bluuebunny/crossref_metadata_2025 huggingface dataset:
Autophagy is required for the rejection of self-incompatible pollen in two accessions of transgenicArabidopsis thaliana
Authors: Macgregor Stuart R. , Lee Hyun Kyung , Nelles Hayley , Johnson Daniel C. , Zhang Tong , Ma Chaozhi , Goring Daphne R.
Published: August 2020

Successful reproduction in the Brassicaceae is mediated by a complex series of interactions between the pollen and the pistil, and some species have an additional layer of regulation with the self-incompatibility trait. While the initial activation of the self-incompatibility pathway by the pollen S-locus protein11/S-locus cysteine-rich peptide and the stigmaSReceptor Kinase is well characterized, the downstream mechanisms causing self-pollen rejection are still not fully understood. In previous studies, we had detected the presence of autophagic bodies with self-incompatible pollinations inArabidopsis lyrataand transgenicA. thalianalines, but it was not known if autophagy was essential for self-pollen rejection. Here, we investigated the requirement of autophagy in this response by crossing mutations in the essentialAUTOPHAGY7(ATG7) orAUTOPHAGY5(ATG5) genes into two different transgenic self-incompatibleA. thalianalines in the Col-0 and C24 accessions. By using these previously characterized transgenic lines that expressA. lyrataandA. halleriself-incompatibility genes, we demonstrated that disrupting autophagy can weaken their self-incompatible responses in the stigma. When theatg7oratg5mutations were present, an increased number of self-incompatible pollen were found to hydrate and form pollen tubes that successfully fertilized the self-incompatible pistils. Additionally, we confirmed the presence of GFP-ATG8a labelled autophagosomes in the stigmatic papillae following self-incompatible pollinations. Together, these findings support the requirement of autophagy in the self-incompatibility response and add to the growing understanding of the cellular events that take place in the stigma to reject self-pollen.

One Sentence Summary

In self-incompatible transgenicArabidopsis thalianalines, autophagy is an integral part of the cellular responses in the stigma to efficiently block fertilization by self-incompatible pollen.
Gene disruption by structural mutations drives selection in US rice breeding over the last century
Authors: Vaughn Justin N. , Korani Walid , Stein Joshua C. , Edwards Jeremy D. , Peterson Daniel G. , Simpson Sheron A. , Youngblood Ramey C. , Grimwood Jane , Ware Doreen H. , McClung Anna M. , Scheffler Brian E.
Published: August 2020

The genetic basis of general plant vigor is of major interest to food producers, yet the trait is recalcitrant to genetic mapping because of the number of loci involved, their small effects, and linkage. Observations of heterosis in many crops suggests that recessive, malfunctioning versions of genes are a major cause of poor performance, yet we have little information on the mutational spectrum underlying these disruptions. To address this question, we generated a long-read assembly of a tropicaljaponicarice (Oryza sativa) variety, Carolina Gold, which allowed us to identify structural mutations (>50 bp) and orient them with respect to their ancestral state using the outgroup,Oryza glaberrima. Supporting prior work, we find substantial genome expansion is thesativabranch. While transposable elements (TEs) account for the largest share of size variation, the majority of events are not directly TE-mediated. Tandem duplications are the most common source of insertions and are highly enriched among 50-200bp mutations. To explore the relative impact of various mutational classes on crop fitness, we then track these structural events over the last century of US rice improvement using 101 resequenced varieties. Within this material, a pattern of temporary hybridization between medium and long-grain varieties was followed by recent divergence. During this long-term selection, structural mutations that impact gene exons have been removed at a greater rate than intronic indels and single-nucleotide mutations. These results support the use ofab initioestimates of mutational burden, based on structural data, as an orthogonal predictor in genomic selection.

