Biology subjects

Liang, X.

Publications and source records attributed to Liang, X..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Large Serine Integrase Off-target Discovery and Validation for Therapeutic Genome Editing

While numerous technologies for the characterization of potential off-target editing by CRISPR/Cas9 have been described, the development of new technologies and analytical methods for off-target recombination by Large Serine Integrases (LSIs) are required to advance the application of LSIs for therapeutic gene integration. Here we describe a suite of off-target recombination discovery technologies and a hybrid capture validation approach as a comprehensive framework for off-target characterization of LSIs. HIDE- Seq (High-throughput Integrase-mediated DNA Event Sequencing) is a PCR-free unbiased genome-wide biochemical assay capable of discovering sites with LSI- mediated free DNA ends (FDEs) and off-target recombination events. Cryptic-Seq is a PCR-based unbiased genome-wide biochemical or cellular-based assay that is more sensitive than HIDE-Seq but is limited to the discovery of sites with off-target recombination. HIDE-Seq and Cryptic-Seq discovered 38 and 44,311 potential off-target sites respectively. 2,455 sites were prioritized for validation by hybrid capture NGS in LSI- edited K562 cells and off-target integration was detected at 52 of the sites. We benchmarked the sensitivity of our LSI off-target characterization framework against unbiased whole genome sequencing (WGS) on LSI-edited samples, and off-target integration was detected at 5 sites with an average genome coverage of 40x. This reflects a greater than 10-fold increase in sensitivity for off-target detection compared to WGS, however only 4 of the 5 sites detected by WGS were also validated by hybrid capture NGS. The dissemination of these technologies will help advance the application of LSIs in therapeutic genome editing by establishing methods and benchmarks for the sensitivity of off-target detection.

genomics↗

bioRxiv2024

Amidase and Lysozyme Dual Functions in TseP Reveal a New Family of Chimeric Effectors in the Type VI Secretion System

Peptidoglycan (PG) serves as an essential target for antimicrobial development. An overlooked reservoir of antimicrobials lies in the form of PG-hydrolyzing enzymes naturally produced for polymicrobial competition, particularly those associated with the type VI secretion system (T6SS). Here we report that a T6SS effector TseP, from Aeromonas dhakensis, represents a family of effectors with dual amidase-lysozyme activities. In vitro PG-digestion coupled with LC-MS analysis revealed the N-domains amidase activity, which is neutralized by either catalytic mutations or the presence of the immunity protein TsiP. The N-domain, but not the C-domain, of TseP is sufficient to restore T6SS secretion in T6SS-defective mutants, underscoring its critical structural role. Using pull-down and secretion assays, we showed that these two domains interact directly with a carrier protein VgrG2 and can be secreted separately. Homologs in Aeromonas hydrophila and Pseudomonas syringae exhibited analogous dual functions. Additionally, N- and C-domains display distinctive GC contents, suggesting an evolutionary fusion event. By altering the surface charge through structural-guided design, we engineered the TsePC4+ effector that successfully lyses otherwise resistant Bacillus subtilis cells, enabling the T6SS to inhibit B. subtilis in a contact-independent manner. This research uncovers TseP as a new family of bifunctional chimeric effectors targeting PG, offering a potential strategy to harness these proteins in the fight against antimicrobial resistance. Significance StatementAntimicrobial resistance urgently demands global interventions, and the bacteria cell wall remains a promising target. Our research introduces a novel family of bifunctional, cell-wall-damaging T6SS effectors. More importantly, we demonstrate an effective strategy to enable an otherwise ineffective enzyme to target both Gram-negative and Gram-positive bacteria. Our findings highlight a promising path forward using cell-wall-damaging effectors, a largely untapped resource, in the fight against antimicrobial resistance.

microbiology↗

bioRxiv2024

Biochar Modulates Wheat Root Metabolome and Rhizosphere Microbiome in a Feedstock-dependent Manner

