bluuebunny/crossref_metadata_2025 huggingface dataset:
Structures of Langya virus fusion protein ectodomain in pre and post fusion conformation
Authors: May Aaron J. , Pothula Karunakar Reddy , Janowska Katarzyna , Acharya Priyamvada
Published: April 2023

Langya virus (LayV) is a paramyxovirus in theHenipavirusgenus, closely related to the deadly Nipah and Hendra viruses, that was identified in August 2022 through disease surveillance following animal exposure in eastern China. Paramyxoviruses present two glycoproteins on their surface, known as attachment and fusion proteins, that mediate entry into cells and constitute the primary antigenic targets for immune response. Here, we determine cryo-EM structures of the uncleaved LayV fusion protein (F) ectodomain in pre- and post-fusion conformations. The LayV-F protein exhibits pre- and post-fusion architectures that, despite being highly conserved across paramyxoviruses, show differences in their surface properties, in particular at the apex of the prefusion trimer, that may contribute to antigenic variability. While dramatic conformational changes were visualized between the pre- and post-fusion forms of the LayV-F protein, several domains remained invariant, held together by highly conserved disulfides. The LayV-F fusion peptide is buried within a highly conserved, hydrophobic, interprotomer pocket in the pre-fusion state and is notably less flexible than the rest of the protein, highlighting its “spring-loaded” state and suggesting that the mechanism of pre-to-post transition must involve perturbations to the pocket and release of the fusion peptide. Together, these results offer a structural basis for how the Langya virus fusion protein compares to its Henipavirus relatives and propose a mechanism for the initial step of pre- to post-fusion conversion that may apply more broadly to paramyxoviruses.

Importance

The Henipavirus genus is quickly expanding into new animal hosts and geographic locations. This study compares the structure and antigenicity of the Langya virus fusion protein to other henipaviruses, which has important vaccine or therapeutic development implications. Furthermore, the study proposes a new mechanism to explain the early steps of the fusion initiation process that can be more broadly applied to theParamyxoviridaefamily.
Organic matter influence on ooid formation: New insights into classic examples (Great Salt Lake, USA; Triassic Germanic Basin, Germany)
Authors: Pei Yu , Suarez-Gonzalez Pablo , Duda Jan-Peter , Reitner Joachim
Published: April 2023

Ooids are particles composed of a tangential or radial cortex growing around a nucleus. They are common in carbonate deposits of almost any geological age and provide insights into environmental conditions. However, abiotic or biotic factors influencing their formation remain unclear. This study aims to advance our understanding of ooid formation with a multi- analytical approach (e.g., FE-SEM, Raman spectroscopy, μ-XRF) to classic examples from Great Salt Lake (GSL; USA) and the Lower Triassic Germanic Basin (GB; Germany). Both deposits represent hypersaline shallow-water environments where ooids are closely associated with microbial mats. GSL ooids are dominantly 0.2–1 mm in size, ellipsoidal to subspherical in shape, composed of aragonite, and contain organic matter (OM). GB ooids are mainly ≤4 mm in size, spherical to subspherical in shape, composed of calcite, and currently contain little OM. Despite the differences, both ooids have the same cortex structures, likely reflecting similar formation processes. Some GSL ooids formed around detrital grains while others exhibit micritic particles in their nuclei. In GB ooids, detrital nuclei are rare, despite the abundance of siliciclastic particles of various sizes in the host rocks. GB deposits also include “compound ooids”, i.e., adjacent ooids that coalesced with each other during growth, suggesting staticin-situdevelopment, which is supported by the lack of detrital grains as nuclei. GB ooids also grew into laminated microbial crusts with identical microstructures, further indicating a static formation. Such microbial crusts typically form through mineral precipitation associated with OM (e.g., extracellular polymeric substances), suggesting a similar formation pathway for ooids. The inferred key-role of OM is further supported by features in radial ooids from the GSL, which commonly exhibit, from their nuclei towards their surface, increasing OM contents and decreasing calcification.
Kir7.1 is the physiological target for hormones and steroids that regulate uteroplacental function
Authors: Haoui Monika , Vergara Citlalli , Lishko Polina V.
Published: April 2023

Preterm birth is a multifactorial syndrome that is detrimental to the well-being of both the mother and the newborn. During normal gestation, the myometrium is maintained in a quiescent state by the action of progesterone. As a steroid hormone, progesterone is thought to modify uterine and placental morphology by altering gene expression, but a nongenomic mode of action has long been suspected. Here we reveal that progesterone activates both human and murine inwardly rectifying potassium channel Kir7.1, which is expressed in mammalian myometrial smooth muscle and placental pericytes during late gestation. Kir7.1 is also activated by compounds used to prevent premature labor, including the progestogens 17-alpha-hydroxyprogesterone caproate and dydrogesterone, revealing an unexpected mode of action for these drugs. Our results reveal that Kir7.1 is the molecular target of a number of endogenous and synthetic steroids that control uterine excitability and placental function, and is therefore a promising therapeutic target to control utero-placental physiology and support healthy pregnancy.
Flexibility and sensitivity in gene regulation out of equilibrium
Authors: Mahdavi Sara , Salmon Gabriel L. , Daghlian Patill , Garcia Hernan G. , Phillips Rob
Published: April 2023