Significance Statement

Some crop varieties have superior performance across years and environments. In hybrids, harmful mutations in one parent are masked by the ancestral alleles in the other parent, resulting in increased vigor. Unfortunately, these mutations are very difficult to identify precisely because, individually, they only have a small effect. In this study, we use long-read sequencing to characterize the entire mutational spectrum between two rice varieties. We then track these mutations through the last century of rice breeding. We show that large structural mutations in exons are selected against at a greater rate than any other mutational class. These findings illuminate the nature of deleterious alleles and will guide attempts to predict variety vigor based solely on genomic information.
Embryo CHH hypermethylation is mediated by RdDM and is autonomously directed inBrassica rapa
Authors: Chakraborty Tania , Kendall Timmy , Grover Jeffrey W. , Mosher Rebecca A.
Published: August 2020

Background

RNA directed DNA methylation (RdDM) initiates cytosine methylation in all contexts, and maintains asymmetric CHH methylation (where H is any base other than G). Mature plant embryos show one of the highest levels of CHH methylation, and it has been suggested that RdDM is responsible for this hypermethylation. Because loss of RdDM inBrassica rapacauses seed abortion, embryo methylation might play a role in seed development. RdDM is required in the maternal sporophyte, suggesting that small RNAs from the maternal sporophyte might translocate to the developing embryo, triggering DNA methylation that prevents seed abortion. This raises the question whether embryo hypermethylation is autonomously regulated by the embryo itself or influenced by the maternal sporophyte.

Results

Here, we demonstrate thatB. rapaembryos are hypermethylated in both euchromatin and heterochromatin and that this process requires RdDM. Contrary to current models,B. rapaembryo hypermethylation is not correlated with demethylation of the endosperm. We also show that maternal somatic RdDM is not sufficient for global embryo hypermethylation, and we find no compelling evidence for maternal somatic influence over embryo methylation at any locus. Decoupling of maternal and zygotic RdDM leads to successful seed development despite loss of embryo CHH hypermethylation.

Conclusions

We conclude that embryo CHH hypermethylation is conserved, autonomously controlled, and not required for embryo development. Furthermore, maternal somatic RdDM, while required for seed development, does not directly influence embryo methylation patterns.
Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases
Authors: Kato Kazuki , Ahmad Sadeem , Zhu Zixiang , Young Janet M. , Mu Xin , Park Sehoon , Malik Harmit S. , Hur Sun
Published: August 2020

RNA helicases and ubiquitin E3 ligases mediate many critical functions within cells, but their actions have been studied largely in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryo-electron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity-driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass-spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses thus reveal not only conserved substrates for TRIM proteins but also unexpectedly deep evolutionary connections between TRIM proteins and RNA helicases, thereby linking ubiquitin and RNA biology throughout animal evolution.
Estimating fecundity and density dependence from mark-recapture data for making population projections
Authors: Şen Bilgecan , Akçakaya H. Reşit
Published: August 2020

Forecasting changes in size and distributions of populations is at the forefront of ecological sciences in the 21stcentury. Such forecasts require robust estimators of fecundity, survival and density-dependence. While survival estimation is the main focus of mark-recapture modelling, fecundity and density dependence are rarely the subject of these models. Here, we demonstrate that these parameters can be simultaneously estimated in a Bayesian framework using only robust design mark-recapture data. Using simulated capture histories, we show that this framework (which we named CJS-pop) can estimate vital rates and their density dependence with little bias. When CJS-pop is applied to capture history data from Brown Creeper (Certhia americana), it provides estimates of fecundity that is expected from the breeding biology of this species. Finally, we illustrate that density dependence, even when estimated with uncertainty in the CJS-pop framework, regularizes population dynamics and reduces the frequent population extinctions and explosions observed under density-independent models. While CJS-pop as a whole is a useful addition to the current mark-recapture modelling toolbox, we argue that the independent components of this framework in estimating fecundity and density dependence can be integrated to other CJS frameworks, potentially creating models capable of population projections.
Growth-laws and invariants from ribosome biogenesis in lower Eukarya
Authors: Kostinski Sarah , Reuveni Shlomi
Published: August 2020