BackgroundBiochar is a multifunctional soil conditioner capable of enhancing soil health and plant productivity, but the underlying mechanisms remain elusive. Here we tackled this question using wheat as a model plant and through the lens of the rhizosphere, a vital soil-plant interface continuum. We systematically examined the effects of four types of biochar (corn stover, cattle manure, pine sawdust, or wheat straw) applied at two rates (0.25% or 2.5%, w/w). ResultsEmploying untargeted metabolomics and 16S rRNA gene sequencing, we revealed both common and unique modulating effects of the tested biochar treatments on wheat root metabolites and rhizosphere microbiome structure and functioning. Biochar modulated numerous metabolic pathways in wheat roots, where amino acid metabolism was the most common one, leading to cascade effects on the dynamics of a wide range of secondary metabolites, including many plant signaling molecules (e.g., flavonoid compounds, brassinosteroids) that are known to be involved in plant-microbe interactions. All biochar treatments increased rhizosphere microbial diversity, altered community composition, enhanced microbial interactions, and resulted in functional changes. Increased Burkholderiales (denitrifying bacteria) abundance and decreased Thermoplasmata (archaeal methanogens) abundance could explain biochars widely reported effects on nitrous oxide and methane mitigation, respectively. Biochar enhanced positive correlations among microbes and network complexity, particularly modularity, suggesting local adaptation through mutualism and/or synergism and the formation of modules of functionally interrelated taxa. A large number of diverse keystone taxa from both dominant and non-dominant phyla emerged after biochar treatments, including those known to be involved in methane, nitrogen, and sulfur cycling. Besides common alterations, treatment-specific alterations also occurred, and biochar type (i.e., feedstock choice) exerted greater influence than application rate. Wheat biochar applied at a 0.25% rate showed the strongest and distinct modulating effects, resulting in orchestrated changes in both root metabolites and rhizosphere microbiome, especially those relevant to plant-microbe interactions and likely beneficial to the host plant (e.g., upregulated biosynthesis of zeatin and down-regulated limonene degradation). ConclusionsOur work contributes to a mechanistic understanding of how biochar modulates the soil-plant continuum and provides new insights into the potential of top-down rhizosphere microbiome engineering through biochar-based reprogramming of root-microbe interactions.

microbiology↗

bioRxiv2024

Load-dependent and load-independent effects on longitudinal motor training in human continuous hand movements

Motor learning involves complex interactions between the cognitive and sensorimotor systems, which is susceptible to different levels of task load. While the mechanism underlying load-dependent regulations in cognitive functions has been extensively investigated, their influence on downstream execution in motor skill learning remains less understood. The current study extends the understanding of how load levels affect motor learning by a longitudinal functional near-infrared spectroscopy (fNIRS) study in which 30 participants (15 females) engaged in extensive practice on a two-dimensional continuous hand tracking task with varying task difficulties. We propose the index of difficulty (ID) as a quantitative estimate of task difficulty, which is positively correlated with psychometric measure of subjective workload level. Results shows that as behavioral performance improved over time, participants adopted a direction-specific and load-independent (i.e., consistent across different load levels) control strategy, shifting from feedback-dominant to feedforward-dominant control in the vertical direction as training progressed. Crucially, we provide robust evidence of the learning-induced alteration in load-dependent cortical activation patterns, suggesting that effective motor skill learning may lead to shift towards an inverted-U relationship between activation and load level in the pre-motor and supplementary motor areas. In addition, brain-behavior relationship in the frontoparietal network was strengthened after training. Taken together, our findings provide new insights into the learning-induced plasticity in brain and behavior associated with load-dependent and load-independent contributions to motor skill learning.

neuroscience↗

bioRxiv2024

Enhanced purinergic signaling in the paraventricular hypothalamus induces hyperphagic obesity and insulin resistance

