bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial fusion mediated by mitofusin 1 regulates macrophage mycobactericidal activity by enhancing autophagy
Authors: Ning Yuping , Cai Yi , Dai Youchao , Mo Siwei , Werz Oliver , Chen Xinchun
Published: December 2020

Mitochondria as a highly dynamic organelle continuously changes morphology and position during its life cycle. Mitochondrial dynamics including fission and fusion play a critical role in maintaining functional mitochondria for ATP production, which is directly linked to host defense against Mtb infection. However, how macrophages regulate mitochondrial dynamics duringMycobacterium tuberculosis(Mtb) infection remains elusive. In this study, we found that Mtb infection induced mitochondrial fusion through enhancing the expression of mitofusin 1 (MFN1), which resulted in increased ATP production. SilencingMFN1inhibited mitochondrial fusion and subsequently reduced ATP production, which, in turn, severely impaired macrophage mycobactericidal activity by inhibiting autophagy. Impairment of mycobactericidal activity and autophagy was replicated using oligomycin, an inhibitor of ATP synthase. In summary, our study revealed MFN1-mediated mitochondrial fusion is essential for macrophage mycobactericidal activity through the regulation of ATP dependent autophagy. MFN1-mediated metabolism pathway might be targets for development of host direct therapy (HDT) strategy against TB.

Importance

How mitochondrial dynamic is regulated in attempt to fight against Mtb remains elusive. Our study revealed that the fission/fusion dynamics of mitochondria during Mtb infection is regulated by MFN1, through which mitochondrial respiration and autophagy activity are affected. Our findings suggested that intervention of energy metabolism by targeting MFN1 might be a strategy against Mtb infection.
Regulation of pancreatic fibrosis by acinar cell-derived exosomal miR-130a-3p via targeting of stellate cell PPAR-γ
Authors: Wang Qiang , Wang Hao , Jing Qingxu , Yang Yang , Xue Dongbo , Hao Chen jun , Zhang Weihui
Published: December 2020

As endogenous miRNA carriers,exosomes play a role in the pathophysiological process of various diseases. However, their functions and regulation mechanisms in pancreatic fibrosis remain unclear. In this study, an RNA microarray was used to detect differentially expressed exosomal miR-130a-3p in AR42J cells after taurolithocholate (TLC) treatment. mRNA-seq was used to screen the differentially expressed PPAR-γ after pancreatic stellate cell (PSC) activation. Fluorescence labeling of exosomes and dynamic tracing showed that exosomes can fuse with the cell membrane of PSCs and transport miR-130a-3p into PSCs. A luciferase reporter gene assay was used to confirm that miR-130a can bind to PPAR-γ to inhibit PPAR-γ expression. In vitro and in vivo functional experiments were performed for gain-of-function studies and loss-of-function studies, respectively. These studies showed that acinar cell-derived exosomal miR-130a-3p promotes PSC activation and collagen formation through targeting of cellular PPAR-γ. Knockdown of miR-130a-3p significantly improved pancreatic fibrosis. Notably, miR-130a-3p knockdown reduced serum levels of hyaluronic acid (HA) and β-amylase and increased the C-peptide to protect endocrine and exocrine pancreatic functions and the function of endothelial cells. The exosomal miR-130a-3p/PPAR-γ axis participates in the activation of PSCs and the mechanism of CP fibrosis, thus providing a potential new target for the treatment of chronic pancreatitis fibrosis.
Spatio-temporal Dynamics of the Patterning ofArabidopsisFlower Meristem
Authors: Díaz José , Álvarez-Buylla Elena R.
Published: December 2020

The qualitative model presented in this work recovers the onset of the four fields that correspond to those of each floral organ whorl ofArabidopsisflower, suggesting a mechanism for the generation of the positional information required for the differential expression of the A, B and C identity genes according to the ABC model for organ determination during early stages of flower development. Our model integrates a previous model for the emergence of WUS pattern in the apical meristem, and shows that this pre-pattern is a necessary but not sufficient condition for the posterior information of the four fields predicted by the ABC model. Furthermore, our model predicts that LFY diffusion along the L1 layer of cells is not a necessary condition for the patterning of the floral meristem.
Multiple lineages of Streptomyces produce antimicrobials within passalid beetle galleries across eastern North America
Authors: de Cassia Pessotti Rita , Hansen Bridget L. , Reaso Jewel N. , Ceja-Navarro Javier A. , El-Hifnawi Laila , Brodie Eoin L. , Traxler Matthew F.
Published: December 2020