Cells adapt to environments and tune gene expression by controlling the concentrations of proteins and their kinetics in regulatory networks. In both eukaryotes and prokaryotes, experiments and theory increasingly attest that these networks can and do consume bio-chemical energy. How does this dissipation enable cellular behaviors unobtainable in equilibrium? This open question demands quantitative models that transcend thermodynamic equilibrium. Here we study the control of a simple, ubiquitous gene regulatory motif to explore the consequences of departing equilibrium in kinetic cycles. Employing graph theory, we find that dissipation unlocks nonmonotonicity and enhanced sensitivity of gene expression with respect to a transcription factor’s concentration. These features allow a single transcription factor to act as both a repressor and activator at different levels or achieve outputs with multiple concentration regions of locally-enhanced sensitivity. We systematically dissect how energetically-driving individual transitions within regulatory networks, or pairs of transitions, generates more adjustable and sensitive phenotypic responses. Our findings quantify necessary conditions and detectable consequences of energy expenditure. These richer mathematical behaviors—feasibly accessed using biological energy budgets and rates—may empower cells to accomplish sophisticated regulation with simpler architectures than those required at equilibrium.

Significance Statement

Growing theoretical and experimental evidence demonstrates that cells can (and do) spend biochemical energy while regulating their genes. Here we explore the impact of departing from equilibrium in simple regulatory cycles, and learn that beyond increasing sensitivity, dissipation can unlock more flexible input-output behaviors that are otherwise forbidden without spending energy. These more complex behaviors could enable cells to perform more sophisticated functions using simpler systems than those needed at equilibrium.
Modelling human KCNT1-epilepsy inDrosophila: a seizure phenotype and drug responses
Authors: Hussain Rashid , Lim Chiao Xin , Shaukat Zeeshan , Islam Anowarul , Caseley Emily A. , Lippiat Jonathan D. , Rychkov Grigori Y. , Ricos Michael G. , Dibbens Leanne M.
Published: April 2023

Mutations in theKCNT1potassium channel cause severe forms of epilepsy which are resistant to current treatments.In vitrostudies have shown thatKCNT1-epilepsy mutations are gain of function, significantly increasing K+current amplitudes. To investigate ifDrosophilacan be used to model humanKCNT1epilepsy, we generatedDrosophila melanogasterlines carrying humanKCNT1with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which was sensitive to drugs currently used to treat patients withKCNT1-epilepsy. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows thatDrosophilacan be used to model humanKCNT1-epilepsy and potentially used as a tool to assess new treatments forKCNT1epilepsy.
Universal baleen whale microsatellite panel for individual identification and power to detect parentage
Authors: Suárez-Menéndez Marcos , Bérubé Martine , Bachmann Lutz , Best Peter , Heide-Jørgensen Mads Peter , Lesage Veronique , Oosting Tom , Prieto Rui , Ramp Christian , Robbins Jooke , Sears Richard , Silva Mónica A. , Tollis Marc , Vermeulen Els , Víkingsson Gísli A. , Wiig Øystein , Palsbøll Per J.
Published: April 2023

Highly polymorphic single tandem repeat loci (STR, also known as microsatellite loci) remain a familiar, cost efficient class of markers for genetic analyses in ecology, behavior and conservation. We characterize a new universal set of ten STR loci (from 28 potential candidate loci) in seven baleen whale species, which are optimized for PCR amplification in two multiplex reactions along with a Y chromosome marker for sex determination. The optimized, universal set of STR loci provides an ideal starting point for new studies in baleen whales aimed at individual-based and population genetic studies, and facilitates data sharing among research groups. Data from the new STR loci were combined with genotypes from other published STR loci to assess the power to assign parentage (paternity) using exclusion in four species: fin whales, humpback whales, blue whales and bowhead whales. We argue that parentage studies should present a power analysis to demonstrate that the specific data are sufficiently informative to assign parentage with statistical rigor.
Spatial transcriptome of developmental mouse brain reveals temporal dynamics of gene expressions and heterogeneity of the claustrum
Authors: Hara Yuichiro , Kumamoto Takuma , Yoshizawa-Sugata Naoko , Hirai Kumiko , Xianghe Song , Kawaji Hideya , Ohtaka-Maruyama Chiaki
Published: April 2023

During the development of the mammalian cerebral cortex, numerous neurons are arranged in a six-layer structure with an inside-out fashion to form the neocortex and wire neural circuits. This process includes cell proliferation, differentiation, migration, and maturation, supported by precise genetic regulation. To understand this sequence of processes at the cellular and molecular levels, it is necessary to characterize the fundamental anatomical structures by gene expression. However, markers established in the adult brain sometimes behave differently in the fetal brain, actively changing during development. Spatial transcriptomes yield genome-wide gene expression profiles from each spot patterned on tissue sections, capturing RNA molecules from fresh-frozen sections and enabling sequencing analysis while preserving spatial information. However, a deeper understanding of this data requires computational estimation, including integration with single-cell transcriptome data and aggregation of spots on the single-cell cluster level. The application of such analysis to biomarker discovery has only begun recently, and its application to the developing fetal brain is largely unexplored. In this study, we performed a spatial transcriptome analysis of the developing mouse brain to investigate the spatiotemporal regulation of gene expression during development. Using these data, we conducted an integrated study with publicly available mouse data sets, the adult brain’s spatial transcriptome, and the fetal brain’s single-cell transcriptome. Our data-driven analysis identified novel molecular markers of the choroid plexus, piriform cortex, thalamus, and claustrum. In addition, we revealed that the internal structure of the embryonic claustrum is composed of heterogeneous cell populations.
SorLA restricts TNFα release from microglia to shape glioma-supportive brain microenvironment
Authors: Kaminska Paulina , Ovesen Peter L. , Jakiel Mateusz , Obrebski Tomasz , Schmidt Vanessa , Bieniek Magdalena , Anink Jasper , Paterczyk Bohdan , Draminski Michal , Jensen Anne Mette Gissel , Piatek Sylwia , Andersen Olav M. , Aronica Eleonora , Willnow Thomas E. , Kaminska Bozena , Dabrowski Michal J. , Malik Anna R.
Published: April 2023