Body energy homeostasis is tightly regulated by hypothalamic neural circuits. However, their remodeling upon metabolic challenges remains incompletely characterized, thus complicating the development of safe medications against the surge of metabolic diseases. Oxytocin (OXT) neurons in the paraventricular nucleus of the hypothalamus (PVH) are one of the key effectors regulating energy balance. In this work, we report that high-fat diet (HFD) feeding in mice evokes spatiotemporally selective ATP release from astrocytes (Inflares) in the PVH, accompanied by the expression of hematopoietic lineage-specific ADP/ATP receptor P2Y12 on local OXT (PVHOXT) neurons. Strikingly, increased purinergic signaling leads to impaired responsiveness of PVHOXT neurons accompanied by hyperphagic obesity and insulin resistance in mice. Conversely, loss of P2Y12 in PVHOXT neurons attenuates metabolic phenotypes, illustrating that such remodeling is both necessary and sufficient to induce metabolic phenotypes. Inflares were also induced by hyperglycemia, while emergence of P2Y12 on OXT neurons of patients with diabetes mellitus suggests an evolutionary conserved purinergic response to various metabolic challenges and its potential as a drug target. Accordingly, nasal administration of clinically approved doses of P2Y12 inhibitors counteracts obesity in mice and non-human primates, paving the way for application of these compounds in patients with metabolic disorders.

neuroscience↗

bioRxiv2024

The continuous differentiation of multiscale structural gradients from childhood to adolescence correlates with the maturation of cortical morphology and functional specialization

From childhood to adolescence, the structural organization of the human brain undergoes dynamic and regionally heterogeneous changes across multiple scales, from synaptic pruning to the reorganization of large-scale anatomical wiring. However, during this period, the developmental process of multiscale structural architecture, its association with cortical morphological changes, and its role in the maturation of functional organization remain largely unknown. Here, we utilized a longitudinal multimodal imaging dataset including 276 children aged 6 to 14 years to investigate the developmental process of multiscale cortical wiring. We used an in vivo model of cortical wiring that combines features of white matter tractography, cortico-cortical proximity, and microstructural similarity to construct a multiscale brain structural connectome. By employing the gradient mapping method, the gradient space derived from the multiscale structural connectome effectively recapitulated the sensory-association axis and anterior-posterior axis. Our findings revealed a continuous expansion of the multiscale structural gradient space during development, with the principal gradient increasingly distinguishing between primary and transmodal regions. This age-related differentiation coincided with regionally heterogeneous changes in cortical morphology. Furthermore, our study revealed that developmental changes in coupling between multiscale structural and functional connectivity were correlated with functional specialization refinement, as evidenced by changes in the participation coefficient. We also found that the differentiation of the principal multiscale structural gradient was associated with improved cognitive abilities, such as enhanced working memory and attention performance, and potentially supported by molecular processes related to synaptic functions. These findings advance our understanding of the intricate maturation process of brain structural organization and its implications for cognitive performance.

neuroscience↗

bioRxiv2024

c-di-GMP modulates ribosome assembly by inhibiting rRNA methylation

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial secondary messenger, with diverse functions, many of which are yet to be uncovered. Stemming from an Escherichia coli proteome microarray, we found that c-di-GMP bound to 23S rRNA methyltransferases (RlmI and RlmE). rRNA methylation assays showed that c-di-GMP inhibits RlmI activity, thereby modulating ribosome assembly. Based on molecular dynamic simulation and mutagenesis studies, we found that c-di-GMP binds to RlmI at residues R64, R103, G114, and K201. Structural simulation revealed that c-di-GMP quenches RlmI activity by inducing the closure of the catalytic pocket. Furthermore, we revealed that c-di-GMP promotes antibiotic tolerance by regulating RlmI activity, which played a role in antibiotic-resistant strains. Finally, the binding and methylation assays showed that the effect of c-di-GMP on RlmI is conserved, at least in various pathogenic bacteria. This study discovered an unexpected functional role of c-di-GMP in regulating ribosome assembly by inhibiting rRNA methylases. This study identified an unexpected but crucial member among the c-di-GMP effectors. HighlightsO_LIc-di-GMP regulates ribosome assembly in Escherichia coli. C_LIO_LIc-di-GMP inhibits rRNA methylation activity of RlmI by inducing catalytic pocket closure. C_LIO_LIc-di-GMP promotes antibiotic resistance by regulating ribosome assembly. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/597503v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@52baforg.highwire.dtl.DTLVardef@176f86dorg.highwire.dtl.DTLVardef@1afed11org.highwire.dtl.DTLVardef@f53687_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