Some insects form symbioses in which actinomycetes provide defense against pathogens by making antimicrobials. The range of chemical strategies employed across these associations, and how these strategies relate to insect lifestyle, remains underexplored. We assessed subsocial passalid beetles of the species Odontotaenius disjunctus, and their frass (fecal material) which is an important food resource within their galleries, as a model insect/actinomycete system. Through chemical and phylogenetic analyses, we found that O. disjunctus frass collected across eastern North America harbored multiple lineages of Streptomyces and diverse antimicrobials. Metabolites detected in frass displayed synergistic and antagonistic inhibition of a fungal entomopathogen, Metarhizium anisopliae, and multiple streptomycete isolates inhibited this pathogen when co-cultivated directly in frass. These findings support a model in which the lifestyle of O. disjunctus accommodates multiple Streptomyces lineages in their frass, resulting in a rich repertoire of antimicrobials that likely insulates their galleries against pathogenic invasion.
Pluripotent stem cell derived models of neurological diseases reveal early transcriptional heterogeneity
Authors: Sorek Matan , Oweis Walaa , Nissim-Rafinia Malka , Maman Moria , Simon Shahar , Hession Cynthia C. , Adiconis Xian , Simmons Sean K. , Sanjana Neville , Shi Xi , Lu Congyi , Pan Jen Q. , Xu Xiaohong , Pouladi Mahmoud A. , Ellerby Lisa M. , Zhang Feng , Levin Joshua Z. , Meshorer Eran
Published: December 2020

Background

Many neurodegenerative diseases (NDs) develop only later in life, when cells in the nervous system lose their structure or function. In genetic forms of NDs, this late onset phenomenon remains largely unexplained.

Results

Analyzing single cell RNA sequencing (scRNA-seq) from Alzheimer’s disease (AD) patients, we find increased transcriptional heterogeneity in AD excitatory neurons. We hypothesized that transcriptional heterogeneity precedes ND pathologies. To test this idea experimentally, we used juvenile forms (72Q; 180Q) of Huntington’s disease (HD) iPSCs, differentiated them into committed neuronal progenitors, and obtained single cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking-down several differentially-variable genes resulted in increased aggregate formation, a pathology associated with HD. We further validated the increased transcriptional heterogeneity in CHD8+/-cells, a model for autism spectrum disorder.

Conclusions

Overall, our results suggest that although NDs develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.
THE DYNAMIC INTERPLAY BETWEEN HOMEODOMAIN TRANSCRIPTION FACTORS AND CHROMATIN ENVIRONMENT REGULATES PRONEURAL FACTOR OUTCOMES
Authors: Ang Cheen Euong , Olmos Victor Hipolito , Zhou Bo , Lee Qian Yi , Sinha Rahul , Narayanaswamy Aadit , Mall Moritz , Chesnov Kirill , Sudhof Thomas , Wernig Marius
Published: December 2020

Generation of neurons of vast diversity involves early spatial and temporal patterning of the neuronal precursors by morphogenic gradients and combinatorial expression of transcription factors. While the proneuronal function of the basic-helix-loop-helix (bHLH) transcription factor Ngn2 is well established, its role in neuronal subtype specification remains unclear. Here, we found that coexpressing NGN2 with the forebrain homeobox factor EMX1 converts human pluripotent stem cells into a highly homogeneous glutamatergic forebrain neurons without partial cholinergic and monoaminergic gene programs observed in cells infected with NGN2 only. Our molecular characterization revealed that transcriptional output and genomic targeting of Ngn2 is altered by co-factors such as EMX1 explaining the more focused subtype specification. Ngn2 function is less modified by the chromatin environment and does not affect regionalization of pre-patterned neural progenitors. These results enable improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

Highlights

NGN2 converts human ES cells into glutamatergic neurons some of which co-express a partial cholinergic program

NGN2 directly binds to and activates ISL1 in ES cells which together with PHOX2A/B induce cholinergic genes

Anterior-posterior regionalization affects NGN2 binding and transcriptional output but does not focus subtype specification

Forebrain homeobox factors including EMX1 and FOXG1 redirect NGN2 chromatin binding and repress posterior and cholinergic genes, resulting in homogeneous forebrain excitatory neurons
Learning interpretable latent autoencoder representations with annotations of feature sets
Authors: Rybakov Sergei , Lotfollahi Mohammad , Theis Fabian J. , Wolf F. Alexander
Published: December 2020

Existing methods for learning latent representations for single-cell RNA-seq data are based on autoencoders and factor models. However, representations learned by autoencoders are hard to interpret and representations learned by factor models have limited flexibility. Here, we introduce a framework for learning interpretable autoencoders based on regularized linear decoders. It decomposes variation into interpretable components using prior knowledge in the form of annotated feature sets obtained from public databases. Through this, it provides an alternative to enrichment techniques and factor models for the task of explaining observed variation with biological knowledge. Benchmarking our model on two single-cell RNA-seq datasets, we demonstrate how our model outperforms an existing factor model regarding scalability while maintaining interpretability.
CXCR6 by increasing retention of memory CD8 T cells in the ovarian tumor microenvironment promotes immunosurveillance and control of ovarian cancer
Authors: Muthuswamy Ravikumar , McGray AJ Robert , Battaglia Sebastiano , He Wenjun , Miliotto Anthony , Eppolito Cheryl , Lichty Brian , Shrikant Protul , Odunsi Kunle
Published: December 2020