Eukarya and Bacteria are the most evolutionarily distant domains of life, which is reflected by differences in their cellular structure and physiology. For example, Eukarya feature membrane-bound organelles such as nuclei and mitochondria, whereas Bacteria have none. The greater complexity of Eukarya renders them difficult to study from both an experimental and theoretical perspective. However, encouraged by a recent experimental result showing that budding yeast (a unicellular eukaryote) obeys the same proportionality between ribosomal proteome fractions and cellular growth rates as Bacteria, we derive a set of relations describing eukaryotic growth from first principles of ribosome biogenesis. We recover the observed ribosomal protein proportionality, and then continue to obtain two growth-laws for the number of RNA polymerases synthesizing ribosomal RNA per ribosome in the cell. These growth-laws, in turn, reveal two invariants of eukaryotic growth, i.e. quantities predicted to be conserved by Eukarya regardless of growth conditions. The invariants, which are the first of their kind for Eukarya, clarify the coordination of transcription and translation kinetics as required by ribosome biogenesis, and link these kinetic parameters to cellular physiology. We demonstrate application of the relations to the yeastS. cerevisiaeand find the predictions to be in good agreement with currently available data. We then outline methods to quantitatively deduce several unknown kinetic and physiological parameters. The analysis is not specific toS. cerevisiaeand can be extended to other lower (unicellular) Eukarya when data become available. The relations may also have relevance to certain cancer cells which, like bacteria and yeast, exhibit rapid cell proliferation and ribosome biogenesis.
Cell growth model with stochastic gene expression helps understand the growth advantage of metabolic exchange and auxotrophy
Authors: Dutta Dibyendu , Saini Supreet
Published: August 2020

During cooperative growth, microbes often experience higher fitness, due to sharing of resources by metabolic exchange and herd protection through biofilm structures. However, the trajectory of evolution of competitive species towards cooperation is not known. Moreover, existing models (based on optimisation of steady-state resources or fluxes) are often unable to explain the growth advantage for the cooperating species, even for simple reciprocally cross-feeding auxotrophic pairs. We present an abstracted model of cell growth that considers the stochastic burst-like gene expression of biosynthetic pathways of limiting biomass precursor metabolites, and directly connects their cellular levels to growth and division using a “metabolic sizer/adder” rule. Our model recapitulates Monod’s law and yields the experimentally observed right-skewed long-tailed distribution of cell doubling times. The model further predicts the growth effect of secretion and uptake of metabolites, by linking it to changes in the internal metabolite levels. The model also explains why auxotrophs may grow faster when provided the metabolite they cannot produce, and why a pair of reciprocally cross-feeding auxotrophs can grow faster than prototrophs. Overall, our framework allows us to predict the growth effect of metabolic interactions in microbial communities and also sets the stage to study the evolution of these interactions.

Importance

Cooperative behaviours are highly prevalent in the wild, but we do not understand how it evolves. Metabolic flux models can demonstrate the viability of metabolic exchange as cooperative interactions, but steady-state growth models cannot explain why cooperators grow faster. We present a stochastic model that connects growth to the cell’s internal metabolite levels and quantifies the growth effect of metabolite exchange and auxotrophy. We show that a reduction in gene expression noise explains why cells that import metabolites or become auxotrophs can grow faster, and also why reciprocal cross-feeding of metabolites between complementary auxotrophs allow them to grow faster. Our framework can simulate the growth of interacting cells, which will enable us to understand the possible trajectories of the evolution of cooperationin silico.
Toxoplasma gondiiinfections are associated with boldness towards lions in wild hyena hosts
Authors: Gering Eben , Laubach Zachary M. , Weber Patricia , Hussey Gisela Soboll , Lehmann Kenna D. S. , Montgomery Tracy M. , Turner Julie W. , Perng Wei , Pioon Malit O. , Holekamp Kay E. , Getty Thomas
Published: August 2020

Toxoplasma gondiiis widely reported to manipulate the behavior of its non-definitive hosts in ways that promote lethal interactions with the parasite’s definitive feline hosts. Nonetheless, there is a lack of data on the association betweenT. gondiiinfection and costly behavioral interactions with felids in nature. Here, we report that three decades of field observations revealT. gondiiinfected hyena cubs approach lions more closely than uninfected peers and have higher rates of lion mortality. Our findings support the hypothesis thatT. gondii’smanipulation of host boldness is an extended phenotype that promotes parasite transmission from intermediate hosts to feline predators. While upregulating hyena boldness toward lions might achieve this, it may also reflect a collateral influence of manipulative traits that evolved in other hosts (e.g., rodents). In either case, our findings corroborate the potential impacts of a globally distributed and generalist parasite (T. gondii) on fitness-related interaction with felids in a wild host.