CAMSAP3 Forms Dimers via its lapha-helix Domain that Directly Stabilize Non-centrosomal Microtubule Minus Ends

Microtubules are vital components of the cytoskeleton. Their plus ends are dynamic and respond to changes in cell morphology, while the minus ends are stable and serve a crucial role in microtubule seeding and maintaining spatial organization. In mammalian cells, the calmodulin-regulated spectrin-associated proteins (CAMSAPs), play a key role in directly regulating the dynamics of non-centrosomal microtubules minus ends. However, the molecular mechanisms are not yet fully understood. Our study reveals that CAMSAP3 forms dimers through its C-terminal -helix; this dimerization not only enhances the microtubule-binding affinity of the CKK domain but also enables the CKK domain to regulate the dynamics of microtubules. Furthermore, CAMSAP3 also specializes in decorating at the minus end of microtubules through the combined action of the microtubule-binding domain (MBD) and the C-terminal -helix, thereby achieving dynamic regulation of the minus ends of microtubules. These findings are crucial for advancing our understanding and treatment of diseases associated with non-centrosomal microtubules. SignificanceOur study reveals the molecular mechanism of how CAMSAP3, a key regulator of non-centrosomal microtubule dynamics, directly regulates the dynamics of non-centrosomal microtubule minus ends through CKK domain. CAMSAP3 forms dimers through its C-terminal -helix, which enhances the CKK domain of CAMSAP3 binding to microtubule minus ends and confers stability of them. This finding is not limited to CAMSAP3, but can also be applied to the understanding of the regulation of non-centrosomal microtubule minus end stability by CAMSAP family proteins. Our findings deepen our comprehension of cellular structure and function, offering insights into the role of microtubules in cellular integrity and disease. This study fills a significant knowledge gap and lays the foundation for future research into the complex balance of microtubule dynamics required for cellular health and disease prevention.

cell biology↗

bioRxiv2024

Multi-task benchmarking of spatially resolved gene expression simulation models

Computational methods for spatially resolved transcriptomics (SRT) are frequently developed and assessed through data simulation. The effectiveness of these evaluations relies on the simulation methods ability to accurately reflect experimental data. However, a systematic evaluation framework for spatial simulators is lacking. Here, we present SpatialSimBench, a comprehensive evaluation framework that assesses 13 simulation methods using 10 distinct STR datasets. We introduce simAdaptor, a tool that extends single-cell simulators to incorporate spatial variables, thus enabling them to simulate spatial data. SimAdaptor enables SpatialSimBench to be "back-wards" compatible. That is, it facilitates direct comparison between spatially aware simulators and existing non-spatial single-cell simulators through the adaption. Through SpatialSimBench, we demonstrate the feasibility of leveraging existing single-cell simulators for SRT data and highlight performance differences among methods. Additionally, we evaluate the simulation methods based on a total of 35 metrics across data property estimation, various downstream analysis and scalability. In total, we generated 4550 results from 13 simulation methods, 10 spatial datasets and 35 metrics. Our findings reveal that model estimation can be impacted by distribution assumptions and dataset characteristics. In summary, our evaluation and the evaluation framework will provide guidelines for selecting appropriate methods for specific scenarios and informing future method development.

bioinformatics↗

bioRxiv2024

CRMP/UNC-33 maintains neuronal microtubule arrays by promoting individual microtubule rescue