Purpose

Resident memory CD8 T cells owing to their ability to reside and persist in peripheral tissues, impart adaptive sentinel activity and amplify local immune response, have beneficial implications for tumor surveillance and control. The current study aims to clarify the less known chemotactic mechanisms that govern the localization, retention, and residency of memory CD8 T cells in the ovarian tumor microenvironment.

Experimental Design

RNA/FACS based profiling of chemokine receptor expression in CD8+resident memory T cells in human ovarian cancer and analyze their association with survival. Analyze chemokine receptor role in anti-tumor response and control by resident memory T cells using prophylactic mice models of ovarian cancer, treated with adoptive transfer of OT1 T cells and vaccination with maraba virus-OVA to target Ovalbumin expressing tumor.

Results

Chemokine receptor profiling of CD8+CD103+resident memory TILs in ovarian cancer patients revealed high expression of CXCR6. Analysis of the TCGA ovarian cancer database revealed CXCR6 to be associated with CD103 and increased patient survival. Functional studies in mouse models of ovarian cancer revealed that CXCR6 is a marker of resident, but not circulatory tumor-specific memory CD8 T cells. Knockout of CXCR6 in tumor-specific CD8 T cells showed reduced retention in tumor tissues leading to diminished resident memory responses and poor control of ovarian cancer

Conclusions

CXCR6 by promoting increased retention in tumor tissues serves a critical role in resident memory T cell-mediated immunosurveillance and control of ovarian cancer. Future studies warrant exploiting CXCR6 to promote resident memory response in cancers.
A Novel Role for Nucleolin in Splice Site Selection
Authors: Shefer Kinneret , Boulos Ayub , Gotea Valer , Chaim Yair Ben , Sperling Joseph , Elnitski Laura , Sperling Ruth
Published: December 2020

Latent 5’ splice sites are highly abundant in human introns, yet, are apparently not normally used. Splicing at most of these sites would incorporate in-frame stop codons generating nonsense mRNAs. Importantly, under stress and in cancer, splicing at latent sites is activated generating nonsense mRNAs from thousands of genes. Previous studies point to an unresolved RNA quality control mechanism that suppresses latent splicing independently of NMD. They further demonstrated a pivotal role for initiator-tRNA in this mechanism, through its interaction with the AUG codon, independent of its role in protein translation. To further elucidate this mechanism, here we searched for nuclear proteins directly bound to initiator-tRNA in the nucleus. We identified nucleolin (NCL), a multifunctional, abundant, and conserved protein, as a novel regulator of splice site selection. Starting with UV crosslinking, we show that NCL is directly and specifically interacting with initiator-tRNA in the nucleus, but not in the cytoplasm. In support of NCL involvement in this mechanism, we show activation of latent splicing in hundreds of transcripts upon NCL knockdown, disrupting gene transcripts involved in several important cellular pathways and cell metabolism functions (e.g. transcription factors, oncogenes, kinases, splicing factors, translation factors, and genes affecting cell motility, proliferation, and cellular trafficking). We thus propose NCL, a component of the endogenous spliceosome, through its direct interaction with initiator-tRNA and its effect on latent splicing as the first documented protein of a nuclear quality control mechanism that regulates splice site selection to protect cells from latent splicing that would generate defective mRNAs.
Triggering closure of a sialic acid TRAP transporter substrate binding protein through binding of natural or artificial substrates
Authors: Peter Martin F. , Gebhardt Christian , Glaenzer Janin , Schneberger Niels , de Boer Marijn , Thomas Gavin H. , Cordes Thorben , Hagelueken Gregor
Published: December 2020

The pathogensVibrio choleraeandHaemophilus influenzaeuse tripartite ATP-independent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues. They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open- to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter fromV. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.
Non-invasive Differentiation of M1 and M2 Activation in Macrophages using Hyperpolarized13C MRS of Pyruvate and DHA at 1.47 Tesla
Authors: Qiao Kai , Le Page Lydia M. , Chaumeil Myriam M.
Published: December 2020