SorLA, encoded by the geneSORL1, is an intracellular sorting receptor of the VPS10P domain receptor gene family. Although SorLA is best recognized for its ability to shuttle target proteins between intracellular compartments in neurons, recent data suggest that also its microglial expression can be of high relevance for the pathogenesis of brain diseases, including glioblastoma (GBM). Here we interrogated the impact of SorLA on the functional properties of glioma-associated microglia and macrophages (GAMs). In the GBM microenvironment, GAMs are re-programmed and in turn lose the ability to elicit anti-tumor responses. Instead, they acquire glioma-supporting phenotype, which is a key mechanism promoting glioma progression. Our analysis of scRNA-seq data from GBM patients revealed that the pro-tumorigenic and pro-inflammatory properties of GAMs are linked to high and lowSORL1expression, respectively. Using cell models, we confirm that SorLA levels are differentially regulated by the presence of glioma cells and by inflammatory cues. We further show that SorLA acts as a sorting receptor for the pro-inflammatory cytokine TNFα to restrain its secretion from microglia. As a consequence, loss of SorLA enhanced the pro-inflammatory potential of microglia, having a remarkable impact on glioma progression. In a murine model of glioma, SorLA-deficient mice develop smaller tumors and show hallmarks of anti-tumor response including altered microglia morphology, enhanced necroptosis, and massive neutrophil influx into the tumor parenchyma. Our findings indicate that SorLA is a key player in shaping the phenotype of GAMs, and its depletion can unlock an anti-tumor response.

Significance statement

Our study provides insight into the mechanisms shaping the tumor microenvironment in glioblastoma (GBM), the most prevalent and aggressive brain malignancy in adults. Poor prognosis in GBM largely results from the properties of the glioma milieu that blocks the anti-tumor response. We show that SorLA restricts release of the pro-inflammatory cytokine TNFα from microglia, thereby hampering their anti-glioma response. SorLA depletion reinforces the pro-inflammatory properties of tumor microenvironment and inhibits glioma growth. These findings have significant implications for our understanding of glioma biology, indicating SorLA-TNFα interaction as a potential target in GBM therapies. They also offer a new perspective on SorLA activities in microglia which emerge as highly relevant not only for the pathogenesis GBM, but also of other brain diseases such as Alzheimer’s disease.
Structural bases for the Charcot–Marie–Tooth disease induced by single amino acid substitutions of myelin protein zero
Authors: Sakakura Masayoshi , Tanabe Mikio , Mori Masaki , Takahashi Hideo , Mio Kazuhiro
Published: April 2023

Myelin protein zero (MPZ or P0) is a major transmembrane protein expressed in peripheral compact myelin and functions to glue membranes to form multiple layered membranes characteristic of myelin. Intermembrane adhesion is mediated by homophilic interactions between the extracellular domains (ECDs) of MPZ molecules. Single amino acid substitutions in an ECD cause demyelinating neuropathy, known as Charcot–Marie–Tooth disease (CMT); however, the mechanisms by which such substitutions induce the disease are not well understood. To address this issue, we constructed a novel assay to evaluate the membrane-stacking activity of ECD using ECD-immobilized nanodiscs. Using this novel “nanomyelin” system, we found that octameric (8-meric) ECDs with a stacked-rings-like configuration are responsible for membrane adhesion. Two inter-ECD interactions,cisand head-to-head, are essential to constituting the 8-mer and, consequently, to gluing the membranes. This result was further reinforced by the observation that the CMT-related N87H substitution at thecisinterface abolished membrane-adhesion activity. In contrast, the CMT-related D32G and E68V variants of ECD retained membrane-stacking activity, whereas their thermal stability was reduced compared to that of the WT. Reduced thermal stability may lead to impairment of the long-term stability of ECD and the layered membranes of myelin.
SUBTLE: An unsupervised platform with temporal link embedding that maps animal behavior
Authors: Kwon Jea , Kim Sunpil , Kim Dong-Kyum , Joo Jinhyeong , Kim SoHyung , Cha Meeyoung , Lee C. Justin
Published: April 2023

While huge strides have recently been made in language-based machine learning, the ability of artificial systems to comprehend the sequences that comprise animal behavior has been lagging behind. In contrast, humans instinctively recognize behaviors by finding similarities in behavioral sequences. Here, we develop an unsupervised behavior-mapping framework, SUBTLE (spectrogram-UMAP-based temporal-link embedding), to capture comparable behavioral repertoires from 3D action skeletons. To find the best embedding method, we devise a temporal proximity index as a metric to gauge temporal representation in the behavioral embedding space. The method achieves the best performance compared to current embedding strategies. Its spectrogram-based UMAP clustering not only identifies subtle inter-group differences but also matches human-annotated labels. SUBTLE framework automates the tasks of both identifying behavioral repertoires like walking, grooming, standing, and rearing, and profiling individual behavior signatures like subtle inter-group differences by age. SUBTLE highlights the importance of temporal representation in the behavioral embedding space for human-like behavioral categorization.

