Mononuclear cell dynamics in <i>M</i>. <i>tuberculosis</i> infection provide opportunities for therapeutic intervention

by Brian A. Norris, Joel D. Ernst

Mycobacterium tuberculosis causes chronic infection of mononuclear phagocytes, especially resident (alveolar) macrophages, recruited macrophages, and dendritic cells. Despite the importance of these cells in tuberculosis (TB) pathogenesis and immunity, little is known about the population dynamics of these cells at the sites of infection. We used a combination of congenic monocyte adoptive transfer, and pulse-chase labeling of DNA, to determine the kinetics and characteristics of trafficking, differentiation, and infection of mononuclear phagocytes during the chronic, adaptive immune phase of M. tuberculosis infection in mice. We found that Ly6Chi monocytes traffic rapidly to the lungs, where a subpopulation become Ly6Clo and remain in the lung vascular space, while the remainder migrate into the lung parenchyma and differentiate into Ly6Chi dendritic cells, CD11b+ dendritic cells, and recruited macrophages. As in humans with TB, M. tuberculosis-infected mice have increased numbers of blood monocytes; this is due to increased egress from the bone marrow, and not delayed egress from the blood. Pulse-chase labeling of dividing cells and flow cytometry analysis revealed a T1/2 of ~15 hrs for Ly6Chi monocytes, indicating that they differentiate rapidly upon entry to the parenchyma of infected lungs; in contrast, cells that differentiate from Ly6Chi monocytes turn over more slowly, but diminish in frequency in less than one week. New cells (identified by pulse-chase labeling) acquire bacteria within 1–3 days of appearance in the lungs, indicating that bacteria regularly encounter new cellular niches, even during the chronic stage of infection. Our findings that mononuclear phagocyte populations at the site of M. tuberculosis infection are highly dynamic provide support for specific approaches for host-directed therapies directed at monocytes, including trained immunity, as potential interventions in TB, by replacing cells with limited antimycobacterial capabilities with newly-recruited cells better able to restrict and kill M. tuberculosis.

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The Zn<sub>2</sub>Cys<sub>6</sub>-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in <i>Aspergillus fumigatus</i>

by Nanbiao Long, Thomas Orasch, Shizhu Zhang, Lu Gao, Xiaoling Xu, Peter Hortschansky, Jing Ye, Fenli Zhang, Kai Xu, Fabio Gsaller, Maria Straßburger, Ulrike Binder, Thorsten Heinekamp, Axel A. Brakhage, Hubertus Haas, Ling Lu

Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.

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Order of removal of conventional and nonconventional introns from nuclear transcripts of <i>Euglena gracilis</i>

by Natalia Gumińska, Magdalena Płecha, Bożena Zakryś, Rafał Milanowski

Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5′ end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.

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Filamentation of asparagine synthetase in <i>Saccharomyces cerevisiae</i>

by Shanshan Zhang, Kang Ding, Qing-Ji Shen, Suwen Zhao, Ji-Long Liu

Asparagine synthetase (ASNS) and CTP synthase (CTPS) are two metabolic enzymes crucial for glutamine homeostasis. A genome-wide screening in Saccharomyces cerevisiae reveal that both ASNS and CTPS form filamentous structures termed cytoophidia. Although CTPS cytoophidia were well documented in recent years, the filamentation of ASNS is less studied. Using the budding yeast as a model system, here we confirm that two ASNS proteins, Asn1 and Asn2, are capable of forming cytoophidia in diauxic and stationary phases. We find that glucose deprivation induces ASNS filament formation. Although ASNS and CTPS form distinct cytoophidia with different lengths, both structures locate adjacently to each other in most cells. Moreover, we demonstrate that the Asn1 cytoophidia colocalize with the Asn2 cytoophidia, while Asn2 filament assembly is largely dependent on Asn1. In addition, we are able to alter Asn1 filamentation by mutagenizing key sites on the dimer interface. Finally, we show that ASN1D330V promotes filamentation. The ASN1D330V mutation impedes cell growth in an ASN2 knockout background, while growing normally in an ASN2 wild-type background. Together, this study reveals a connection between ASNS and CTPS cytoophidia and the differential filament-forming capability between two ASNS paralogs.

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3 minutes to precisely measure morphogen concentration

by Tanguy Lucas, Huy Tran, Carmina Angelica Perez Romero, Aurélien Guillou, Cécile Fradin, Mathieu Coppey, Aleksandra M. Walczak, Nathalie Dostatni

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of the establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the major Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to distinguish subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the hb promoter region, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that a simple model based only on the cooperative binding of Bicoid is not sufficient to describe the spatiotemporal dynamics of early hb expression.

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CXXC1 is not essential for normal DNA double-strand break formation and meiotic recombination in mouse

by Hui Tian, Timothy Billings, Petko M. Petkov

In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break formation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here, we investigated whether CXXC1 has a similar function in mouse meiosis. We created two Cxxc1 conditional knockout mouse models to deplete CXXC1 generally in germ cells, and before the onset of meiosis. Surprisingly, male knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on PRDM9 hotspot trimethylation, double strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between PRDM9-activated recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1.