Macrophage activation, first generalized to the M1/M2 dichotomy, is a complex and central process of the innate immune response. Simply, M1 describes the classical pro-inflammatory activation, leading to tissue damage, and M2 the alternative activation promoting tissue repair. Given the central role of macrophages in multiple diseases, the ability to non-invasively differentiate between M1 and M2 activation states would be highly valuable for monitoring disease progression and therapeutic responses. Since M1/M2 activation patterns are associated with differential metabolic reprogramming, we hypothesized that hyperpolarized13C magnetic resonance spectroscopy (HP13C MRS), an innovative metabolic imaging approach, could distinguish between macrophage activation states noninvasively. The metabolic conversions of HP [1-13C]pyruvate to HP [1-13C]lactate and HP [1-13C]dehydroascorbic acid to HP [1-13C]ascorbic acid were monitored in live M1 and M2 activated J774a.1 macrophages non-invasively by HP13C MRS on a 1.47 Tesla NMR system. Our results show that both metabolic conversions were significantly increased in M1 macrophages compared to M2 and non-activated cells. Biochemical assays and high resolution1H MRS were also performed to investigate the underlying changes in enzymatic activities and metabolite levels linked to M1/M2 activation. Altogether, our results demonstrate the potential of HP13C MRS for monitoring macrophage activation states non-invasively.
The indoor environment - a potential source for intact human-associated anaerobes
Authors: Pausan Manuela-Raluca , Blohs Marcus , Mahnert Alexander , Moissl-Eichinger Christine
Published: December 2020

Background

People in westernised countries spend most of their time indoors. A healthy human microbiome relies on the interaction with and exchange of microbes that takes place between the human body and its environment. For this reason, the built environment might represent a potent source of commensal microbes. Anaerobic microbes are of particular interest, as researchers have not yet sufficiently clarified how the human microbiome acquires oxygen-sensitive microbes, such as obligate or facultative anaerobes.

Methods

We sampled ten households and used propidium monoazide to assess the viability of the collected prokaryotes. We compared the microbiome profiles based on 16S rRNA gene sequencing and confirmed our results by genetic and cultivation-based analyses.

Results

Quantitative and qualitative analysis revealed that most of the microbial taxa are of human origin. Less than 25% of the prokaryotic signatures found in built environment (BE) samples originate from intact – and thus potentially living – cells, indicating that aerobic and stress resistant taxa display an apparent survival advantage. Although the dominant microbial fraction identified on the bathroom floors is composed of aerobes, we confirmed the presence of strictly anaerobic taxa, including methanogenic archaea, in PMA-treated samples. As methanogens are regarded as highly sensitive to aerobic conditions, oxygen-tolerance experiments were performed with human-associated isolates to validate their survival. These results show that these taxa have a limited but substantial ability to survive in the BE. We determined that human-associated methanogens can survive oxic conditions for at least 6 h.

Conclusions

This study enabled us to collect strong evidence that supports the hypothesis that obligate anaerobic taxa can survive in the BE for a limited amount of time. This suggests that the BE serves as a potential source of anaerobic human commensals.
In vitroanalysis of colistin and ciprofloxacin antagonism ofPseudomonas aeruginosaphage PEV2 infection activities
Authors: Danis-Wlodarczyk Katarzyna , Cai Alice , Chen Anna , Gittrich Marissa , Sullivan Matthew B. , Wozniak Daniel J. , Abedon Stephen T.
Published: December 2020

Phage therapy is a century-old technique employing viruses (phages) to treat bacterial infections. In the clinic, phage therapy often is used in combination with antibiotics. Antibiotics, however, interfere with critical bacterial activities, such as DNA and protein synthesis, which also are required for phage infection processes. Resulting antagonistic impacts of antibiotics on phages nevertheless are not commonly determined in association with phage therapy studies using standard, planktonic approaches. Here we assess the antagonistic impact of two antibiotics, colistin and ciprofloxacin, on the bactericidal, bacteriolytic, and new virion production activities ofPseudomonas aeruginosapodovirus PEV2, using a broth culture, optical density-based ‘lysis profile’ assay. Though phage-antibiotic combinations were more potent in reducing cell viability than phages or antibiotics alone, colistin substantially interfered with phage PEV2 bacteriolytic and virion-production activities at minimum inhibitory concentration (MIC). Ciprofloxacin, by contrast, had no such impact at 1x MIC or 3x MIC. At higher but still clinically relevant concentrations (9× MIC) burst sizes were still significant (~30 phages/infected bacterium). We corroborated these lysis profile results by more traditional measurements (colony forming units, plaque forming units, one-step growth experiments) and two otherP. aeruginosaphages. To our knowledge this is the first study in which detailed antibiotic impact onP. aeruginosaphage infection activities has been determined under conditions similar to those used to determine antibiotic MICs and could point especially to ciprofloxacin as a minimally antagonistic phage therapy co-treatment ofP. aeruginosainfections.
Allosteric activation of T-cell antigen receptor signalling by quaternary structure relaxation
Authors: Lanz Anna-Lisa , Masi Giulia , Porciello Nicla , Cohnen André , Cipria Deborah , Prakaash Dheeraj , Bálint Štefan , Raggiaschi Roberto , Galgano Donatella , Cole David K. , Lepore Marco , Dushek Omer , Dustin Michael L. , Sansom Mark S. P. , Kalli Antreas C. , Acuto Oreste
Published: December 2020

