioid Receptors in Mesolimbic Terminals Mediate Escalation of Cocaine Consumption
Authors: Gordon-Fennell L. , Farero R.D. , Burgeno L.M. , Murray N.L. , Abraham A.D. , Soden M.E. , Stuber G.D. , Chavkin C. , Zweifel L.S. , Phillips P.E.M.
Published: December 2023

Increases in drug consumption over time, also known as escalation, is a key behavioral component of substance use disorder (SUD) that is related to potential harm to users, such as overdose. Studying escalation also allows researchers to investigate the transition from casual drug use to more SUD-like drug use. Understanding the neurobiological systems that drive this transition will inform therapeutic treatments in the aim to prevent increases in drug use and the development of SUD. The kappa opioid receptor (KOR) system is typically known for its role in negative affect, which is commonly found in SUD as well. Furthermore, the KOR system has also been implicated in drug use and importantly, modulating the negative effects of drug use. However, the specific neuronal subpopulation expressing KOR involved has not been identified. Here, we first demonstrated that pharmacologically inhibiting KOR in the nucleus accumbens core (NAcC), as a whole, blocks cocaine escalation under long-access self-administration conditions. We then demonstrated that KOR expressed on ventral tegmental area (VTA) neurons but not NAcC neurons is sufficient for blocking cocaine escalation by utilizing a novel virally-mediated CRISPR-SaCas9 knock-out of theoprk1gene. Together, this suggests that activation of KOR on VTA terminals in the NAcC drives the transition to the SUD-like phenotype of escalation of cocaine consumption.
Characterization of peroxidasin expression in histologically normal human adult and fetal kidney tissue
Authors: Brandão Isabel , Silva Roberto , Conde Eduardo , Gomes Bárbara , Sampaio Paula , Braga Ana Costa , Almeida Jorge Reis , Soares Alencastre Inês , Paulo Oliveira João
Published: December 2023

Peroxidasin (PXDN) is an enzyme of the peroxidase family that plays a critical role in extracellular matrix (ECM) formation and tissue development. In the kidney, very few studies using animal cells suggested that PXDN may have a role in the maintenance of the structural and functional integrity of the renal ECM. Still, the normal expression and (patho)physiological roles of PDXN in the mature and developing human kidney remain unknown.

In this work we used fluorescent immunohistochemistry and advanced microscopy and image processing methodologies to perform a quantitative characterization of PXDN expression in the different anatomic compartments of histologically normal kidney tissue, obtained from adult nephrectomy specimens, and from postmortem fetal and neonatal kidney specimens (excluding death by kidney disease and genitourinary developmental anomalies).

Results show that PXDN is expressed in all mature kidney compartments, but at significantly higher levels in tubular epithelial cells, particularly in the distal tubules. A similar PXDN expression pattern was observed in the developing kidney, as early as the in the comma-shaped-body stage. In the fetal kidney, PXDN was diffusely expressed in the branches of the ureteric bud but not in the cells of the metanephric blastema.

This is the first demonstration that PXDN is differentially expressed in histologically normal human renal parenchyma, exhibiting a remarkably consistent pattern of predominant tubular expression since the early stages of metanephrogenesis This data suggests a relevant, compartment-specific, role of PXDN in nephrogenesis and in the kidney physiology, highlighting a main role in tubular functions, that is worth further investigation.
Phosphorylation regulates tau’s phase separation behavior and interactions with chromatin
Authors: Abasi Lannah S. , Elathram Nesreen , Movva Manasi , Deep Amar , Corbett Kevin D. , Debelouchina Galia T.
Published: December 2023

