Miracle-dz/UniFAR-FTUs huggingface dataset:
Role of Ring6 in the function of the E. coli MCE protein LetB

LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that ΔRing3 and ΔRing6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the ΔRing6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein. Highlights Deleting MCE domains 3 or 6 from LetB confers increased resistance to membrane-perturbing agents Cryo-EM structure of ΔRing6 LetB mutant highlights the modular nature of MCE domains Mutations along LetB pore-lining loops modulate detergent resistance
A gut commensal niche regulates stable association of a multispecies microbiota

The intestines of animals are typically colonized by a complex, relatively stable microbiota that influences health and fitness, but the underlying mechanisms of colonization remain poorly understood. As a typical animal, the fruit fly, Drosophila melanogaster, is associated with a consistent set of commensal bacterial species, yet the reason for this consistency is unknown. Here, we use gnotobiotic flies, microscopy, and microbial pulse-chase protocols to show that a commensal niche exists within the proventriculus region of the Drosophila foregut that selectively binds bacteria with exquisite strain-level specificity. Primary colonizers saturate the niche and exclude secondary colonizers of the same strain, but initial colonization by Lactobacillus physically remodels the niche to favor secondary colonization by Acetobacter. Our results provide a mechanistic framework for understanding the establishment and stability of an intestinal microbiome. One-Sentence Summary A strain-specific set of bacteria inhabits a defined spatial region of the Drosophila gut that forms a commensal niche.
Evaluating the reliability, validity, and utility of overlapping networks: Implications for network theories of cognition

Brain network definitions typically assume non- or minimal overlap, ignoring regions’ connections to multiple networks. However, new methods are emerging that emphasize network overlap. Here, we investigated the reliability and validity of one assignment method, the mixed membership algorithm, and explored its potential utility for identifying gaps in existing network models of cognition. We first assessed between-sample reliability of overlapping assignment with a split-half design; a bootstrapped Dice similarity analysis demonstrated good agreement between the networks from the two subgroups. Next, we assessed whether overlapping networks captured expected nonoverlapping topographies; overlapping networks captured portions of one to three nonoverlapping topographies, which aligned with canonical network definitions. Following this, a relative entropy analysis showed that a majority of regions participated in more than one network, as is seen biologically, and many regions did not show preferential connection to any one network. Finally, we explored overlapping network membership in regions of the Dual-Networks model of cognitive control, showing that almost every region was a member of multiple networks. Thus, the mixed membership algorithm produces consistent and biologically plausible networks, which presumably will allow for the development of more complete network models of cognition.
ZBP1 induces inflammatory signaling via RIPK3 and promotes SARS-CoV-2-induced cytokine expression

COVID-19 caused by the SARS-CoV-2 virus remains a threat to global health. The disease severity is mediated by cell death and inflammation, which regulate both the antiviral and the pathological innate immune responses. ZBP1, an interferon-induced cytosolic nucleic acid sensor, facilitates antiviral responses via RIPK3. Although ZBP1-mediated cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway that depends on ubiquitination and RIPK3’s scaffolding ability independently of cell death. In human cells, ZBP1 associates with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. RIPK1 and ZBP1 are ubiquitinated to promote TAK1- and IKK-mediated inflammatory signaling. Additionally, RIPK1 recruits the p43/41-caspase-8-p43-FLIP heterodimer to suppress RIPK3 kinase activity, which otherwise promotes inflammatory signaling in a kinase activity-dependent manner. Lastly, we show that ZBP1 contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK1-RIPK3-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspase-8. Our results suggest the ZBP1 pathway contributes to inflammation in response to SARS-CoV-2 infection.
Hippocampal-Prefrontal θ Coupling Develops as Mice Become Proficient in Associative Odorant Discrimination Learning