The mechanism of T cell antigen receptor (TCR-CD3) signalling remains elusive. Here, we identified mutations in the transmembrane region of TCRβ or CD3ζ that augmented pMHC-induced signalling, not explicable by enhanced ligand binding, lateral diffusion, clustering or co-receptor function. Using a novel biochemical assay and molecular dynamics simulation, we demonstrated that the gain-of-function mutations loosened interaction between TCRαβ and CD3ζ. We found that, similar to the activating mutations, pMHC binding reduced TCRαβ cohesion with CD3ζ. This event occurred prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrated that soluble monovalent pMHC alone induced signalling and reduced TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to licence CD3ζ phosphorylation and initiate signal propagation.
Light spectra modify nitrogen assimilation and nitrogen content inQuercus variabilisBlume seedling components: A bioassay with15N pulses
Authors: Gao Jun , Zhang Jinsong , He Chunxia , Wang Qirui
Published: December 2020

The light spectra that reach plants change across different shading conditions, may alter the pattern of nitrogen (N) uptake and assimilation by understory regenerations that are also exposed to N deposition. We conducted a bioassay on Chinese cork oak (Quercus variabilisBlume) seedlings subjected to five-month N pulsing with15NH4Cl (10.39 atom %) at 120 mg15N plant-1under the blue (48.5% blue, 33.7% green, and 17.8% red), red (14.6% blue, 71.7% red, 13.7% green), and green (17.4% blue, 26.2% red, 56.4% green) spectra provided by light-emitting diodes (LEDs). Half of the seedlings were fed twice a week using a 250 ppm N solution with added phosphorus, potassium, and micro-nutrients, while the other half received only distilled water. Neither treatment affected growth of height, diameter, or leaf area. Compared to the red light spectrum, the blue light treatment increased chlorophyll and soluble protein contents and glutamine synthetase (GS) activity, root N concentration, and N derived from the pulses. The green light spectrum induced more biomass to allocate to the roots and a higher percentage of N derived from internal reserves compared to the other two spectra. The15N pulses demonstrated no interaction with spectra but weakened the reliance on N remobilization from acorns, strengthened biomass allocation to shoots, and induced higher chlorophyll content, GS activity, and N concentration. In conclusion, the red light spectrum should be avoided forQ. variabilisregenerations whose biomass allocation to underground organs are weakened under this condition.
Perturbations both trigger and delay seizures due to generic properties of slow-fast relaxation oscillators
Authors: Pérez-Cervera Alberto , Hlinka Jaroslav
Published: December 2020

The mechanism underlying the emergence of seizures is one of the most important unresolved issues in epilepsy research. In this paper, we study how perturbations, exogenous of endogenous, may promote or delay seizure emergence. To this aim, due to the increasingly adopted view of epileptic dynamics in terms of slow-fast systems, we perform a theoretical analysis of the phase response of a generic relaxation oscillator. As relaxation oscillators are effectively bistable systems at the fast time scale, it is intuitive that perturbations of the non-seizing state with a suitable direction and amplitude may cause an immediate transition to seizure. By contrast, and perhaps less intuitively, smaller amplitude perturbations have been found to delay the spontaneous seizure initiation. By studying the isochrons of relaxation oscillators, we show that this is a generic phenomenon, with the size of such delay depending on the slow flow component. Therefore, depending on perturbation amplitudes, frequency and timing, a train of perturbations causes an occurrence increase, decrease or complete suppression of seizures. This dependence lends itself to analysis and mechanistic understanding through methods outlined in this paper. We illustrate this methodology by computing the isochrons, phase response curves and the response to perturbations in several epileptic models possessing different slow vector fields. While our theoretical results are applicable to any planar relaxation oscillator, in the motivating context of epilepsy they elucidate mechanisms of triggering and abating seizures, thus suggesting stimulation strategies with effects ranging from mere delaying to full suppression of seizures.