One Sentence Summary

Unsupervised behavior-mapping from 3D action skeletons achieves superior performance, captures behavioral repertoires, and identifies inter-group differences, emphasizing how temporal representation is critical in the behavioral embedding space.
Identification of differentially recognized T cell epitopes in the spectrum ofMtbinfection
Authors: Panda Sudhasini , Morgan Jeffrey , Cheng Catherine , Saito Mayuko , Gilman Robert H. , Ciobanu Nelly , Crudu Valeriu , Catanzaro Donald G , Catanzaro Antonino , Rodwell Timothy , Perera Judy S.B. , Chathuranga Teshan , Gunasena Bandu , DeSilva Aruna D. , Peters Bjoern , Sette Alessandro , Lindestam Arlehamn Cecilia S.
Published: April 2023

Tuberculosis caused byMycobacterium tuberculosisis one of the leading causes of death from a single infectious agent. Identifying dominant epitopes and comparing their reactivity in different tuberculosis (TB) infection states can help design diagnostics and vaccines. We performed a proteome-wide screen of 20,610Mtbderived peptides in 21 Active TB (ATB) patients 3-4 months post-diagnosis of pulmonary TB (mid-treatment) using an IFNγ and IL-17 Fluorospot assay. Responses were mediated exclusively by IFNγ and identified a total of 137 unique epitopes, with each patient recognizing, on average, 8 individual epitopes and 22 epitopes (16%) recognized by 2 or more participants. Responses were predominantly directed against antigens part of the cell wall and cell processes category. Testing 517 peptides spanning TB vaccine candidates and ESAT- 6 and CFP10 antigens also revealed differential recognition between ATB participants mid-treatment and healthy IGRA+ participants of several vaccine antigens. An ATB-specific peptide pool consisting of epitopes exclusively recognized by participants mid-treatment, allowed distinguishing participants with active pulmonary TB from healthy interferon-gamma release assay (IGRA)+/- participants from diverse geographical locations. Analysis of longitudinal samples indicated decreased reactivity during treatment for pulmonary TB. Together, these results show that a proteome-wide screen of T cell reactivity identifies epitopes and antigens that are differentially recognized depending on theMtbinfection stage. These have potential use in developing diagnostics and vaccine candidates and measuring correlates of protection.
Comparative gene expression analysis of differentiated terminal and lateral haustoria of the obligate root parasitic plantPhelipanche ramosa(Orobanchaceae)
Authors: Brun Guillaume , Leman Julia K. H. , Wicke Susann
Published: April 2023

Abstract

Branched broomrape (Phelipanche ramosa, Orobanchaceae) is the most important parasitic weed of oilseed and vegetable crops in Europe. Deciphering the parasite’s basic biology and genetics underpinning adaptations to parasitism are essential for effective weed management. Here, we compared the gene expression profiles of differentiated terminal haustoria, formed immediately after the germination ofP. ramosawith those of the parasite’s adventitious roots and lateral haustoria, which develop on the latter as it grows. Principal component analysis and functional annotation of differentially expressed genes indicate a greater transcriptional similarity between adventitious roots and lateral haustoria compared to terminal haustoria. Genes involved in hydrogen peroxide catabolic processes and mucilage metabolic processes were more abundant in lateral haustoria compared to adventitious roots, indicating that secondary host attachment involves secretion of adhesive compounds, cell wall modification, and a termination of the developmental program associated with root growth. In terminal haustoria, phytohormonal signaling pathways for post-germination growth and meristem development were the most prominent expression profile difference to lateral haustoria. Besides this, wide similarity of expressed genes between lateral and terminal haustoria suggest overlapping pathways underlying haustorium differentiation and a conserved functional relevance of both haustoria types at maturity. Together, our study provides first insights into the transcriptional landscapes of the primary organs securingP.ramosa’s nutritional success. However, more research is needed to investigate whether the parasite’s various haustorium types differ in their sensitivities to environmental stimuli and whether transcriptional divergence in terminal and lateral haustoria ofP. ramosareflect differences regarding their developmental origin.
Impact of light on task-evoked pupil responses during cognitive tasks
Authors: Campbell Islay , Beckers Elise , Sharifpour Roya , Berger Alexandre , Paparella Ilenia , Balda Aizpurua Jose Fermin , Koshmanova Ekaterina , Mortazavi Nasrin , Sherif Siya , Vandewalle Gilles
Published: April 2023