Abstract Learning and memory requires coordinated activity between different regions of the brain. Here, we studied the interaction between infralimbic medial prefrontal cortex (mPFC) and hippocampal dorsal CA1 during associative odorant discrimination learning in the mouse. We found that as the animal learns to discriminate odorants in a go-no go task, the coupling of high-frequency neural oscillations to the phase of θ oscillations (θ-referenced phase-amplitude coupling or tPAC) changes in a manner that results in divergence between rewarded and unrewarded odorant-elicited changes in the θ phase-referenced power (tPRP) for β and γ oscillations. In addition, in the proficient animal there was a decrease in the coordinated oscillatory activity between CA1 and mPFC in the presence of the unrewarded odorant. Furthermore, the changes in tPAC resulted in a marked increase in the accuracy for decoding contextual odorant identity from tPRP when the animal became proficient. Finally, we studied the role of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα), a protein involved in learning and memory, in oscillatory neural processing in this task. We find that the accuracy for decoding the contextual odorant identity from tPRP decreases in CaMKIIα knock-out mice and that this accuracy correlates with behavioral performance. These results implicate a role for tPAC and CaMKIIα in olfactory go-no go associative learning in the hippocampal-prefrontal circuit.
Differential effects of intra-modal and cross-modal reward value on visual perception: ERP evidence

Stimuli associated with high reward modulate perception and such value-driven effects have been shown to originate from the modulation of the earliest stages of sensory processing in the brain. In natural environments objects comprise multiple features (imagine a rolling soccer ball, with its black and white patches and the swishing sound made during its motion), where each feature may signal different associations with previously encountered rewards. How perception of such an object is affected by the value associations of its constituent parts is unknown. The present study compares intra- and cross-modal value-driven effects on behavioral and electrophysiological correlates of visual perception. Human participants first learned the reward associations of visual and auditory cues. Subsequently, they performed a visual orientation discrimination task in the presence of previously rewarded visual or auditory cues (intra- and cross-modal cues, respectively) that were concurrently presented with the target stimulus. During the conditioning phase, when reward associations were learned and reward cues were the target of the task, reward value of both modalities enhanced the electrophysiological correlates of sensory processing in visual cortex. During the post-conditioning phase, when reward delivery was halted and previously rewarded stimuli were task-irrelevant, cross-modal value-enhanced behavioral measures of visual sensitivity whereas intra-modal value led to a trend for suppression. A similar pattern of modulations was found in the simultaneously recorded event-related potentials (ERPs) of posterior electrodes. We found an early (90-120 ms) suppression of ERPs evoked by high-value, intra-modal stimuli. Cross-modal cues led to a later value-driven modulation, with an enhancement of response positivity for high-compared to low-value stimuli starting at the N1 window (180-250 ms) and extending to the P3 (300-600 ms) responses of the posterior electrodes. These results indicate that visual cortex is modulated by the reward value of visual as well as auditory cues. Previously rewarded, task-irrelevant cues from the same or different sensory modality have a different effect on visual perception, as intra-modal high-value cues may interfere with the target processing, whereas cross-modal high-value cues boost the perception of the target.
Dcp2 C-terminal cis-binding elements control selective targeting of the decapping enzyme by forming distinct decapping complexes

A single Dcp1-Dcp2 decapping enzyme targets diverse classes of yeast mRNAs for decapping-dependent 5’ to 3’ decay, but the molecular mechanisms controlling selective mRNA targeting by the enzyme remain elusive. Through extensive genetic analyses we uncover cis-regulatory elements in the Dcp2 C-terminal domain that control selective targeting of the decapping enzyme by forming distinct decapping complexes. Two Upf1-binding motifs target the decapping enzyme to NMD substrates, and a single Edc3-binding motif targets both Edc3 and Dhh1 substrates. Pat1-binding leucine-rich motifs target Edc3 and Dhh1 substrates under selective conditions. Although it functions as a unique targeting component of specific complexes, Edc3 is a common component of multiple complexes. Xrn1 also has a specific Dcp2 binding site, allowing it to be directly recruited to decapping complexes. Collectively, our results demonstrate that Upf1, Edc3, and Pat1 function as regulatory subunits of the holo-decapping enzyme, controlling both its targeting specificity and enzymatic activation. Highlights Loss of Dcp2 cis-binding elements causes selective stabilization of distinct decapping substrates Dcp2 cis-binding elements promote the assembly of target-specific decapping complexes in vivo Xrn1 binds to Dcp2, and both Edc3 and Xrn1 are common components of multiple decapping complexes Upf1, Edc3, and Pat1 function as unique targeting subunits of the yeast holo-decapping enzyme
Biomass formation and sugar release efficiency of Populus modified by altered expression of a NAC transcription factor