Author summary

Despite its simplicity, the modelling of epileptic dynamics as a slow-fast transition between low and high activity states mediated by some slow feedback variable is a relatively novel albeit fruitful approach. This study is the first, to our knowledge, characterizing the response of such slow-fast models of epileptic brain to perturbations by computing its isochrons. Besides its numerical computation, we theoretically determine which factors shape the geometry of isochrons for planar slow-fast oscillators. As a consequence, we introduce a theoretical approach providing a clear understanding of the response of perturbations of slow-fast oscillators. Within the epilepsy context, this elucidates the origin of the contradictory role of interictal epileptiform discharges in the transition to seizure, manifested by both pro-convulsive and anti-convulsive effect depending on the amplitude, frequency and timing. More generally, this paper provides theoretical framework highlighting the role of the of the slow flow component on the response to perturbations in relaxation oscillators, pointing to the general phenomena in such slow-fast oscillators ubiquitous in biological systems.
Lactate receptor HCAR1 regulates neurogenesis and microglia activation after neonatal hypoxia-ischemia
Authors: Kennedy Lauritz H. , Glesaaen Emilie R. , Palibrk Vuk , Pannone Marco , Wang Wei , Al-Jabri Ali H.J. , Suganthan Rajikala , Meyer Niklas , Lin Xiaolin , Bergersen Linda H. , Bjørås Magnar , Rinholm Johanne E.
Published: December 2020

Neonatal cerebral hypoxia-ischemia (HI) is the leading cause of death and disability in newborns with the only current treatment option being hypothermia. An increased understanding of the pathways that facilitate tissue repair after HI can aid the development of better treatments. Here we have studied the role of lactate receptor HCAR1 (Hydroxycarboxylic acid receptor 1) in tissue repair after HI in mice. We show that HCAR1 knockout (KO) mice have reduced tissue regeneration compared with wildtype (WT) mice. Further, proliferation of neural progenitor cells and microglial activation were impaired after HI. Transcriptome analysis showed a strong transcriptional response to HI in the subventricular zone of WT mice involving about 7300 genes. In contrast, the HCAR1 KO mice showed a very modest response to HI, involving about 750 genes. Notably, fundamental processes involved in tissue repair such as cell cycle and innate immunity were dysregulated in HCAR1 KO. Taken together, our data suggest that HCAR1 is a key transcriptional regulator of the pathways that promote tissue regeneration after HI. Thus, HCAR1 could be a promising therapeutic target in the treatment of neonatal HI and other forms of brain ischemia.
Single-cell intracellular epitope and transcript detection revealing signal transduction dynamics
Authors: Rivello Francesca , van Buijtenen Erik , Matuła Kinga , van Buggenum Jessie A.G.L. , Vink Paul , van Eenennaam Hans , Mulder Klaas W. , Huck Wilhelm T. S.
Published: December 2020

Current high-throughput single-cell multi-omics methods cannot concurrently map changes in (phospho)protein levels and the associated gene expression profiles. We present QuRIE-seq (Quantification of RNA and Intracellular Epitopes by sequencing) and use multi-factor omics analysis (MOFA+) to map signal transduction over multiple timescales. We demonstrate that QuRIE-seq can trace the activation of the B-cell receptor pathway at the minute and hour time-scale and provide insight into the mechanism of action of an inhibitory drug, Ibrutinib.
Oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis and white matter degeneration
Authors: Cleveland Abigail H. , Romero-Morales Alejandra , Azcona Laurent Alfonso , Herrero Melisa , Nikolova Viktoriya D. , Moy Sheryl , Elroy-Stein Orna , Gama Vivian , Gershon Timothy R.
Published: December 2020

Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion ofBcl-xLin these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion ofMcl-1produces no phenotype. Here, we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wideMcl-1deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion ofBaxorBakrescued white matter degeneration, implicating the intrinsic apoptotic pathway inMcl-1-dependence.BaxandBakco-deletions rescued different aspects of theMcl-1-deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced ineif2b5-mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.
Deep learning enables rapid and robust analysis of fluorescence lifetime imaging in photon-starved conditions
Authors: Chen Yuan-I , Chang Yin-Jui , Liao Shih-Chu , Nguyen Trung Duc , Yang Jianchen , Kuo Yu-An , Hong Soonwoo , Liu Yen-Liang , Rylander H. Grady , Santacruz Samantha R. , Yankeelov Thomas E. , Yeh Hsin-Chih
Published: December 2020

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study the molecular states in the complex cellular environment as the lifetime readings are not biased by the fluorophore concentration or the excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termedflimGANE(fluorescence lifetimeimaging based onGenerativeAdversarialNetworkEstimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions. We demonstrated our model is not only 258 times faster than the most popular time-domain least-square estimation (TD_LSE) method but also provide more accurate analysis in barcode identification, cellular structure visualization, Förster resonance energy transfer characterization, and metabolic state analysis. With its advantages in speed and reliability,flimGANEis particularly useful in fundamental biological research and clinical applications, where ultrafast analysis is critical.
Long-term persistence of crAss-like phage crAss001 is associated with phase variation inBacteroides intestinalis
Authors: Shkoporov Andrey N. , Khokhlova Ekaterina V. , Stephens Niamh , Hueston Cara , Seymour Samuel , Hryckowian Andrew J. , Scholz Dimitri , Ross R. Paul , Hill Colin
Published: December 2020

The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of classical lysogeny, these viruses demonstrate unexplained long-term persistence in the human gut microbiome, dominating the virome in some individuals. Here we demonstrate that rapid phase variation of alternate capsular polysaccharides plays an important role in dynamic equilibrium between phage sensitivity and resistance inB. intestinaliscultures, allowing phage and bacteria to multiply in parallel. The data also suggests the role of concomitant phage persistence mechanisms associated with delayed lysis of infected cells, such as carrier state infection. From an ecological and evolutionary standpoint this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other “benign” forms of phage infection when the host is stably present at high abundance.

