Mol Microbiol. 2023 Dec 22. doi: 10.1111/mmi.15211. Online ahead of print.

ABSTRACT

An essential process in transmission of the malaria parasite to the Anopheles vector is the conversion of mature gametocytes into gametes within the mosquito gut, where they egress from the red blood cell (RBC). During egress, male gametocytes undergo exflagellation, leading to the formation of eight haploid motile microgametes, while female gametes retain their spherical shape. Gametocyte egress depends on sequential disruption of the parasitophorous vacuole membrane and the host cell membrane. In other life cycle stages of the malaria parasite, phospholipases have been implicated in membrane disruption processes during egress, however their importance for gametocyte egress is relatively unknown. Here, we performed comprehensive functional analyses of six putative phospholipases for their role during development and egress of Plasmodium falciparum gametocytes. We localize two of them, the prodrug activation and resistance esterase (PF3D7_0709700) and the lysophospholipase 1 (PF3D7_1476700), to the parasite plasma membrane. Subsequently, we show that disruption of most of the studied phospholipase genes does neither affect gametocyte development nor egress. The exception is the putative patatin-like phospholipase 3 (PF3D7_0924000), whose gene deletion leads to a delay in male gametocyte exflagellation, indicating an important, albeit not essential, role of this enzyme in male gametogenesis.

PMID:38131156 | DOI:10.1111/mmi.15211

JBMR Plus. 2023 Nov 8;7(12):e10810. doi: 10.1002/jbm4.10810. eCollection 2023 Dec.

ABSTRACT

Astronauts have an increased risk of back pain and disc herniation upon returning to Earth. Thus, it is imperative to understand the effects of spaceflight and readaptation to gravity on the musculoskeletal tissues of the spine. Here we investigated whether ~6 months of spaceflight led to regional differences in bone loss within the vertebral body. Additionally, we evaluated the relationships between vertebral bone density and paraspinal muscle morphology before flight, after flight, and after readaptation on Earth. We measured vertebral trabecular bone mineral density (Tb.BMD), paraspinal muscle cross-sectional area (CSA), and muscle density in 17 astronauts using computed tomography (CT) images of the lumbar spine obtained before flight (before flight, n = 17), after flight (spaceflight, n = 17), and ~12 months of readaptation to gravitational loading on Earth (follow-up, n = 15). Spaceflight-induced declines in Tb.BMD were greater in the superior region of the vertebral body (-6.7%) than the inferior (-3.1%, p = 0.052 versus superior region) and transverse regions (-4.3%, p = 0.057 versus superior region). After a year of readaptation to Earth's gravity, Tb.BMD in the transverse region remained significantly below preflight levels (-4.66%, p = 0.0094). Paraspinal muscle CSA and muscle density declined -1.0% (p = 0.005) and -0.83% (p = 0.001) per month of spaceflight, respectively. Ultimately, bone loss in the superior vertebral body, along with fatty infiltration of paraspinal muscles and incomplete recovery even after a year of readaptation on Earth, may contribute to spinal pathology in long-duration astronauts. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

PMID:38130751 | PMC:PMC10731107 | DOI:10.1002/jbm4.10810

Mol Microbiol. 2023 Dec 22. doi: 10.1111/mmi.15201. Online ahead of print.

ABSTRACT

Legionella pneumophila, the causative agent of a life-threatening pneumonia, intracellularly replicates in a specialized compartment in lung macrophages, the Legionella-containing vacuole (LCV). Secreted proteins of the pathogen govern important steps in the intracellular life cycle including bacterial egress. Among these is the type II secreted PlaA which, together with PlaC and PlaD, belongs to the GDSL phospholipase family found in L. pneumophila. PlaA shows lysophospholipase A (LPLA) activity which increases after secretion and subsequent processing by the zinc metalloproteinase ProA within a disulfide loop. Activity of PlaA contributes to the destabilization of the LCV in the absence of the type IVB-secreted effector SdhA. We here present the 3D structure of PlaA which shows a typical α/β-hydrolase fold and reveals that the uncleaved disulfide loop forms a lid structure covering the catalytic triad S30/D278/H282. This leads to reduction of substrate access before activation; however, the catalytic site gets more accessible when the disulfide loop is processed. After structural modeling, a similar activation process is suggested for the GDSL hydrolase PlaC, but not for PlaD. Furthermore, the size of the PlaA substrate-binding site indicated preference toward phospholipids comprising ~16 carbon fatty acid residues which was verified by lipid hydrolysis, suggesting a molecular ruler mechanism. Indeed, mutational analysis changed the substrate profile with respect to fatty acid chain length. In conclusion, our analysis revealed the structural basis for the regulated activation and substrate preference of PlaA.