Light has many non-image-forming functions including modulation of pupil size and stimulation of alertness and cognition. Part of these non-image-forming effects may be mediated by the brainstem locus coeruleus. The processing of sensory inputs can be associated with a transient pupil dilation that is likely driven in part by the phasic activity of the locus coeruleus. Here, we aimed to characterise the task-evoked pupil response associated with auditory inputs under different light levels and across two cognitive tasks. We continuously monitored the pupil of 20 young healthy participants (24.05y ±4.0; 14 women) while they completed an attentional and an emotional auditory task whilst exposed to repeated 30-to-40s-blocks of light interleaved with darkness periods. Blocks could either consist of monochromatic orange light [0.16 melanopic Equivalent Daylight Illuminance (EDI) lux] or blue-enriched white light of three different levels [37, 92, 190 melanopic EDI lux; 6500K]. For the analysis 15 and then 14 participants were included in the attentional and emotional tasks respectively. Generalized Linear Mixed Models showed a significant main effect of light level on the task-evoked pupil responses triggered by the attentional and emotional tasks (p≤.0001). The impact of light was different for the target vs. non-target stimulus of the attentional task but was not different for the emotional and neutral stimulus of the emotional task. Despite a smaller sustained pupil size during brighter light blocks, a higher light level triggers a stronger task-evoked pupil response to auditory stimulation, presumably through the recruitment of the locus coeruleus.
Multimodal mismatch responses in associative but not primary visual cortex support hierarchical predictive coding in cortical networks
Authors: Van Derveer Alice B , Ross Jordan M. , Hamm Jordan P.
Published: April 2023

A key function of the mammalian neocortex is to process sensory data in the context of current and past stimuli. Primary sensory cortices, such as V1, respond weakly to stimuli that typical in their context but strongly to novel stimuli, an effect known as “deviance detection”. How deviance detection occurs in associative cortical regions that are downstream of V1 is not well-understood. Here we investigated parietal associative area (PTLp) responses to auditory, visual, and audio-visual mismatches with two-photon calcium imaging and local field potential recordings. We employed basic unisensory auditory and visual oddball paradigms as well as a novel multisensory oddball paradigm, involving typical parings (VaAc or VbAd) presented at p=.88 with rare “deviant” pairings (e.g. VaAd or VbAc) presented at p=.12. We found that PTLp displayed robust deviance detection responses to auditory-visual mismatches, both in individual neurons and in population theta and gamma-band oscillations. In contrast, V1 neurons displayed deviance detection only to visual deviants in a unisensory context, but not to auditory or auditory-visual mismatches. Taken together, these results accord with a predictive processing framework for cortical responses, wherein modality specific prediction errors (i.e. deviance detection responses) are computed in functionally specified cortical areas and feed-forward to update higher brain regions.
Capsules and their traits shape phage susceptibility and plasmid conjugation efficiency
Authors: Haudiquet Matthieu , Nucci Amandine , Bris Julie Le , Bonnin Rémy A. , Domingo-Calap Pilar , Rendueles Olaya , Rocha Eduardo P.C.
Published: April 2023

Bacterial evolution is affected by mobile genetic elements such as phages and conjugative plasmids, which may provide novel adaptive traits but also incur in fitness costs. Infection by these elements is affected by the bacterial capsule. Yet, its importance has been difficult to quantify and characterise because of the high diversity of bacterial genomes regarding confounding mechanisms such as anti-viral systems. We swapped capsule loci betweenKlebsiella pneumoniaestrains to quantify their effect on transfer of conjugative plasmids and phages independently of the genetic background. Capsule swaps systematically invert phage susceptibility, demonstrating that serotypes are key determinants of phage infection. Capsule types also affect conjugation efficiency in both donor and recipient cells depending on the serotype, a mechanism shaped by the capsule volume and depending on the structure of the conjugative pilus. Comparative genomics confirmed that more permissive serotypes in the lab correspond to the strains acquiring more conjugative plasmids in nature. The pili least sensitive to capsules (F-like) are also the most frequent in the species’ plasmids, and are the only ones associated with both antibiotic resistance and virulence factors, driving the convergence between virulence and antibiotics resistance in the population. These results show how the traits of cellular envelopes define slow and fast lanes of infection by mobile genetic elements, with implications for population dynamics and horizontal gene transfer.

Graphical abstract
Chromatin context-dependent regulation and epigenetic manipulation of prime editing
Authors: Li Xiaoyi , Chen Wei , Martin Beth K. , Calderon Diego , Lee Choli , Choi Junhong , Chardon Florence M. , McDiarmid Troy , Kim Haedong , Lalanne Jean-Benoît , Nathans Jenny F. , Shendure Jay
Published: April 2023

Prime editing is a powerful means of introducing precise changes to specific locations in mammalian genomes. However, the widely varying efficiency of prime editing across target sites of interest has limited its adoption in the context of both basic research and clinical settings. Here, we set out to exhaustively characterize the impact of thecis-chromatin environment on prime editing efficiency. Using a newly developed and highly sensitive method for mapping the genomic locations of a randomly integrated “sensor”, we identify specific epigenetic features that strongly correlate with the highly variable efficiency of prime editing across different genomic locations. Next, to assess the interaction oftrans-acting factors with thecis-chromatin environment, we develop and apply a pooled genetic screening approach with which the impact of knocking down various DNA repair factors on prime editing efficiency can be stratified bycis-chromatin context. Finally, we demonstrate that we can dramatically modulate the efficiency of prime editing through epigenome editing,i.e.altering chromatin state in a locus-specific manner in order to increase or decrease the efficiency of prime editing at a target site. Looking forward, we envision that the insights and tools described here will broaden the range of both basic research and therapeutic contexts in which prime editing is useful.
Functional whole genome screen of nutrient-starvedMycobacterium tuberculosisidentifies genes involved in antibiotic tolerance
Authors: Matern William M. , Harris Harley T. , Danchik Carina , McDonald Marissa , Patel Gopi , Srivastava Aashish , Ioerger Thomas R. , Bader Joel S. , Karakousis Petros C.
Published: April 2023