Tau is a microtubule-associated protein often found in neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer’s disease (AD). Beyond this context, mounting evidence suggests that tau localizes into the nucleus, where it may play a role in DNA protection and heterochromatin regulation. Models of tau depletion or pathology show loss of genetically silent heterochromatin, aberrant expression of heterochromatic genes, and transposable element activation. The molecular mechanisms behind these observations are currently unclear. Usingin vitrobiophysical experiments, here we demonstrate that tau can undergo liquid-liquid phase separation (LLPS) with DNA, mononucleosomes, and reconstituted nucleosome arrays under low salt conditions. Low concentrations of tau promote chromatin compaction and protect DNA from digestion. While the material state of samples at physiological salt is dominated by chromatin oligomerization, tau can still associate strongly and reversibly with nucleosome arrays. These properties are driven by tau’s strong interactions with linker and nucleosomal DNA, while magic angle spinning (MAS) solid-state NMR experiments show that tau binding does not drastically alter nucleosome structure and dynamics. In addition, tau co-localizes into droplets formed by nucleosome arrays and phosphorylated HP1α, a key heterochromatin constituent thought to function through an LLPS mechanism. Importantly, LLPS and chromatin interactions are disrupted by aberrant tau hyperphosphorylation. These biophysical properties suggest that tau may directly impact DNA and chromatin accessibility and that loss of these interactions could contribute to the aberrant nuclear effects seen in tau pathology.
Single-cell and spatial multiomic inference of gene regulatory networks using SCRIPro
Authors: Chang Zhanhe , Xu Yunfan , Dong Xin , Gao Yawei , Wang Chenfei
Published: December 2023

The accurate reconstruction of gene regulation networks (GRNs) from sparse and noisy single-cell or spatial multi-omics data remains a challenge. Here, we present SCRIPro, a comprehensive computational framework that robustly infers GRNs for both single-cell and spatial multi-omics data. SCRIPro first addresses sample sparseness by a density clustering approach. SCRIPro assesses transcriptional regulator (TR) importance through chromatin reconstruction andin silicodeletion, referencing 1,292 human and 994 mouse TRs. It combines TR-target importance scores with expression levels for precise GRN reconstruction. Finally, we benchmarked SCRIPro on diverse datasets, it outperforms existing motif-based methods and accurately reconstructs cell type-specific, stage-specific, and region-specific GRNs.
Nanoscale profiling of evolving intermolecular interactions in ageing FUS condensates
Authors: Miller Alyssa , Toprakcioglu Zenon , Qamar Seema , St. George-Hyslop Peter , Ruggeri F. Simone , Knowles Tuomas P. J. , Vendruscolo Michele
Published: December 2023

In addition to the native state, proteins can form liquid-like condensates, viscoelastic condensates, such as gels, as well as solid-like condensates, such as amyloid fibrils, crystals and amorphous materials. The material properties of these condensates play important roles in their cellular functions, with aberrant liquid-to-solid phase transitions having been implicated in neurodegenerative diseases. However, the molecular changes and resultant material properties across the whole phase space of condensates are complex and yet to be fully understood. The extreme sensitivity to their environment, which enables their biological function, is also what makes protein condensates particularly challenging experimental targets. Here, we provide a characterisation of the ageing behaviour of the full-length fused in sarcoma (FUS) protein. We achieve this goal by using a microfluidic sample deposition technology to enable the application of surface-based techniques to the study of biological condensates. We first demonstrate that we maintain relevant structural features of condensates in physiologically-relevant conditions on surfaces. Then, using a combination of atomic force microscopy and vibrational spectroscopy, we characterise the spatio-temporal changes in the structure and mechanical properties of the condensates to reveal local phase transitions in individual condensates. We observe that initially dynamic, fluid-like condensates undergo a global increase in elastic response conferred by an increase in the density of cation-π intermolecular interactions. Solid-like structures form first at condensate-solvent interfaces, before heterogeneously propagating throughout the aged fluid core. These solid structures are composed of heterogenous, non-amyloid β-sheets, which are stabilised by hydrogen-bonding interactions not observed in the fluid state. Overall, this study identifies the molecular conformations associated with different physical states of FUS condensates, establishing a technology platform to understand the role of phase behaviour in condensate function and dysfunction.
In vitrodifferentiation of mouse pluripotent stem cells into glucocorticoid-producing adrenocortical cells
Authors: Oikonomakos Ioannis , Tedesco Melina , Motamedi Fariba Jian , Peitzsch Mirko , Nef Serge , Bornstein Stefan , Schedl Andreas , Steenblock Charlotte , Neirijnck Yasmine
Published: December 2023