PMID:38130174 | DOI:10.1111/mmi.15201

BMC Infect Dis. 2023 Dec 21;23(1):902. doi: 10.1186/s12879-023-08904-7.

ABSTRACT

BACKGROUND: Coxiella burnetii, an intracellular pathogen, serves as the causative agent of zoonotic Q fever. This pathogen presents a significant threat due to its potential for airborne transmission, environmental persistence, and pathogenicity. The current whole-cell vaccine (WCV) utilized in Australia to combat Q fever exhibits notable limitations, including severe adverse reactions and limited regulatory approval for human use. This research employed the reverse vaccinology (RV) approach to uncover antigenic proteins and epitopes of C. burnetii, facilitating the development of more potent vaccine candidates.

METHODS: The potential immunogenic proteins derived from C. burnetii RSA493/Nine Mile phase I (NMI) were extracted through manual, automated RV, and virulence factor database (VFDB) methods. Web tools and bioinformatics were used to evaluate physiochemical attributes, subcellular localization, antigenicity, allergenicity, human homology, B-cell epitopes, MHC I and II binding ratios, functional class scores, adhesion probabilities, protein-protein interactions, and molecular docking.

RESULTS: Out of the 1850 proteins encoded by RSA493/NMI, a subset of 178 demonstrated the potential for surface or membrane localization. Following a series of analytical iterations, 14 putative immunogenic proteins emerged. This collection included nine proteins (57.1%) intricately involved in cell wall/membrane/envelope biogenesis processes (CBU_0197 (Q83EW1), CBU_0311 (Q83EK8), CBU_0489 (Q83E43), CBU_0939 (Q83D08), CBU_1190 (P39917), CBU_1829 (Q83AQ2), CBU_1412 (Q83BU0), CBU_1414 (Q83BT8), and CBU_1600 (Q83BB2)). The CBU_1627 (Q83B86 ) (7.1%) implicated in intracellular trafficking, secretion, and vesicular transport, and CBU_0092 (Q83F57) (7.1%) contributing to cell division. Additionally, three proteins (21.4%) displayed uncharacterized functions (CBU_0736 (Q83DJ4), CBU_1095 (Q83CL9), and CBU_2079 (Q83A32)). The congruent results obtained from molecular docking and immune response stimulation lend support to the inclusion of all 14 putative proteins as potential vaccine candidates. Notably, seven proteins with well-defined functions stand out among these candidates.

CONCLUSIONS: The outcomes of this study introduce promising proteins and epitopes for the forthcoming formulation of subunit vaccines against Q fever, with a primary emphasis on cellular processes and the virulence factors of C. burnetii.

PMID:38129801 | DOI:10.1186/s12879-023-08904-7

BMC Bioinformatics. 2023 Dec 21;24(1):492. doi: 10.1186/s12859-023-05599-0.

ABSTRACT

BACKGROUND: Flux Balance Analysis (FBA) is a key metabolic modeling method used to simulate cellular metabolism under steady-state conditions. Its simplicity and versatility have led to various strategies incorporating transcriptomic and proteomic data into FBA, successfully predicting flux distribution and phenotypic results. However, despite these advances, the untapped potential lies in leveraging gene-related connections like co-expression patterns for valuable insights.

RESULTS: To fill this gap, we introduce ICON-GEMs, an innovative constraint-based model to incorporate gene co-expression network into the FBA model, facilitating more precise determination of flux distributions and functional pathways. In this study, transcriptomic data from both Escherichia coli and Saccharomyces cerevisiae were integrated into their respective genome-scale metabolic models. A comprehensive gene co-expression network was constructed as a global view of metabolic mechanism of the cell. By leveraging quadratic programming, we maximized the alignment between pairs of reaction fluxes and the correlation of their corresponding genes in the co-expression network. The outcomes notably demonstrated that ICON-GEMs outperformed existing methodologies in predictive accuracy. Flux variabilities over subsystems and functional modules also demonstrate promising results. Furthermore, a comparison involving different types of biological networks, including protein-protein interactions and random networks, reveals insights into the utilization of the co-expression network in genome-scale metabolic engineering.