Teaser

CrAss-like phage persistence inBacteroidesis associated with capsule phase-variation and additional unexplored mechanisms.
The Crystal Structures of Bacillithiol Disulfide Reductase YpdA Reveal Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductases
Authors: Hammerstad Marta , Gudim Ingvild , Hersleth Hans-Petter
Published: December 2020

Low G+C Gram-positive Firmicutes, such as the clinically important pathogensStaphylococcus aureusandBacillus cereus,use the low-molecular weight (LMW) thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial in maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely fromS. aureusandB. cereus.Our analyses reveal a uniquely organized biological tetramer; however, the monomeric subunit has high structural similarity to other flavin disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in proximity to the FAD cofactor. We also report enzymatic activity for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.
Spoken language comprehension activates the primary visual cortex
Authors: Seydell-Greenwald Anna , Wang Xiaoying , Newport Elissa , Bi Yanchao , Striem-Amit Ella
Published: December 2020

Primary visual cortex (V1) is generally thought of as a low-level sensory area that primarily processes basic visual features. However, in congenitally blind individuals, V1 is involved in language processing, with no evidence of major changes in anatomical connectivity that could explain this seemingly drastic functional change. This is at odds with current accounts of neural plasticity, which emphasize the role of connectivity and conserved function in determining a neural tissue’s role even after atypical early experiences. To reconcile what appears to be unprecedented functional reorganization with known accounts of plasticity limitations, we tested whether V1 also responds to spoken language in sighted individuals. Using fMRI, we found that V1 in normally sighted individuals was indeed activated by comprehensible speech as compared to a reversed speech control condition, in a left-lateralized and focal manner. Activation in V1 for language was also significant and comparable for abstract and concrete words, suggesting it was not driven by visual imagery. Last, this activation did not stem from increased attention to the auditory onset of words, excluding general attention accounts. Together these findings suggest that V1 responds to verbal information even in sighted individuals, potentially to predict visual input. This capability might be the basis for the strong V1 language activation observed in people born blind, re-affirming the notion that plasticity is guided by pre-existing connectivity and abilities in the typically developed brain.

Significance statement

How flexible is the human brain? Studies of congenitally blind individuals showed that language activates the primary visual cortex. This has been interpreted as evidence for unprecedented functional plasticity from a low-level visual to a language area. To reconcile these findings with known limitations of plasticity based on intrinsic physiology and connectivity, we tested if similar activation can be found in sighted participants. We show that left-lateralized primary visual cortex is activated by spoken language comprehension in sighted individuals, . This suggests that plasticity even in complete blindness from birth is not limitless and is guided by pre-existing connectivity and abilities in the typically-developed brain.
A thermodynamic model of protein structure evolution explains empirical amino acid rate matrices
Authors: Norn Christoffer , André Ingemar , Theobald Douglas L.
Published: December 2020

Proteins evolve under a myriad of biophysical selection pressures that collectively control the patterns of amino acid substitutions. Averaged over time and across proteins, these evolutionary pressures are sufficiently consistent to produce global substitution patterns that can be used to successfully find homologues, infer phylogenies, and reconstruct ancestral sequences. Although the factors which govern the variation of protein substitution rates has received much attention, the influence of thermodynamic stability constraints remains unresolved. Here we develop a simple model to calculate amino acid rate matrices from evolutionary dynamics controlled by a fitness function that reports on the thermodynamic effects of amino acid mutations in protein structures. This hybrid biophysical and evolutionary model accounts for nucleotide transition/transversion rate bias, multi-nucleotide codon changes, the number of codons per amino acid, and thermodynamic protein stability. We find that our theoretical model accurately recapitulates the complex pattern of empirical rates observed in common global amino acid substitution matrices used in phylogenetics. These results suggest that selection for thermodynamically stable proteins, coupled with nucleotide mutation bias filtered by the structure of the genetic code, is the primary global driver behind the amino acid substitution patterns observed in proteins throughout the tree of life.
Molecular Transfer Model for pH effects on Intrinsically Disordered Proteins: Theory and Applications
Authors: Mugnai Mauro L. , Thirumalai D.
Published: December 2020