Directed differentiation of pluripotent stem cells into specialized cell types represents an invaluable tool for a wide range of applications. Here, we have exploited single-cell transcriptomic data to develop a step-wisein vitrodifferentiation system from mouse embryonic stem cells into adrenocortical cells. We show that during development the adrenal primordium is embedded in an extracellular matrix containing tenascin and fibronectin. Culturing cells on fibronectin during differentiation increased the expression of the steroidogenic marker NR5A1. Furthermore, 3D cultures in the presence of PKA-pathway activators led to the formation of aggregates composed of different cell types expressing adrenal progenitor or steroidogenic markers, including the adrenocortical specific enzymeCyp21a1. Importantly,in vitrodifferentiated cells secreted thezona Fasciculataspecific corticosterone in response to cAMP activators, but not gonadal hormones, thus confirming the specificity of differentiation towards the adrenal lineage.

Highlights

Mouse adrenal progenitors differentiate in a tenascin/fibronectin-rich extracellular matrix

Stepwise differentiation of ES cells yields NR5A1+ adrenocortical cells

3D aggregates produce glucocorticoids in response to cAMP signalling
Plant genetic bases explaining microbiota diversity shed light into a novel holobiont generalist gene theory
Authors: Maillet Loeiz , Norest Manon , Kautsky Adam , Geraci Anna , Oddo Elisabetta , Troia Angelo , Guillerm-Erckelboudt Anne-Yvonne , Falentin Cyril , Rousseau-Gueutin Mathieu , Chèvre Anne-Marie , Istace Benjamin , Cruaud Corinne , Belser Caroline , Aury Jean-Marc , Schicchi Rosario , Frachon Léa , Bartoli Claudia
Published: December 2023

Plants as animals are strictly associated with a cortege of microbial communities influencing their health, fitness and evolution. Therefore, scientists refer to all living organisms as holobionts; complex genetic units that coevolve simultaneously. This is what has been recently proposed as the hologenome theory of evolution. This exciting and attractive theory has important implications on animal and plant health; however, it still needs consistent proof to be validated. Indeed, holobionts are still poorly studied in their natural habitats where coevolution and natural selective processes occur. Compared to animals and crops, wild plant populations are an excellent and unique model to explore the hologenome theory. These sessile holobionts have strictly coevolved with their microbiota for decades and natural selection and adaptive processes acting on wild plants are likely to regulate the plant-microbe interactions. Here we conducted for the first time a microbiota survey, plant genome sequencing and Genome-Environmental Analysis (GEA) of 26 natural populations of the non-model plant speciesBrassica rapa. We collected plants over two seasons in Italy and France, and analyzed the microbiota on two plant compartments (root and rhizosphere). We identified that plant compartment and season modulateB. rapamicrobiota. More importantly, when conducting GEA we evidenced neat peaks of association correlating with both fungal and bacterial microbiota. Surprisingly, we found 13 common genes between fungal and bacterial diversity descriptors that we referred under the name of Holobiont Generalist Genes (HGG). These genes might strongly regulate the diversity and composition of plant microbiota at the inter-kingdom level.

Significance Statement

The novel hologenome concept claims that hosts and their associated microbes (considered as holobionts) are a unique evolutionary unit on which natural selection acts. Thus, the hologenome theory assumes that hosts and microbiomes simultaneously coevolve. This novel vision of universal evolutionary entities is promising for both animal and plant health purposes. However, it is still quietly controversial as it suffers from a lack of tangible evidence. How can we enrich the debate on holobionts? How can we translate this concept in discoveries that can change farming practices? Our study is filling the gaps of the hologenome theory by showing that certain genes under natural selection and regulating plant microbiota are generalist in response to fungal and bacterial communities.
Structural analysis of the dynamic ribosome-translocon complex
Authors: Lewis Aaron J. O. , Zhong Frank , Keenan Robert J. , Hegde Ramanujan S.
Published: December 2023

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-EM and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61’s lateral gate, widening Sec61’s central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.
Three-step mechanism of promoter escape by RNA polymerase II
Authors: Zhan Yumeng , Grabbe Frauke , Oberbeckmann Elisa , Dienemann Christian , Cramer Patrick
Published: December 2023