CONCLUSION: ICON-GEMs introduce an innovative constrained model capable of simultaneous integration of gene co-expression networks, ready for board application across diverse transcriptomic data sets and multiple organisms. It is freely available as open-source at https://github.com/ThummaratPaklao/ICOM-GEMs.git .

PMID:38129786 | DOI:10.1186/s12859-023-05599-0

Sci Rep. 2023 Dec 20;13(1):22820. doi: 10.1038/s41598-023-50158-5.

ABSTRACT

Bunyaviruses constitute a large and diverse group of viruses encompassing many emerging pathogens, such as Rift Valley fever virus (family Phenuiviridae), with public and veterinary health relevance but with very limited medical countermeasures are available. For the development of antiviral strategies, the identification and validation of virus-specific targets would be of high value. The cap-snatching mechanism is an essential process in the life cycle of bunyaviruses to produce capped mRNAs, which are then recognized and translated into viral proteins by the host cell translation machinery. Cap-snatching involves cap-binding as well as endonuclease functions and both activities have been demonstrated to be druggable in related influenza viruses. Here, we explore the suitability of the phenuivirus cap-binding function as a target in medium- and high-throughput drug discovery approaches. We developed a range of in vitro assays aiming to detect the interaction between the cap-binding domain (CBD) and the analogue of its natural cap-ligand m7GTP. However, constricted by its shallow binding pocket and low affinity for m7GTP, we conclude that the CBD has limited small molecule targeting potential using classical in vitro drug discovery approaches.

PMID:38129678 | DOI:10.1038/s41598-023-50158-5

Genome Res. 2023 Dec 21. doi: 10.1101/gr.277845.123. Online ahead of print.

ABSTRACT

Mammalian sperm show an unusual and heavily compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, nucleosome localization, histone modification, and chromosome folding. Here, we revisit these studies in light of recent reports that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis, we readily recapitulate multiple prominent genome-wide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, and appropriate lysis conditions, are together required to reveal a sperm chromatin state distinct from most previous reports. Using ATAC-seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with early development and transcriptional control. Histone modification and chromosome folding profiles also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies show that our knowledge of chromosome packaging in mammalian sperm remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm.

PMID:38129076 | DOI:10.1101/gr.277845.123

Kidney Int. 2023 Dec 19:S0085-2538(23)00869-4. doi: 10.1016/j.kint.2023.11.026. Online ahead of print.

ABSTRACT

Kidney transplant (KTx) biopsies showing transplant glomerulopathy (TG) (glomerular basement membrane double contours (cg) > 0)) and microvascular inflammation (MVI) in the absence of C4d staining and donor-specific antibodies (DSA)s do not fulfill the criteria for chronic active antibody-mediated rejection (CA-AMR) diagnosis and do not fit into any other Banff category. To investigate this, we initiated a multicenter intercontinental study encompassing 36 cases, comparing the immunomic and transcriptomic profiles of 14 KTx biopsies classified as cg+MVI DSA-/C4d- with 22 classified as CA-AMR DSA+/C4d+ through novel transcriptomic analysis using the NanoString Banff-Human Organ Transplant (B-HOT) panel and subsequent orthogonal subset analysis using two innovative 5-marker multiplex immunofluorescent panels. Nineteen genes were differentially expressed between the two study groups. Samples diagnosed with CA-AMR DSA+/C4d+ showed a higher glomerular abundance of natural killer cells and higher transcriptomic cell type scores for macrophages in an environment characterized by increased expression of complement-related genes (i.e., C5AR1) and higher activity of angiogenesis, interstitial fibrosis tubular atrophy, CA-AMR, and DSA-related pathways when compared to samples diagnosed with cg+MVI DSA-/C4d-. Samples diagnosed with cg+MVI DSA-/C4d- displayed a higher glomerular abundance and activity of T-cells (CD3+, CD3+CD8+, and CD3+CD8-). Thus, we show that using novel multiomic techniques, KTx biopsies with cg+MVI DSA-/C4d- have a prominent T-cell presence and activity, putting forward the possibility that these represent a more T-cell-dominant phenotype.