Mycobacterium tuberculosis(Mtb), the causative agent of tuberculosis (TB), poses a global health challenge and is responsible for over a million deaths each year. Current treatment is lengthy and complex, and new, abbreviated regimens are urgently needed.Mtbadapts to nutrient starvation, a condition experienced during host infection, by shifting its metabolism and becoming tolerant to the killing activity of bactericidal antibiotics. An improved understanding of the mechanisms mediating antibiotic tolerance inMtbcan serve as the basis for developing more effective therapies. We performed a forward genetic screen to identify candidateMtbgenes involved in tolerance to the two key first-line antibiotics, rifampin and isoniazid, under nutrient-rich and nutrient-starved conditions. In nutrient-rich conditions, we found 220 mutants with differential antibiotic susceptibility (218 in the rifampin screen and 2 in the isoniazid screen). FollowingMtbadaptation to nutrient starvation, 82 mutants showed differential antibiotic susceptibility (80 in the rifampin screen and 2 in the isoniazid screen). Using targeted mutagenesis, we validated the rifampin-hypersusceptible phenotype under nutrient starvation inMtbmutants lacking the following genes:ercc3,moeA1,rv0049, andrv2179c. These findings shed light on potential therapeutic targets, which could help shorten the duration and complexity of antitubercular regimens.

Importance

Treatment ofMtbinfection requires a long course of combination antibiotics, likely due to subpopulations of tolerant bacteria exhibiting decreased susceptibility to antibiotics. Identifying and characterizing the genetic pathways involved in antibiotic tolerance is expected to yield therapeutic targets for the development of novel TB treatment-shortening regimens.
Functional transcriptional signatures for tumor-type-agnostic phenotype prediction
Authors: Weistuch Corey , Murgas Kevin A. , Zhu Jiening , Norton Larry , Dill Ken A. , Tannenbaum Allen R. , Deasy Joseph O.
Published: April 2023

Cancer transcriptional patterns exhibit both shared and unique features across diverse cancer types, but whether these patterns are sufficient to characterize the full breadth of tumor phenotype heterogeneity remains an open question. We hypothesized that cancer transcriptional diversity mirrors patterns in normal tissues optimized for distinct functional tasks. Starting with normal tissue transcriptomic profiles, we use non-negative matrix factorization to derive six distinct transcriptomic phenotypes, called archetypes, which combine to describe both normal tissue patterns and variations across a broad spectrum of malignancies. We show that differential enrichment of these signatures correlates with key tumor characteristics, including overall patient survival and drug sensitivity, independent of clinically actionable DNA alterations. Additionally, we show that in HR+/HER2-breast cancers, metastatic tumors adopt transcriptomic signatures consistent with the invaded tissue. Broadly, our findings suggest that cancer often arrogates normal tissue transcriptomic characteristics as a component of both malignant progression and drug response. This quantitative framework provides a strategy for connecting the diversity of cancer phenotypes and could potentially help manage individual patients.
Differential neural circuit vulnerability to β-amyloid and tau pathologies in novel Alzheimer’s disease mice
Authors: Capilla-López Maria Dolores , Deprada Angel , Andrade-Talavera Yuniesky , Martínez-Gallego Irene , Coatl-Cuaya Heriberto , Rodríguez-Alvarez José , Rodríguez-Moreno Antonio , Parra-Damas Arnaldo , Saura Carlos A.
Published: April 2023

Alzheimer’s disease (AD) progresses with memory loss and neuropsychiatric symptoms associated with cell specific vulnerability in memory- and emotion-related neural circuits. Neuropathological and synaptic changes are key factors influencing the clinical progression to dementia, but how they cooperate to cause memory and emotional disturbances is largely unknown. Here, we employed pathological, behavioral, expansion microscopy, electrophysiology and transcriptomic approaches to evaluate the effects of amyloid-β (Aβ) and tau on neuropathological progression, synaptic function, and memory and emotional symptoms in amyloid precursor protein (APP), Tau and double novel APP/Tau transgenic mice expressing the mutant human amyloid precursor protein (APPSw,Ind) and/or microtubule-associated protein tau (MAPT) in excitatory neurons. APP/Tau mice of both sexes show spatial learning and memory deficits associated with synaptic tau accumulation and reduced synaptic proteins and neurotransmission in the hippocampus. By contrast, male and female APP/Tau mice exhibit innate anxious behavior and impaired fear memory extinction linked to Aβ pathology and with absence of synaptic tau in the basolateral amygdala (BLA). Intriguingly, APP/Tau mice show NMDA-dependent long-term potentiation (LTP) deficits in the hippocampus but not in the amygdala. Bulk RNA sequencing reveals region-specific but also common transcriptional changes in response to Aβ/tau pathology, including downregulation of synapse transmission and ion channel activity genes. Importantly, we detected 65 orthologs of human AD risk genes identified in GWAS (e.g.,APOE,BIN1,CD33,CLU,PICALM,PLCG2,PTK2B,TREM2,SORL1,USP6NL) differentially expressed in the hippocampus and/or BLA of APP/Tau mice, indicating that this APP/Tau model exhibits transcriptional alterations linked to known molecular determinants of AD development. In conclusion, simultaneous development of Aβ and tau neuropathologies in this double APP/Tau transgenic mouse model reproduces synaptic, behavioral, and molecular alterations associated with AD pathophysiology in a region-specific manner. Our findings highlight region-specific pathological effects of Aβ and tau in excitatory neuronal circuits mediating emotional and memory processing, providing evidence that both factors and their molecular cascades should be considered in future AD preventive and therapeutic strategies.