One Sentence Summary

Wild hyenas infected with the parasiteT. gondiishow evidence of costly behavioral manipulation when interacting with lions.
Underlying microevolutionary processes parallel macroevolutionary patterns in ancient Neotropical Mountains
Authors: Dantas-Queiroz Marcos Vinicius , da Costa Cacossi Tami , Leal Bárbara Simões Santos , Chaves Cleber Juliano Neves , Vasconcelos Thais N. C. , de Melo Versieux Leonardo , Palma-Silva Clarisse
Published: August 2020

Aim

The exceptional species-richness associated with mountains worldwide is linked to the fragmented topography of these areas, responsible for constantly isolating populations during periods of climatic fluctuations. Consequently, endemism and spatial turnover in mountains are very high and few species are widespread among entire mountain ranges, precluding population-level studies that help understanding how macroevolutionary patterns were shaped. Here, we used the bromeliadVriesea oligantha, a species endemic to, but widespread in, one of the most species-rich ancient montane areas in the globe, the Espinhaco Range, to test how environmental changes over time may have acted on the evolutionary history of this taxon, contributing to understanding how montane macroevolutionary patterns were shaped. Through analyses of plastidial and nuclear DNA ofV. oligantha, we dated its origin and intraspecific diversification, and estimated the genetic diversity, structure and migration rates among populations. Using climatic and geographic variables, we modeled suitable areas for the present and the past, estimating corridors between isolated populations. We also used demographic analyses to estimate ancient population dynamics ofV. oligantha. Finally, we tested whether climatic variables or geographical distance explain the observed population structure. The origin and intraspecific diversification ofV. oliganthaare related to early climatic oscillations during the Plio-Pleistocene. This species has a high population structure due to its low pollen and seed dispersibility. The analysis of species distribution modeling estimated corridors between populations in the past, whereas the structure ofV. oligantharesults from both models of isolation by distance and isolation by environment. The phylogeographic patterns ofVriesea oliganthareflect previously recognized spatial and temporal macroevolutionary patterns in the Espinhaco Range, providing insights into how microevolutionary processes may have given rise to this astonishing mountain biodiversity.
A biophysical and structural analysis of DNA binding by oligomeric hSSB1 (NABP2/OBFC2B)
Authors: El-Kamand Serene , Jergic Slobodan , Lawson Teegan , Kariawasam Ruvini , Richard Derek J. , Cubeddu Liza , Gamsjaeger Roland
Published: August 2020

The oxidative modification of DNA can result in the loss of genome integrity and must be repaired to maintain overall genomic stability. We have recently demonstrated that human single stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays a crucial role in the removal of 8-oxo-7,8-dihydro- guanine (8-oxoG), the most common form of oxidative DNA damage. The ability of hSSB1 to form disulphide-bonded tetramers and higher oligomers in an oxidative environment is critical for this process. In this study, we have used nuclear magnetic resonance (NMR) spectroscopy and surface plasmon resonance (SPR) experiments to determine the molecular details of ssDNA binding by oligomeric hSSB1. We reveal that hSSB1 oligomers interact with single DNA strands containing damaged DNA bases; however, our data also show that oxidised bases are recognised in the same manner as undamaged DNA bases. We further demonstrate that oxidised hSSB1 interacts with ssDNA with a significantly higher affinity than its monomeric form confirming that oligomeric proteins such as tetramers can bind directly to ssDNA. NMR experiments provide evidence that oligomeric hSSB1 is able to bind longer ssDNA in both binding polarities using a distinct set of residues different to those of the related SSB fromEscherichia coli.
Chemokines as phosphatidylserine-bound ‘find-me’ signals in apoptotic cell clearance
Authors: Pontejo Sergio M. , Murphy Philip M.
Published: August 2020