PMID:38128610 | DOI:10.1016/j.kint.2023.11.026

J Infect Dis. 2023 Dec 21:jiad595. doi: 10.1093/infdis/jiad595. Online ahead of print.

ABSTRACT

BACKGROUND: A better understanding of the dynamics of HIV reservoirs in CD4+ T cells of people with HIV (PWH) receiving antiretroviral therapy (ART) is crucial for developing therapies to eradicate the virus.

METHODS: We conducted a study involving 28 aviremic PWH receiving ART with high and low levels of HIV DNA. We analyzed immunologic and virologic parameters and their association with the HIV reservoir size.

RESULTS: The frequency of CD4+ T cells carrying HIV DNA was associated with higher pre-ART plasma viremia, lower pre-ART CD4+ T cell counts, and lower pre-ART CD4/CD8 ratios. During ART, the High group maintained elevated levels of intact HIV proviral DNA, cell-associated HIV RNA, and inducible virion-associated HIV RNA. HIV sequence analysis showed no evidence for preferential accumulation of defective proviruses nor higher frequencies of clonal expansion in the High versus Low group. Phenotypic and functional T-cell analyses did not show enhanced immune-mediated virologic control in the Low versus High group. Of considerable interest, pre-ART innate immunity was significantly higher in the Low versus High group.

CONCLUSIONS: Our data suggest that innate immunity at the time of ART initiation may play an important role in modulating the dynamics and persistence of viral reservoirs in PWH.

PMID:38128541 | DOI:10.1093/infdis/jiad595

Mol Cell. 2023 Dec 13:S1097-2765(23)00978-4. doi: 10.1016/j.molcel.2023.11.032. Online ahead of print.

ABSTRACT

SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3' UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development.

PMID:38128540 | DOI:10.1016/j.molcel.2023.11.032

Cell Syst. 2023 Dec 16:S2405-4712(23)00332-0. doi: 10.1016/j.cels.2023.12.002. Online ahead of print.

ABSTRACT

Stem cells differentiate into distinct fates by transitioning through a series of transcriptional states. Current computational approaches allow reconstruction of differentiation trajectories from single-cell transcriptomics data, but it remains unknown to what degree differentiation can be predicted across biological processes. Here, we use transfer learning to infer differentiation processes and quantify predictability in early embryonic development and adult hematopoiesis. Overall, we find that non-linear methods outperform linear approaches, and we achieved the best predictions with a custom variational autoencoder that explicitly models changes in transcriptional variance. We observed a high accuracy of predictions in embryonic development, but we found somewhat lower agreement with the real data in adult hematopoiesis. We demonstrate that this discrepancy can be explained by a higher degree of concordant transcriptional processes along embryonic differentiation compared with adult homeostasis. In summary, we establish a framework for quantifying and exploiting predictability of cellular differentiation trajectories.

PMID:38128536 | DOI:10.1016/j.cels.2023.12.002

EBioMedicine. 2023 Dec 20;99:104907. doi: 10.1016/j.ebiom.2023.104907. Online ahead of print.

ABSTRACT

BACKGROUND: Diagnosing Cushing's syndrome (CS) is highly complex. As the diagnostic potential of urinary steroid metabolome analysis by gas chromatography-mass spectrometry (GC-MS) in combination with systems biology has not yet been fully exploited, we studied a large cohort of patients with CS.

METHODS: We quantified daily urinary excretion rates of 36 steroid hormone metabolites. Applying cluster analysis, we investigated a control group and 168 patients: 44 with Cushing's disease (CD) (70% female), 18 with unilateral cortisol-producing adrenal adenoma (83% female), 13 with primary bilateral macronodular adrenal hyperplasia (PBMAH) (77% female), and 93 ruled-out CS (73% female).