Woody biomass is an important feedstock for biofuel production. Manipulation of wood properties that enable efficient conversion of biomass to biofuel reduces cost of biofuel production. Wood cell wall composition is regulated at several levels that involve expression of transcription factors such as wood-/secondary cell wall-associated NAC domains (WND or SND). In Arabidopsis thaliana, SND1 regulates cell wall composition through activation of its down-stream targets such as MYBs. The functional aspects of SND1 homologs in the woody Populus have been studied through transgenic manipulation. In this study, we investigated the role of PdWND1B, Populus SND1 sequence ortholog, in wood formation using transgenic manipulation through over-expression or silencing under the control of a vascular-specific 4-coumarate-CoA ligase (4CL) promoter. As compared to control plants, PdWND1B-RNAi plants were shorter in height, with significantly reduced stem diameter and dry biomass, whereas there were no significant differences in growth and productivity of PdWND1B over-expression plants. Conversely, PdWND1B over-expression lines showed a significant reduction in cellulose and increase in lignin content, whereas there was no significant impact on lignin content of down-regulated lines. Stem carbohydrate composition analysis revealed a decrease in glucose, mannose, arabinose, and galactose, but an increase in xylose in the over-expression lines. Transcriptome analysis revealed upregulation of several downstream transcription factors and secondary cell wall related structural genes in the PdWND1B over-expression lines that corresponded to significant phenotypic changes in cell wall chemistry observed in PdWND1B overexpression lines. Relative to the control, glucose release and ethanol production from stem biomass was significantly reduced in over-expression lines but appeared enhanced in the RNAi lines. Our results show that PdWND1B is an important factor determining biomass productivity, cell wall chemistry and its conversion to biofuels in Populus.
The distribution of fitness effects of spontaneous mutations in Chlamydomonas reinhardtii inferred using frequency changes under experimental evolution

The distribution of fitness effects (DFE) for new mutations is fundamental for many aspects of population and quantitative genetics. In this study, we have inferred the DFE in the single-celled alga Chlamydomonas reinhardtii by estimating changes in the frequencies of 254 spontaneous mutations under experimental evolution and equating the frequency changes of linked mutations with their selection coefficients. We generated seven populations of recombinant haplotypes by crossing seven independently derived mutation accumulation lines carrying an average of 36 mutations in the homozygous state to a mutation-free strain of the same genotype. We then allowed the populations to evolve under natural selection in the laboratory by serial transfer in liquid culture. We observed substantial and repeatable changes in the frequencies of many groups of linked mutations, and, surprisingly, as many mutations were observed to increase as decrease in frequency. We developed a Bayesian Monte Carlo Markov Chain method to infer the DFE. This computes the likelihood of the observed distribution of changes of frequency, and obtains the posterior distribution of the selective effects of individual mutations, while assuming a two-sided gamma distribution of effects. We infer that the DFE is a highly leptokurtic distribution, and that approximately equal proportions of mutations have positive and negative effects on fitness. This result is consistent with what we have observed in previous work on a different C. reinhardtii strain, and suggests that a high fraction of new spontaneously arisen mutations are advantageous in a simple laboratory environment.
Pyronaridine Protects Against SARS-CoV-2 in Mouse

There are currently relatively few small-molecule antiviral drugs that are either approved or emergency approved for use against SARS-CoV-2. One of these is remdesivir, which was originally repurposed from its use against Ebola and functions by causing early RNA chain termination. We used this as justification to evaluate three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg. Out of these we previously identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. Herein, the in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS CoV-2 infected mice. Furthermore, the pyronaridine treated group reduced lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Notably, pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 µM) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. Interestingly, pyronaridine also selectively inhibits the host kinase CAMK1 (IC50 of 2.4 µM). We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19. One sentence summary There is currently intense interest in discovering small molecules with direct antiviral activity against the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Pyronaridine, an antiviral drug with in vitro activity against Ebola, Marburg and SARS-CoV-2 has now statistically significantly reduced the viral load in mice along with IL-6, TNF-α, and IFN-β ultimately demonstrating a protective effect against lung damage by infection to provide a new potential treatment for testing clinically.
Multivariate pattern analysis reveals location specific aftereffects of 10Hz motor cortex transcranial alternating current stimulation