Graphical abstract

Age-dependent vulnerability of memory and emotional neural circuits in response to tau and Aβ pathologies.
To let go or not to let go: how ParA can impact the release of the chromosomal anchoring inCaulobacter crescentus
Authors: Puentes-Rodriguez Stephanie G. , Norcross J.D. , Mera Paola E.
Published: April 2023

Chromosomal maintenance is vital for the survival of bacteria. InCaulobacter crescentus, chromosome replication initiates atoriand segregation is delayed until the nearby centromere-like regionparSis replicated. Our understanding of how this sequence of events is regulated remains limited. The segregation ofparShas been shown to involve multiple steps including polar release from anchoring protein PopZ, slow movement, and fast ParA-dependent movement to opposite cell pole. In this study, we demonstrate that ParA’s competing attractions from PopZ and from DNA are critical for segregation ofparS. Interfering with this balance of attractions – by expressing a variant ParA-R195E unable to bind DNA and thus favoring interactions exclusively between ParA-PopZ – results in cell death. Our data revealed that ParA-R195E’s sole interactions with PopZ obstruct PopZ’s ability to release the polar anchoring ofparSresulting in cells with multipleparSloci fixed at one cell pole. We show that the inability to separate and segregate multipleparSloci from the pole is specifically dependent on the interaction between ParA and PopZ. Interfering with interactions between PopZ and the partitioning protein ParB, which is the interaction that anchorsparSat the cell pole, does not rescue the ability of cells to separate the fixedparSloci when expressingparA-R195E. Thus, ParA and PopZ appear to have a distinct conversation from ParB yet can impact the release of ParB-parSfrom the anchoring at the cell pole. Collectively, our results reveal that the initial steps in chromosome segregation are highly regulated.
Similar enzymatic functions in distinct bioluminescence systems: Evolutionary recruitment of sulfotransferases in ostracod light organs
Authors: Lau Emily S , Goodheart Jessica A , Anderson Nolan T , Liu Vannie L , Mukherjee Arnab , Oakley Todd H
Published: April 2023

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracodVargula tsujii, transfer sulfate in vivo to the luciferin substrate, vargulin. We find luciferin sulfotransferases of ostracods are not orthologous to known luciferin sulfotransferases of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Re-use of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.
The bi-directional extracellular electron transfer process aids iron cycling byGeoalkalibacter halelectricusin a highly saline-alkaline condition
Authors: Yadav Sukrampal , Sadhotra Chetan , Patil Sunil A.
Published: April 2023

Bi-directional extracellular electron transfer (EET) is crucial to upholding microbial metabolism with insoluble electron acceptors or donors in anoxic environments. Investigating bi-directional EET-capable microorganisms is desired to understand the cell-cell and microbe-mineral interactions and their role in mineral cycling besides leveraging their energy generation and conversion, biosensing, and bio-battery applications. Here, we report on iron cycling by haloalkaliphilicGeoalkalibacter halelectricusvia bi-directional EET under haloalkaline conditions. It efficiently reduces Fe3+-oxide (Fe2O3) to Fe0at a 2.29±0.07 mM/day rate linked to acetate oxidation via outward EET and oxidizes Fe0to Fe3+with a 0.038±0.002 mM/day rate via inward EET to reduce fumarate. Bioelectrochemical cultivation confirmed its outward and inward EET capabilities. It produced 895±23 μA/cm2current by linking acetate oxidation to anode reduction via outward EET and reduced fumarate by drawing electrons from the cathode (−2.5±0.3 μA/cm2) via inward EET. The cyclic voltammograms ofG. halelectricus biofilms revealed redox moieties with different formal potentials, suggesting the involvement of different membrane components in bi-directional EET. The cyclic voltammetry and GC-MS analysis of the cell-free spent medium revealed the lack of soluble redox mediators, suggesting direct electron transfer byG. halelecctricus in achieving bi-directional EET. By reporting on the first haloalkaliphilic bacterium capable of oxidizing and reducing insoluble Fe0and Fe3+-oxide, respectively, this study advances the limited understanding of the metabolic capabilities of extremophiles to respire on insoluble electron acceptors or donors via bi-directional EET and invokes the possible role ofG. halelectricus in iron cycling in barely studied haloalkaline environments.