FINDINGS: Cluster-Analysis delineated five urinary steroid metabotypes in CS. Metabotypes 1, 2 and 3 revealing average levels of cortisol and adrenal androgen metabolites included patients with exclusion of CS or and healthy controls. Metabotype 4 reflecting moderately elevated cortisol metabolites but decreased DHEA metabolites characterized the patients with unilateral adrenal CS and PBMAH. Metabotype 5 showing strong increases both in cortisol and DHEA metabolites, as well as overloaded enzymes of cortisol inactivation, was characteristic of CD patients. 11-oxygenated androgens were elevated in all patients with CS. The biomarkers THS, F, THF/THE, and (An + Et)/(11β-OH-An + 11β-OH-Et) correctly classified 97% of patients with CS and 95% of those without CS. An inverse relationship between 11-deoxygenated and 11-oxygenated androgens was typical for the ACTH independent (adrenal) forms of CS with an accuracy of 95%.

INTERPRETATION: GC-MS based urinary steroid metabotyping allows excellent identification of patients with endogenous CS and differentiation of its subtypes.

FUNDING: The study was funded by the Else Kröner-Fresenius-Stiftung and the Eva-Luise-und-Horst-Köhler-Stiftung.

PMID:38128413 | DOI:10.1016/j.ebiom.2023.104907

Clin Imaging. 2023 Dec 13;106:110031. doi: 10.1016/j.clinimag.2023.110031. Online ahead of print.

ABSTRACT

Awarded the Radiological Society of North America (RSNA) Gold Medal in 2018, Dr. Deborah Levine's research, journalism, and mentorship have left an indelible mark on the radiology field. Her work in ultrasound led to its use as the standard for monitoring benign adnexal cysts. She helped popularize obstetric magnetic resonance imaging (MRI) through her research on its use in placental accreta and fetal abnormalities, which led to the development of the 'Compendium of Fetal MRI' website. This work in research led naturally to a career in journalism, where she eventually became Senior Deputy Editor of Radiology and founded Radiology Select. Concurrently with her personal achievements, Dr. Levine has dedicated herself to the mentorship of her female trainees. She sought various leadership positions to learn more about and advocate for the promotion and support of female leadership in radiology departments. In many ways, Dr. Levine's career and work have transformed radiology as we know it today for both patients and physicians.

PMID:38128405 | DOI:10.1016/j.clinimag.2023.110031

Commun Biol. 2023 Dec 21;6(1):1301. doi: 10.1038/s42003-023-05674-5.

ABSTRACT

Considerable evidence suggests that breast cancer therapeutic resistance and relapse can be driven by polyploid giant cancer cells (PGCCs). The number of PGCCs increases with the stages of disease and therapeutic stress. Given the importance of PGCCs, it remains challenging to eradicate them. To discover effective anti-PGCC compounds, there is an unmet need to rapidly distinguish compounds that kill non-PGCCs, PGCCs, or both. Here, we establish a single-cell morphological analysis pipeline with a high throughput and great precision to characterize dynamics of individual cells. In this manner, we screen a library to identify promising compounds that inhibit all cancer cells or only PGCCs (e.g., regulators of HDAC, proteasome, and ferroptosis). Additionally, we perform scRNA-Seq to reveal altered cell cycle, metabolism, and ferroptosis sensitivity in breast PGCCs. The combination of single-cell morphological and molecular investigation reveals promising anti-PGCC strategies for breast cancer treatment and other malignancies.

PMID:38129519 | DOI:10.1038/s42003-023-05674-5

Elife. 2023 Dec 21;12:e83722. doi: 10.7554/eLife.83722.

ABSTRACT

Private, subjective beliefs about uncertainty have been found to have idiosyncratic computational and neural substrates yet, humans share such beliefs seamlessly and cooperate successfully. Bringing together decision making under uncertainty and interpersonal alignment in communication, in a discovery plus pre-registered replication design, we examined the neuro-computational basis of the relationship between privately held and socially shared uncertainty. Examining confidence-speed-accuracy trade-off in uncertainty-ridden perceptual decisions under social vs isolated context, we found that shared (i.e. reported confidence) and subjective (inferred from pupillometry) uncertainty dynamically followed social information. An attractor neural network model incorporating social information as top-down additive input captured the observed behavior and demonstrated the emergence of social alignment in virtual dyadic simulations. Electroencephalography showed that social exchange of confidence modulated the neural signature of perceptual evidence accumulation in the central parietal cortex. Our findings offer a neural population model for interpersonal alignment of shared beliefs.