The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase (Pol) II and initiation factors are broken to enable promoter escape. Here we reconstitute Pol II promoter escapein vitro, determine high-resolution structures of five transition intermediates, and show that promoter escape occurs in three major steps. First, the growing RNA transcript displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, rewinding of the upstream edge of the growing DNA bubble evicts TFIIA, TFIIB and TBP and repositions parts of TFIIE and TFIIF. Third, binding of DSIF and NELF evicts TFIIE and TFIIH, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of Pol II transcription.
RNA 5ʹ terminal nucleotide determines the strength of the RIG-I/IFN signaling pathway
Authors: Wolczyk Magdalena , Szymanski Jacek , Trus Ivan , Naz Zara , Bolembach Agnieszka , Choudhury Nila Roy , Tame Tola , Könüç Ceren , Nowak Elżbieta , Spanos Christos , Rappsilber Juri , Michlewski Gracjan
Published: December 2023

The interferon (IFN) response is crucial for antiviral activity, but its overstimulation can lead to a wide range of autoimmune disorders. The cytoplasmic pattern recognition receptor RIG-I detects viral RNAs and endogenous polymerase III transcripts carrying a 5′-triphosphate (5′- ppp) or 5′-diphosphate (5′-pp) moiety, triggering an IFN immune response. While many viral RNAs initiate with 5′-ppp-adenosine (5′-pppA) and most endogenous Pol III transcripts in higher eukaryotes start with 5′-ppp-guanosine (5′-pppG), no apparent reason for this bias has been identified so far. Here we demonstrate that RNAs initiating with 5′-pppA trigger a much stronger RIG-I/IFN response than those starting with 5′-pppG. The structures of the matching RNA pairs have been confirmed to be similar, suggesting that functional differences in RIG-I/IFN pathway stimulation cannot be primarily explained by an altered conformation. We show that several GTP-binding proteins interact with 5′-pppG RNAs creating a steric hindrance and protecting these RNAs from activating RIG-I. Our findings suggest that 5′-pppG RNAs may allow some RNA viruses and Pol III transcripts to evade detection by cellular immune sensors. These results provide a new insight into the mechanism of sequence-dependent RIG-I/IFN pathway activation and have significant implications for understanding the antiviral response to RNA viruses and the contribution of Pol III-derived RNAs to autoimmune disorders.
In silicoandin vitrostudies suggest epigallocatechin gallate (EGCG), a polyphenol in green tea, can bind and modulate the aggregation and cytotoxicity of the full-length TDP-43 protein implicated in TDP-43 proteinopathies
Authors: Meshram Vini D. , Balaji Ramkumar , Saravanan Preethi , Subbamanda Yashashwini , Deeksha Waghela , Bajpai Akarsh , Joshi Himanshu , Bhargava Anamika , Patel Basant K.
Published: December 2023

Misfolding and aggregation of TDP-43 protein are implicated in several proteinopathies like ALS and FTLD. Extracellular TDP-43 is also proposed to propagate in a prion-like pathogenic manner to the neighbouring cells. Here, using turbidity and sedimentation assay, we show that a polyphenol in green tea, epigallocatechin gallate (EGCG), can inhibit thein vitroaggregation of the full-length TDP-43 protein. Furthermore, Alexa-Fluor-labelled TDP-43 protein failed to show aggregates in the presence of EGCG in fluorescence microscopy. Also, AFM imaging revealed that EGCG co-incubation with TDP-43 allows formation of only small oligomers in contrast to the larger TDP-43 aggregates formed otherwise. A physical binding of EGCG with TDP-43 was observed using triphenyl tetrazolium chloride (TTC) staining and isothermal titration calorimetry (ITC). ITC also revealed a high-affinity binding site for EGCG on TDP-43 with a Kdvalue of 7.8 µM and a binding free energy of -6.9 kcal/mol. Furthermore,in silicomolecular docking and molecular dynamic simulation (MDS) studies using different available structures of the N-terminal, RRM1-2 and C-terminal domains of TDP-43, predicted a preferable and stable binding of EGCG to the structure of the aggregation prone C-terminal domain (CTD) (PDB ID:7KWZ). Also, EGCG complexed with CTD of TDP-43 yielded a negative ΔG value of -20.29 kcal/mol using MM-PBSA analysis of the MDS data thereby further suggesting a stable complex formation. Also, in MDS, EGCG interacted with the amino acids Phe-313 and Ala-341 of TDP-43, which were previously projected to be important for the recruitment of monomers for the amyloid formation by CTD, thereby suggesting a possible mechanism of EGCG’s inhibition of the TDP-43 aggregation. Notably, while thein vitro-made aggregates of full-length TDP-43 caused mild cytotoxicity to the HEK293 cells, the small oligomers of TDP-43 formed in presence of EGCG did not. In totality, EGCG canin vitrointeract with TDP-43 and inhibit its aggregation, possiblyviainteraction with the amyloidogenic domain, thereby preventing it from assuming cytotoxic conformations. As EGCG is a natural molecule, it could be relevant to the therapeutic quest against the TDP-43 proteinopathies.
Specific inhibition and disinhibition in the higher-order structure of a cortical connectome
Authors: Reimann Michael W. , Santander Daniela Egas , Ecker András , Muller Eilif B.
Published: December 2023