Microtubules (MTs) are intrinsically dynamic polymers. In neurons, staggered individual microtubules form stable, polarized acentrosomal MT arrays spanning the axon and dendrite to support long-distance intracellular transport. How the stability and polarity of these arrays are maintained when individual MTs remain highly dynamic is still an open question. Here we visualize MT arrays in vivo in C. elegans neurons with single microtubule resolution. We find that the CRMP family homolog, UNC-33, is essential for the stability and polarity of MT arrays in neurites. In unc-33 mutants, MTs exhibit dramatically reduced rescue after catastrophe, develop gaps in coverage, and lose their polarity, leading to trafficking defects. UNC-33 is stably anchored on the cortical cytoskeleton and forms patch-like structures along the dendritic shaft. These discrete and stable UNC-33 patches concentrate free tubulins and correlate with MT rescue sites. In vitro, purified UNC-33 preferentially associates with MT tips and increases MT rescue frequency. Together, we propose that UNC-33 functions as a microtubule-associated protein (MAP) to promote individual MT rescue locally. Through this activity, UNC-33 prevents the loss of individual MTs, thereby maintaining the coverage and polarity of MT arrays throughout the lifetime of neurons.

cell biology↗

bioRxiv2024

A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains one of the major hurdles in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles released by cells, that play an important role in intercellular communication. Due to their innate capability of intercellular transfer of proteins, RNA, and various other biological cargos, EVs have emerged as a novel promising strategy for the delivery of macromolecular biotherapeutics, including CRISPR-Cas9 ribonucleoproteins. Here, we present a versatile, modular strategy for the loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins (MCPs) fused to EV-enriched proteins to MS2 aptamers incorporated into single guide RNAs (sgRNAs), in combination with a UV-activated photocleavable linker domain, PhoCl. Combined with the Vesicular stomatitis virus G (VSV-G) protein this modular platform enables efficient loading and subsequent delivery of the Cas9 ribonucleoprotein complex, which shows critical dependence on the incorporation and activation of the photocleavable linker domain. As this approach does not require any direct fusion of Cas9 to EV-enriched proteins, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e, as confirmed by various sensitive fluorescent reporter assays. Taken together, we describe a robust and modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering as well as transcriptional regulation, underlining the potential of EV-mediated strategies for the treatment of genetic diseases.

molecular biology↗

bioRxiv2024

Development of the overlapping network modules in the human brain

Developmental connectomic studies have shown that the modular organization of functional networks in the human brain undergoes substantial reorganization with age to support cognitive growth. However, these studies implicitly assume that each brain region belongs to one and only one specific network module, ignoring the potential spatial overlap between functional modules. How the overlapping functional modular architecture develops and whether this development is related to structural signatures remain unknown. Using longitudinal multimodal structural, functional, and diffusion MRI data from 305 children (aged 6-14 years), we investigated the development of the overlapping modular architecture of functional networks, and further explored their structural associations. Specifically, an edge-centric network model was used to identify the overlapping functional modules, and the nodal overlap in module affiliations was quantified using the entropy measure. We showed a remarkable regional inhomogeneity in module overlap in children, with higher entropy in the ventral attention, somatomotor, and subcortical networks and lower entropy in the visual and default-mode networks. Furthermore, the overlapping modules developed in a linear, spatially dissociable manner from childhood to adolescence, with significantly reduced entropy in the prefrontal cortex and putamen and increased entropy in the parietal lobules. Personalized overlapping modular patterns capture individual brain maturity as characterized by brain age. Finally, the overlapping functional modules can be significantly predicted by integrating gray matter morphology and white matter network properties. Our findings highlight the maturation of overlapping network modules and their structural substrates, thereby advancing our understanding of the principles of connectome development.

neuroscience↗

bioRxiv2024

Functional divergence between the two cerebral hemispheres contributes to human fluid intelligence