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Representations of regular and irregular shapes by deep Convolutional Neural Networks, monkey inferotemporal neurons and human judgments

by Ioannis Kalfas, Kasper Vinken, Rufin Vogels

Recent studies suggest that deep Convolutional Neural Network (CNN) models show higher representational similarity, compared to any other existing object recognition models, with macaque inferior temporal (IT) cortical responses, human ventral stream fMRI activations and human object recognition. These studies employed natural images of objects. A long research tradition employed abstract shapes to probe the selectivity of IT neurons. If CNN models provide a realistic model of IT responses, then they should capture the IT selectivity for such shapes. Here, we compare the activations of CNN units to a stimulus set of 2D regular and irregular shapes with the response selectivity of macaque IT neurons and with human similarity judgements. The shape set consisted of regular shapes that differed in nonaccidental properties, and irregular, asymmetrical shapes with curved or straight boundaries. We found that deep CNNs (Alexnet, VGG-16 and VGG-19) that were trained to classify natural images show response modulations to these shapes that were similar to those of IT neurons. Untrained CNNs with the same architecture than trained CNNs, but with random weights, demonstrated a poorer similarity than CNNs trained in classification. The difference between the trained and untrained CNNs emerged at the deep convolutional layers, where the similarity between the shape-related response modulations of IT neurons and the trained CNNs was high. Unlike IT neurons, human similarity judgements of the same shapes correlated best with the last layers of the trained CNNs. In particular, these deepest layers showed an enhanced sensitivity for straight versus curved irregular shapes, similar to that shown in human shape judgments. In conclusion, the representations of abstract shape similarity are highly comparable between macaque IT neurons and deep convolutional layers of CNNs that were trained to classify natural images, while human shape similarity judgments correlate better with the deepest layers.

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Structural and dynamic basis of substrate permissiveness in hydroxycinnamoyltransferase (HCT)

by Ying-Chih Chiang, Olesya Levsh, Chun Kei Lam, Jing-Ke Weng, Yi Wang

Substrate permissiveness has long been regarded as the raw materials for the evolution of new enzymatic functions. In land plants, hydroxycinnamoyltransferase (HCT) is an essential enzyme of the phenylpropanoid metabolism. Although essential enzymes are normally associated with high substrate specificity, HCT can utilize a variety of non-native substrates. To examine the structural and dynamic basis of substrate permissiveness in this enzyme, we report the crystal structure of HCT from Selaginella moellendorffii and molecular dynamics (MD) simulations performed on five orthologous HCTs from several major lineages of land plants. Through altogether 17-μs MD simulations, we demonstrate the prevalent swing motion of an arginine handle on a submicrosecond timescale across all five HCTs, which plays a key role in native substrate recognition by these intrinsically promiscuous enzymes. Our simulations further reveal how a non-native substrate of HCT engages a binding site different from that of the native substrate and diffuses to reach the catalytic center and its co-substrate. By numerically solving the Smoluchowski equation, we show that the presence of such an alternative binding site, even when it is distant from the catalytic center, always increases the reaction rate of a given substrate. However, this increase is only significant for enzyme-substrate reactions heavily influenced by diffusion. In these cases, binding non-native substrates ‘off-center’ provides an effective rationale to develop substrate permissiveness while maintaining the native functions of promiscuous enzymes.

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Spikeling: A low-cost hardware implementation of a spiking neuron for neuroscience teaching and outreach

by Tom Baden, Ben James, Maxime J. Y. Zimmermann, Phillip Bartel, Dorieke Grijseels, Thomas Euler, Leon Lagnado, Miguel Maravall

Understanding how neurons encode and compute information is fundamental to our study of the brain, but opportunities for hands-on experience with neurophysiological techniques on live neurons are scarce in science education. Here, we present Spikeling, an open source in silico implementation of a spiking neuron that costs £25 and mimics a wide range of neuronal behaviours for classroom education and public neuroscience outreach. Spikeling is based on an Arduino microcontroller running the computationally efficient Izhikevich model of a spiking neuron. The microcontroller is connected to input ports that simulate synaptic excitation or inhibition, to dials controlling current injection and noise levels, to a photodiode that makes Spikeling light sensitive, and to a light-emitting diode (LED) and speaker that allows spikes to be seen and heard. Output ports provide access to variables such as membrane potential for recording in experiments or digital signals that can be used to excite other connected Spikelings. These features allow for the intuitive exploration of the function of neurons and networks mimicking electrophysiological experiments. We also report our experience of using Spikeling as a teaching tool for undergraduate and graduate neuroscience education in Nigeria and the United Kingdom.

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Neural timing of stimulus events with microsecond precision

by Jinhong Luo, Silvio Macias, Torbjørn V. Ness, Gaute T. Einevoll, Kechen Zhang, Cynthia F. Moss

Temporal analysis of sound is fundamental to auditory processing throughout the animal kingdom. Echolocating bats are powerful models for investigating the underlying mechanisms of auditory temporal processing, as they show microsecond precision in discriminating the timing of acoustic events. However, the neural basis for microsecond auditory discrimination in bats has eluded researchers for decades. Combining extracellular recordings in the midbrain inferior colliculus (IC) and mathematical modeling, we show that microsecond precision in registering stimulus events emerges from synchronous neural firing, revealed through low-latency variability of stimulus-evoked extracellular field potentials (EFPs, 200–600 Hz). The temporal precision of the EFP increases with the number of neurons firing in synchrony. Moreover, there is a functional relationship between the temporal precision of the EFP and the spectrotemporal features of the echolocation calls. In addition, EFP can measure the time difference of simulated echolocation call–echo pairs with microsecond precision. We propose that synchronous firing of populations of neurons operates in diverse species to support temporal analysis for auditory localization and complex sound processing.

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