Graphical abstract
SECRET AGENT O-GlcNAc Modifies GIGANTEA: Methods for Mapping of O-Linked β-N-Acetylglucosamine Modification Sites Using Mass Spectrometry
Authors: Kim Young-Cheon , Hartweck Lynn M. , Olszewski Neil E.
Published: April 2023

Arabidopsis thalianahas two glycosyl transferases, SPINDLY (SPY) and SECRET AGENT (SEC), that modify nuclear and cytosolic protein with O-linked fucose and N-acetylglucosamine (GlcNAc), respectively. SPY interacts physically and genetically with GIGANTEA (GI). Previously, we reported that SEC substrates are O-GlcNAc modified when they are co-expressed inE. coli. By analyzing overlapping sub-fragments of GI, we found a region that was modified by SEC. Mutational mapping of the modified region was then performed. Modification was undetectable when threonine 829 was mutated to alanine (T829A) while the T834A and T837A mutations reduced modification suggesting that T829 was the primary or only modification site. Mapping using several enrichment and mass spectrometry methods all detected only modification of T829.
A model for Scc2p stimulation of cohesin's ATPase and its inhibition by acetylation of Smc3p
Authors: Boardman Kevin , Xiang Siheng , Chatterjee Fiona , Mbonu Udochi , Guacci Vincent , Koshland Douglas
Published: April 2023

The evolutionarily conserved cohesin complex mediates sister chromatid cohesion and facilitates mitotic chromosome condensation, DNA repair, and transcription regulation. These biological functions require cohesin's two ATPases, formed by the Smc1p and Smc3p subunits. Cohesin's ATPase activity is stimulated by the Scc2p auxiliary factor. This stimulation is inhibited by Eco1p acetylation of Smc3p at an interface with Scc2p. It was unclear how cohesin's ATPase activity is stimulated by Scc2p or how acetylation inhibits Scc2p, given that the acetylation site is distal to cohesin's ATPase active sites. Here, we identify mutations in budding yeast that suppressed the in vivo defects caused by Smc3p acetyl-mimic and acetyl-defective mutations. We provide compelling evidence that Scc2p activation of cohesin ATPase depends on an interface between Scc2p and a region of Smc1p proximal to cohesin's Smc3p ATPase active site. Furthermore, substitutions at this interface increase or decrease ATPase activity to overcome ATPase modulation by acetyl-mimic and acetyl-null mutations. Using these observations and an existing cryo-EM structure, we propose a model for regulating cohesin ATPase activity. We suggest that Scc2p binding to Smc1p causes the adjacent Smc1p residues and ATP to shift, stimulating Smc3p's ATPase. This stimulatory shift is inhibited through acetylation of the distal Scc2p–Smc3p interface.
A reactor for time-resolved X-ray studies of nucleation and growth during solvothermal synthesis
Authors: Roelsgaard Martin , Kløve Magnus , Christensen Rasmus , Bertelsen Andreas D. , Broge Nils L. N. , Kantor Innokenty , Sørensen Daniel Risskov , Dippel Ann-Christin , Banerjee Soham , Zimmermann Martin V. , Glaevecke Philipp , Gutowski Olof , Jørgensen Mads Ry Vogel , Iversen Bo Brummerstedt
Published: April 2023

Understanding the nucleation and growth mechanisms of nanocrystals under hydro- and solvothermal conditions is key to tailoring functional nanomaterials. High-energy and high-flux synchrotron radiation is ideal for characterization by powder X-ray diffraction and X-ray total scattering in real time. Different versions of batch-type cell reactors have been employed in this work, exploiting the robustness of polyimide-coated fused quartz tubes with an inner diameter of 0.7 mm, as they can withstand pressures up to 250 bar and temperatures up to 723 K for several hours. Reported here are recent developments of the in situ setups available for general users on the P21.1 beamline at PETRA III and the DanMAX beamline at MAX IV to study nucleation and growth phenomena in solvothermal synthesis. It is shown that data suitable for both reciprocal-space Rietveld refinement and direct-space pair distribution function refinement can be obtained on a timescale of 4 ms.
Digital transformation strategies of project-based firms: case study of a large-scale construction company in China
Authors: Cao Dongping , Teng Xuejiao , Chen Yanyu , Tan Dan , Wang Guangbin
Published: April 2023

Purpose

This study aims to explore how project-based firms, which generally organize most of their work around temporary projects in discontinuous and fragmented types of business contexts, proactively formulate and implement digital transformation strategies under institutional pressures in a predigital era.

Design/methodology/approach

An exploratory case study was conducted in a large-scale construction company in China using multiple data collection methods, including semistructured interviews, documentation collection and observation.

Findings

An integrated framework is developed to conceptualize three key dimensions of digital transformation strategies of project-based firms: strategic adaptation for organization-environment fit through balancing the internal efficiency needs with the external legitimacy pressures; proactive business transformation through comprehensively managing the roles of digital technologies in optimizing defined business processes and fostering new business models; and delicate organizational transformation to integrate temporary project-level operation processes with ongoing firm-level business processes.

Originality/value

This study represents an exploratory effort to empirically investigate how project-based firms strategically organize complex digital transformation imperatives in their discontinuous and fragmented business contexts. The findings contribute to deepened understandings of how complex organizational and environmental contexts can be comprehensively managed for systemic business and organizational transformations to leverage the value of emerging digital technologies for project-based organizations.