Hemispheric lateralization is linked to potential cognitive advantages. It is considered a driving force behind the generation of human intelligence. However, establishing quantitative links between the degree of lateralization and intelligence in humans remains elusive. In this study, we propose a framework that utilizes the functional aligned multidimensional representation space derived from hemispheric functional gradients to compute between-hemisphere distances within this space. Applying this framework to a large cohort (N=777 from the Human Connectome Project), we identified high functional divergence across the two hemispheres within the frontoparietal control network. We found that both global divergence between the cerebral hemispheres and regional divergence within the multiple demand network were positively associated with fluid composite score and partially mediated the influence of brain size on individual differences in fluid intelligence. Together, these findings illuminate the profound significance of brain lateralization as a fundamental organizational principle of the human brain, providing direct evidence that hemispheric lateralization supports human fluid intelligence.

neuroscience↗

bioRxiv2024

Soil viruses reduce greenhouse gas emissions and promote microbial necromass accrual

Viral-induced microbial mortality has been proposed as a major contributor in shaping microbial community structure and function, soil carbon (C) accrual and mobilization of plant available nutrients. Yet, how soil viruses influence soil organic C (SOC) turnover and sequestration remains unknown. Here, we performed microcosm experiments with two distinct soils from grassland (GL) and agricultural (AG) sites and interrogated the roles of soil viruses in driving microbial community succession, SOC transformation and sequestration. The results show that soil viruses affected microbial C use efficiency and reduced respiration in microbial communities obtained from both GL and AG soils. Soil viruses affected microbial successional trajectories (via predation of dominant populations) and functional gene profiles triggering a significant decrease in CO2 and N2O emissions. The impact of soil viruses on microbial community composition in GL microcosms was much less pronounced compared with that in AG microcosms, suggesting contrasting virus-host interaction patterns under different environmental settings. Viral infection significantly enhanced microbial necromass accumulation thereby increasing SOC and total nitrogen (TN) content. The results implicate viral-mediated microbial mortality as a key factor influencing the distribution of C between mineralization and soil C storage pathways. We proposed "viral loop" to explain the crucial function of soil viruses in SOC turnover and sequestration.

ecology↗

bioRxiv2024

Genetic Studies Highlight the Role of TET2 and INO80 in DNA Damage Response and Kidney Disease Pathogenesis

Genome-wide association studies (GWAS) have identified over 800 loci associated with kidney function, yet the specific genes, variants, and pathways involved remain elusive. By integrating kidney function GWAS, human kidney expression and methylation quantitative trait analyses, we identified Ten-Eleven Translocation (TET) DNA demethylase 2: TET2 as a novel kidney disease risk gene. Utilizing single-cell chromatin accessibility and CRISPR-based genome editing, we highlight GWAS variants that influence TET2 expression in kidney proximal tubule cells. Experiments using kidney-tubule-specific Tet2 knockout mice indicated its protective role in cisplatin-induced acute kidney injury, as well as chronic kidney disease and fibrosis, induced by unilateral ureteral obstruction or adenine diet. Single-cell gene profiling of kidneys from Tet2 knockout mice and TET2-knock-down tubule cells revealed the altered expression of DNA damage repair and chromosome segregation genes, notably including INO80, another kidney function GWAS target gene itself. Remarkably both TET2-null and INO80-null cells exhibited an increased accumulation of micronuclei after injury, leading to the activation of cytosolic nucleotide sensor cGAS-STING. Genetic deletion of cGAS or STING in kidney tubules or pharmacological inhibition of STING protected TET2 null mice from disease development. In conclusion, our findings highlight TET2 and INO80 as key genes in the pathogenesis of kidney diseases, indicating the importance of DNA damage repair mechanisms.

genetics↗

bioRxiv2024

Topographic, cognitive, and neurobiological profiling of the interdependent structural and functional connectome in the human brain