We present a theoretical method to study how changes in pH shape the heterogeneous conformational ensemble explored by intrinsically disordered proteins (IDPs). The theory is developed in the context of coarse-grained models, which enable a fast, accurate, and extensive exploration of conformational space at a given protonation state. In order to account for pH effects, we generalize the Molecular Transfer Model (MTM), in which conformations are re-weighted using the transfer free energy, which is the free energy necessary for bringing to equilibrium in a new environment a “frozen” conformation of the system. Using the semi-grand ensemble, we derive an exact expression of the transfer free energy, which amounts to the appropriate summation over all the protonation states. Because the exact result is computationally too demanding to be useful for large polyelectrolytes or IDPs, we introduce a mean-field (MF) approximation of the transfer free energy. Using a lattice model, we compare the exact and MF results for the transfer free energy and a variety of observables associated with the model IDP. We find that the precise location of the charged groups (the sequence), and not merely the net charge, determines the structural properties. We demonstrate that some of the limitations previously noted for MF theory in the context of globular proteins are mitigated when disordered polymers are studied. The excellent agreement between the exact and MF results poises us to use the method presented here as a computational tool to study the properties of IDPs and other biological systems as a function of pH.
Structure and dynamics of the ESX-5 type VII secretion system ofMycobacterium tuberculosis
Authors: Bunduc Catalin M. , Fahrenkamp Dirk , Wald Jiri , Ummels Roy , Bitter Wilbert , Houben Edith N.G. , Marlovits Thomas C.
Published: December 2020

Mycobacterium tuberculosiscauses one of the most important infectious diseases in humans, leading to 1.5 million deaths every year. Specialized protein transport systems, called type VII secretion systems (T7SSs), are central for its virulence, but also crucial for nutrient and metabolite transport across the mycobacterial cell envelope. Here we present the first structure of an intact T7SS inner membrane complex ofM. tuberculosis. We show how the 2.32 MDa, 165 transmembrane helices-containing ESX-5 assembly is restructured and stabilized as a trimer of dimers by the MycP5protease. A trimer of MycP5caps a central periplasmic dome-like chamber formed by three EccB5dimers, with the proteolytic sites facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5show disruption of the EccB5periplasmic assembly and increased flexibility, highlighting the importance of this component for complex integrity. Beneath the EccB5-MycP5chamber, dimers of the EccC5ATPase assemble into three four-transmembrane helix bundles, which together seal the potential central secretion channel. Individual cytoplasmic EccC5domains adopt two distinctive conformations, likely reflecting different secretion states. Our work suggests a novel mechanism of protein transport and provides a structural scaffold to aid drug development against the major human pathogen.
A multi-task deep-learning system for predicting membrane associations and secondary structures of proteins
Authors: Li Bian , Mendenhall Jeffrey , Capra John A. , Meiler Jens
Published: December 2020

Accurate prediction of secondary structures and transmembrane segments is often the first step towards modeling the tertiary structure of a protein. Existing methods are either specialized in one class of proteins or developed to predict one type of 1D structural attributes (secondary structure, topology, or transmembrane segment). In this work, we develop a new method for simultaneous prediction of secondary structure, transmembrane segment, and transmembrane topology with no a priori assumption on the class of the input protein sequence. The new method, Membrane Association and Secondary Structures of Proteins (MASSP) predictor, uses multi-tiered neural networks that incorporate recent innovations in machine learning. The first tier is a multi-task multi-layer convolutional neural network (CNN) that learns patterns in image-like input position-specific-scoring matrices (PSSMs) and predicts residue-level 1D structural attributes. The second tier is a long short-term memory (LSTM) neural network that treats the predictions of the first tier from the perspective of natural language processing and predicts the class of the input protein sequence. We curated a non-redundant data set consisting of 54 bitopic, 241 multi-spanning TM-alpha, 77 TM-beta, and 372 soluble proteins, respectively for training and testing MASSP. For secondary structure prediction, the mean three-state accuracy (Q3) of MASSP is 0.830, better than the Q3 of PSIPRED (0.829) and that of SPINE-X (0.813) and substantially better than that of Jufo9D (0.762) and RaptorX-Property (0.741). The mean segment overlap score (SOV) of MASSP is 0.752, gaining at least 7.7% improvement over all the other four methods. For transmembrane topology prediction, MASSP has a performance comparable to OCTOPUS and substantially better than MEMSAT3 and TMHMM2 on TM-alpha proteins, and on TM-beta proteins, MASSP is significantly better than both BOCTOPUS2 and PRED-TMBB2. By integrating prediction of secondary structure and transmembrane segments in a deep-learning framework, MASSP improves performance over previous methods, has broader applicability, and enables proteome scale predictions.