Neurons are thought to act as parts of assemblies with strong internal excitatory connectivity. Conversely, inhibition is often reduced to blanket inhibition with no targeting specificity. We analyzed the structure of excitation and inhibition in the MICrONSmm3dataset, an electron microscopic reconstruction of a piece of cortical tissue. We found that excitation was structured around a feed-forward flow in large non-random neuron motifs with a structure of information flow from a small number of sources to a larger number of potential targets. Inhibitory neurons connected with neurons in specific sequential positions of these motifs, implementing targeted and symmetrical competition between them. None of these trends are detectable in only pairwise connectivity, demonstrating that inhibition is structured by these large motifs. While descriptions of inhibition in cortical circuits range from non-specific blanket-inhibition to targeted, our results describe a form of targeting specificity existing in the higher-order structure of the connectome. These findings have important implications for the role of inhibition in learning and synaptic plasticity.
Cyfip2 controls the acoustic startle threshold through FMRP, actin polymerization, and GABABreceptor function
Authors: Deslauriers Jacob C. , Ghotkar Rohit P. , Russ Lindsey A. , Jarman Jordan A. , Martin Rubia M. , Tippett Rachel G. , Sumathipala Sureni H. , Burton Derek F. , Cole D. Chris , Marsden Kurt C.
Published: December 2023

Animals process a constant stream of sensory input, and to survive they must detect and respond to dangerous stimuli while ignoring innocuous or irrelevant ones. Behavioral responses are elicited when certain properties of a stimulus such as its intensity or size reach a critical value, and such behavioral thresholds can be a simple and effective mechanism to filter sensory information. For example, the acoustic startle response is a conserved and stereotyped defensive behavior induced by sudden loud sounds, but dysregulation of the threshold to initiate this behavior can result in startle hypersensitivity that is associated with sensory processing disorders including schizophrenia and autism. Through a previous forward genetic screen for regulators of the startle threshold a nonsense mutation inCytoplasmic Fragile X Messenger Ribonucleoprotein (FMRP)-interacting protein 2(cyfip2) was found that causes startle hypersensitivity in zebrafish larvae, but the molecular mechanisms by which Cyfip2 establishes the acoustic startle threshold are unknown. Here we used conditional transgenic rescue and CRISPR/Cas9 to determine that Cyfip2 acts though both Rac1 and FMRP pathways, but not the closely related FXR1 or FXR2, to establish the acoustic startle threshold during early neurodevelopment. To identify proteins and pathways that may be downstream effectors of Rac1 and FMRP, we performed a candidate-based drug screen that indicated that Cyfip2 can also act acutely to maintain the startle threshold branched actin polymerization and N-methyl D-aspartate receptors (NMDARs). To complement this approach, we used unbiased discovery proteomics to determine that loss of Cyfip2 alters cytoskeletal and extracellular matrix components while also disrupting oxidative phosphorylation and GABA receptor signaling. Finally, we functionally validated our proteomics findings by showing that activating GABABreceptors, which like NMDARs are also FMRP targets, restores normal startle sensitivity incyfip2mutants. Together, these data reveal multiple mechanisms by which Cyfip2 regulates excitatory/inhibitory balance in the startle circuit to control the processing of acoustic information.
Homological landscape of human brain functional sub-circuits
Authors: Duong-Tran Duy , Kaufmann Ralph , Chen Jiong , Wang Xuan , Garai Sumita , Xu Frederick , Bao Jingxuan , Amico Enrico , Kaplan Alan David , Petri Giovanni , Goni Joaquin , Zhao Yize , Shen Li
Published: December 2023