The structural connectome (SC) is tightly coupled to the functional connectome (FC) in the human brain. Most previous related studies have modeled and analyzed SC or FC as isolated brain networks. However, challenges remain in modeling the interdependent structural-functional connectome and elucidating its cognitive implications and molecular underpinnings. Here, we present a multilayer connectome model composed of SC and FC components and further characterize their interacting topological properties. We found that the interdependent connectome is topographically heterogeneous, with the transmodal cortex exhibiting greater modular variability across layers. This spatial topography reflects cortical hierarchy and evolution and shows high test-retest reliability, reproducibility, and heritability. The interdependent connectome contributes to high-order cognitive processes and is associated with multiple neurotransmitter systems and transcriptional signatures of synaptic transmission. Our results provide insights into the nontrivial interdependencies of SC and FC, highlighting their cognitive significance and the molecular mechanisms underlying the connectome of connectomes.

neuroscience↗

bioRxiv2023

Auditory High Entropy Response (A-HER): A Novel Paradigm for Studying Brain Processing of Uncertain Information

This paper introduces a novel experimental paradigm - Auditory High Entropy Response (A-HER), which maximizes the information entropy of auditory stimulus sequences. This allows us to study how the brain processes complex information, rather than isolated individual events. Our analysis of the frequency response of the frontal theta rhythm induced by A-HER indicated a significant increase in signal-to-noise ratio and repeatability compared to zero-entropy Auditory Steady-State Response (A-SSR) and low-entropy mismatch negativity (MMN). We further investigated whether the A-HER response was induced by stimulus sequence differences or uncertainty, and studied its propagation rules. Different principles between evoked and entrained were found in A-HER and A-SSR. In conclusion, the A-HER paradigm, by maximizing stimulus sequence uncertainty, offers a new approach to analyzing how the brain processes uncertain information. It has potential for diagnosing and researching neurological and mental diseases, and for brain-computer interfaces, thus potentially impacting neuroscience, cognitive science, and psychology.

bioengineering↗

bioRxiv2023

LLPS condensates of Fha initiate the inside-out assembly of the type VI secretion system

The type VI secretion system (T6SS) is one of the most powerful nanomachines employed by Gram-negative pathogens for penetrating diverse cell envelopes, including bacteria and fungi, to deliver potent effectors into target cells. While the membrane-anchored contractile tubular structure of the T6SS is well characterized, the assembly process remains poorly understood. The prevailing model suggests that the assembly of T6SS initiates from its outer-membrane component. Here, we report a distinct model that the cytoplasmic protein Fha initiates T6SS assembly in Acidovorax citrulli, an important plant pathogen. Fha dictates the formation of the inner-membrane complex and the baseplate, and directly interacts with these key components. Importantly, imaging and biochemical assays reveal that Fha undergoes liquid-liquid phase separation (LLPS), forming condensates that selectively recruit essential T6SS proteins, which are otherwise dispersed in cells. Fha also exhibited conserved functions in human pathogens Vibrio cholerae and Pseudomonas aeruginosa. These findings unveil an inside-first LLPS-driven model for T6SS assembly and suggest LLPS might be broadly involved in mediating the assembly of bacterial macromolecular complexes and facilitating interspecies interactions and pathogenesis. Significance statementThe T6SS plays a pivotal role in interspecies competition and host-microbe interactions by delivering toxins to various prokaryotes and eukaryotes. Its crucial function relies on a membrane-anchored macromolecular structure comprising at least 13 conserved components. However, the mechanisms governing the efficient assembly of its diverse cytosolic and membrane-bound components remain elusive. Here, we identify Fha, a conserved cytosolic protein, as a key initiator of T6SS assembly. Fha recruits multiple structural and effector components, forming LLPS condensates. Fha homologs of plant and human pathogens exhibit conserved functions. Our findings not only unveil an inside-first assembly model for the T6SS, initiating from inner-membrane and baseplate components, but also suggest LLPS may have a broader impact on bacterial physiology beyond intracellular activities.

microbiology↗