PMID:38128085 | DOI:10.7554/eLife.83722

Plant J. 2023 Dec 21. doi: 10.1111/tpj.16599. Online ahead of print.

ABSTRACT

Sugar beet and its wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNAs are among the fastest evolving parts of the genome, we investigated, if repeatome innovations and losses are linked to chromosomal differentiation and speciation. We traced genome and chromosome-wide evolution across 13 beet species comprising all sections of the genera Beta and Patellifolia. For this, we combined short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive framework that spans the complete scale from DNA to chromosome to genome. Genome sizes and repeat profiles reflect the separation into three gene pools with contrasting evolutionary patterns. Among all repeats, satellite DNAs harbor most genomic variability, leading to fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia to the formation of patchwork chromosomes in Corollinae/Nanae. We show that repetitive DNAs are causal for the genome expansions and contractions across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools, likely contributing to the barriers in beet breeding. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genomic variability, and chromosomal differentiation and provide a theoretical fundament for understanding barriers in any crop breeding effort.

PMID:38128038 | DOI:10.1111/tpj.16599

Proc Natl Acad Sci U S A. 2023 Dec 26;120(52):e2318274120. doi: 10.1073/pnas.2318274120. Epub 2023 Dec 21.

ABSTRACT

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

PMID:38127982 | DOI:10.1073/pnas.2318274120

PLoS One. 2023 Dec 21;18(12):e0295945. doi: 10.1371/journal.pone.0295945. eCollection 2023.

ABSTRACT

Pine wilt disease (PWD) is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and transmitted by a vector insect, the Monochamus alternatus. The PWN has caused much extensive damage to pine-dominated forest ecosystems. Trunk injection of emamectin benzoate (EB) has been found to be the most useful protective measure against the PWN, due to its low effective dose and long residence time in the field. However, the interactions between EB and the host or the environment remain largely unknown, which limits the efficacy and stability of EB in practical field settings. In this study, we investigated the impact on PWN from EB injection for both adult and young host plants (Pinus massoniana) by taking a multi-omics (phenomics, transcriptomics, microbiome, and metabolomics) approach. We found that EB injection can significantly reduce the amount of PWN in both living adult and young pine trees. Additionally, EB was able to activate the genetic response of P. massoniana against PWN, promotes P. massoniana growth and development and resistance to Pine wilt disease, which requires the presence of PWN. Further, the presence of EB greatly increased the accumulation of reactive oxygen species (ROS) in the host plant in a PWN-dependent manner, possibly by affecting ROS-related microbes and metabolites. Moreover, we uncovered the function of EB limiting the consumption of P. massoniana by the JPS. Based on biochemical and gut microbial data, we found that EB can significantly reduces cellulase activity in JPS, whose transcription factors, sugar metabolism, and the phosphotransferase system are also affected. These results document the impact of EB on the entire PWD transmission chain through multi-omics regarding the dominant pine (P. massoniana) in China and provide a novel perspective for controlling PWD outbreaks in the field.

PMID:38127873 | DOI:10.1371/journal.pone.0295945

Science. 2023 Dec 22;382(6677):eadi5120. doi: 10.1126/science.adi5120. Epub 2023 Dec 22.

ABSTRACT

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.

PMID:38127763 | DOI:10.1126/science.adi5120

STAR Protoc. 2023 Dec 19;5(1):102785. doi: 10.1016/j.xpro.2023.102785. Online ahead of print.

ABSTRACT

An extensive literature describes how pupil size reflects neuromodulatory activity, including the noradrenergic system. Here, we present a protocol for the simultaneous recording of optogenetically identified locus coeruleus (LC) units and pupil diameter in mice under different conditions. We describe steps for building an optrode, performing surgery to implant the optrode and headpost, searching for opto-tagged LC units, and performing dual LC-pupil recording. We then detail procedures for data processing and analysis. For complete details on the use and execution of this protocol, please refer to Megemont et al.1.

PMID:38127625 | DOI:10.1016/j.xpro.2023.102785