Human whole-brain functional connectivity networks have been shown to exhibit both local/quasilocal (e.g., set of functional sub-circuits induced by node or edge attributes) and non-local (e.g., higher-order functional coordination patterns) properties. Nonetheless, the non-local properties of topological strata induced by local/quasilocal functional sub-circuits have yet to be addressed. To that end, we proposed a homological formalism that enables the quantification of higher-order characteristics of human brain functional sub-circuits. Our results indicated that each homological order uniquely unravels diverse, complementary properties of human brain functional sub-circuits. Noticeably, theH1homological distance between rest and motor task were observed at both whole-brain and sub-circuit consolidated level which suggested the self-similarity property of human brain functional connectivity unraveled by homological kernel. Furthermore, at the whole-brain level, the rest-task differentiation was found to be most prominent between rest and different tasks at different homological orders: i) Emotion task (H0), ii) Motor task (H1), and iii) Working memory task (H2). At the functional sub-circuit level, the rest-task functional dichotomy of default mode network is found to be mostly prominent at the first and second homological scaffolds. Also at such scale, we found that the limbic network plays a significant role in homological reconfiguration across both task- and subject-domain which sheds light to subsequent investigations on the complex neuro-physiological role of such network. From a wider perspective, our formalism can be applied, beyond brain connectomics, to study non-localized coordination patterns of localized structures stretching across complex network fibers.
OmniClustifyXMBD: Uncover putative cell states within multiple single-cell omics datasets
Authors: Yang Fan , Zhou Yitao , Zeng Feng
Published: December 2023

Clustering plays a pivotal role in characterizing cell states in single-cell omics data. Nonetheless, there is a noticeable gap in clustering algorithms tailored for unveiling putative cell states across datasets containing samples with diverse phenotypes. To bridge this gap, we implement an innovative method termed OmniClustifyXMBD, which integrates adaptive signal isolation with cell clustering. The adaptive signal isolation effectively disentangles gene expression variations linked to distinct factors within individual cells. This separation restores cells to their inherent states, free from external influences. Concurrently, a clustering algorithm built upon a deep variational Gaussian mixture model is devised to identify these putative cell states. Experiments showcase the effectiveness of OmniClustifyXMBDin identifying putative cell states while minimizing the influence of various undesired variations, including batch effects and random inter-sample differences. Moreover, OmniClustifyXMBDdemonstrates robustness in its results across different clustering parameters.
Pervasive selective sweeps across human gut microbiomes
Authors: Wolff Richard , Garud Nandita R.
Published: December 2023

The human gut microbiome is composed of a highly diverse consortia of species which are continually evolving within and across hosts. The ability to identify adaptations common to many human gut microbiomes would not only reveal shared selection pressures across hosts, but also key drivers of functional differentiation of the microbiome that may affect community structure and host traits. However, to date there has not been a systematic scan for adaptations that have spread across human gut microbiomes. Here, we develop a novel selection scan statistic named the integrated Linkage Disequilibrium Score (iLDS) that can detect the spread of adaptive haplotypes across host microbiomes via migration and horizontal gene transfer. Specifically, iLDS leverages signals of hitchhiking of deleterious variants with the beneficial variant. Application of the statistic to∼30 of the most prevalent commensal gut species from 24 populations around the world revealed more than 300 selective sweeps across species. We find an enrichment for selective sweeps at loci involved in carbohydrate metabolism—potentially indicative of adaptation to features of host diet—and we find that the targets of selection significantly differ between Westernized and non-Westernized populations. Underscoring the potential role of diet in driving selection, we find a selective sweep absent from non-Westernized populations but ubiquitous in Westernized populations at a locus known to be involved in the metabolism of maltodextrin, a synthetic starch that has recently become a widespread component of Western diets. In summary, we demonstrate that selective sweeps across host microbiomes are a common feature of the evolution of the human gut microbiome, and that targets of selection may be strongly impacted by host diet.