Background Transcranial alternating current stimulation (tACS) may induce frequency-specific aftereffects on brain oscillations in the stimulated location, which could serve as evidence for region-specific neuroplasticity. Aftereffects of tACS on the motor system remain unknown. Objective To find evidence for aftereffects in short EEG segments following tACS to two critical nodes of the motor network, namely, left motor cortex (lMC) and right cerebellum (rCB). We hypothesized that aftereffects of lMC will be stronger in and around lMC compared to both active stimulation of rCB, as well as inactive (“sham”) control conditions. Methods To this end, we employed multivariate pattern analysis (MVPA), and trained a classifier to distinguish between EEG signals following each of the three stimulation protocols. This method accounts for the multitude facets of the EEG signal and thus is more sensitive to subtle modulation of the EEG signal. Results EEG signals in both theta (θ, 4-8Hz) and alpha (α, 8-13Hz) were better classified to lMC-tACS compared to rCB-tACS/sham, in and around lMC-tACS stimulation locations (electrodes FC3 and CP3). This effect was associated with a decrease in power following tACS. Source reconstruction revealed significant differences in premotor cortex but not in primary motor cortex as the computational model suggested. Correlation between classification accuracies in θ and α in lMC-tACS was stronger compared to rCB-tACS/sham, suggesting cross-frequency effects of tACS. Nonetheless, θ/α phase-coupling did not differ between stimulation protocols. Conclusions Successful classification of EEG signals to left motor cortex using MVPA revealed focal tACS aftereffects on the motor cortex, indicative of region-specific neuroplasticity.
Repression of the Hox gene abd-A by ELAV-mediated Transcriptional Interference

Intergenic transcription is a common feature of eukaryotic genomes and performs important and diverse cellular functions. Here, we investigate the iab-8 ncRNA from the Drosophila Bithorax Complex and show that this RNA is able to repress the transcription of genes located at its 3’ end by a sequence-independent, transcriptional interference mechanism. Although this RNA is expressed in the early epidermis and CNS, we find that its repressive activity is limited to the CNS, where in wild-type embryos, it acts on the Hox gene, abd-A located immediately downstream of it. The CNS specificity is achieved through a 3’ extension of the transcript, mediated by the neuronal-specific, RNA-binding protein, ELAV. Loss of ELAV activity eliminates the 3’ extension and results in the ectopic activation of abd-A. Thus, a tissue-specific change in the length of a ncRNA is used to generate a precise pattern of gene expression in a higher eukaryote.
Correlation of mitochondrial TOM core complex stop-and-go and open-closed channel dynamics

The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. Here, we explore the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of β-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. Our approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.
Nup358 regulates remodelling of ER-mitochondrial contact sites and autophagy

The contact sites between ER and mitochondria regulate several cellular processes including inter-organelle lipid transport, calcium homeostasis and autophagy. However, the mechanisms that regulate the dynamics and functions of these contact sites remain unresolved. We show that annulate lamellae (AL), a relatively unexplored subcellular structure representing subdomains of ER enriched with a subset of nucleoporins, are present at ER-mitochondria contact sites (ERMCS). Depletion of one of the AL-resident nucleoporins, Nup358, results in increased contacts between ER and mitochondria. Mechanistically, Nup358 modulates ERMCS dynamics by restricting mTORC2/Akt signalling. Our results suggest that growth factor-mediated remodelling of ERMCS depends on a reciprocal binding of Nup358 and mTOR to the ERMCS tethering complex consisting of VAPB and PTPIP51. Furthermore, Nup358 also interacts with IP3R, an ERMCS-enriched Ca2+ channel, and controls Ca2+ release from the ER. Consequently, depletion of Nup358 leads to elevated cytoplasmic Ca2+ and autophagy via activation of Ca2+/CaMKK2/AMPK axis. Our study thus uncovers a novel role for AL, particularly for Nup358, in regulating mTORC2-mediated ERMCS remodelling and Ca2+-directed autophagy, possibly via independent mechanisms.