Chemokines are positively charged cytokines that attract leukocytes by binding to anionic glycosaminoglycans (GAGs) on endothelial cells for efficient presentation to leukocyte G protein-coupled receptors (GPCRs). The atypical chemokine CXCL16 has been reported to also bind the anionic phospholipid phosphatidylserine (PS), but the biological relevance of this interaction remains poorly understood. Here we demonstrate that PS binding is in fact a widely shared property of chemokine superfamily members that, like GAG binding, induces chemokine oligomerization. PS is an essential phospholipid of the inner leaflet of the healthy cell plasma membrane but it is exposed in apoptotic cells to act as an ‘eat-me’ signal that promotes engulfment of dying cells by phagocytes. We found that chemokines can bind PS in pure form as well as in the context of liposomes and on the surface of apoptotic cells and extracellular vesicles released by apoptotic cells, which are known to act as ‘find-me’ signals that chemoattract phagocytes during apoptotic cell clearance. Importantly, we show that GAGs are severely depleted from the surface of apoptotic cells and that extracellular vesicles extracted from apoptotic mouse thymus bind endogenous thymic chemokines and activate cognate chemokine receptors. Together these results indicate that chemokines tethered to surface-exposed PS may be responsible for the chemotactic and find-me signal activity previously attributed to extracellular vesicles, and that PS may substitute for GAGs as the anionic scaffold that regulates chemokine oligomerization and presentation to GPCRs on the GAG-deficient membranes of apoptotic cells and extracellular vesicles. Here, we present a new mechanism by which extracellular vesicles, currently recognized as essential agents for intercellular communication in homeostasis and disease, can transport signaling cytokines.
Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome
Authors: Lago Sara , Cernilogar Filippo M. , Kazerani Maryam , Moreno Helena Domíniguez , Nadai Matteo , Schotta Gunnar , Richter Sara N.
Published: August 2020

Cell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization1-3. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity4,5. By G4 ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with high G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s strongly emerge as new epigenetic regulators of the transcription machinery.
MitoROS due to loss ofSlc4a11in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy
Authors: Shyam Rajalekshmy , Ogando Diego G. , Choi Moonjung , Bonanno Joseph A.
Published: August 2020

Recent studies fromSlc4a11KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in theSlc4a11KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treatedSlc4a11KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections ofSlc4a11 KOmice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy inSlc4a11KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.
Efficient generation of endogenous protein reporters for mouse preimplantation embryos
Authors: O’Hagan Dan , Ralston Amy
Published: August 2020

Fluorescent proteins and epitope tags can reveal protein localization in cells and animals. However, the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. We present two approaches for higher throughput production of endogenous protein reporters. Our first approach makes use of a split fluorescent protein mNeonGreen2 (mNG2). Knock-in of a small portion of themNG2gene, in frame with gene coding regions of interest was highly efficient in embryos, eliminating the need to establish mouse lines. When complemented by the larger portion of themNG2gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. However, we report a threshold of detection using this approach. By contrast, the V5 epitope enabled high efficiency and higher sensitivity protein reporting. We describe complementary advantages and prospective applications of these two approaches.

Highlights

Split fluorescent protein for in vivo protein localization in living embryos

V5 tagging for in vivo localization of low abundance proteins

Bypassing the need for founder mouse lines for preimplantation studies

Guidelines and strategies for implementation and prospective applications
Priority effects dictate community structure and alter virulence of fungal-bacterial biofilms
Authors: Cheong Alex , Johnson Chad , Wan Hanxiao , Liu Aiping , Kernien John , Gibson Angela , Nett Jeniel , Kalan Lindsay R.
Published: August 2020

A hallmark of chronic infections are polymicrobial biofilms. The forces governing assembly and maturation of these microbial ecosystems are largely unexplored but the consequences on host response and clinical outcome can be significant. In the context of wound healing, formation of a biofilm and a stable microbial community structure is associated with impaired tissue repair resulting in a non-healing chronic wound. These types of wounds can persist for years simmering below the threshold of classical clinical infection or cycling through phases of recurrent infection. In the most severe outcome amputation of lower extremities may occur if spreading infection ensues. Here we take an ecological perspective to study priority effects and competitive exclusion on overall biofilm community structure in a three-membered community of microbes derived from a chronic wound. We find that priority effects occur across both biotic and abiotic substrates, and ecological interactions can alter both fungal physiology and host inflammatory response. We show that bacterial-competition occurs for binding to fungal structures, and some species trigger the yeast-hyphae switch, resulting in enhanced neutrophil killing and inflammation. Collectively, the results presented here facilitate our understanding of fungal-bacterial microbial community dynamics and their effects on, host-microbe interactions, pathogenesis, and ultimately, wound healing.
Auditory beat stimulation modulates memory-related single-neuron activity in the human medial temporal lobe
Authors: Derner M , Chaieb L , Dehnen G , Reber TP , Borger V , Surges R , Staresina BP , Mormann F , Fell J
Published: August 2020

Auditory beats are composed of two sine waves using nearby frequencies, which can either be applied as a superposed signal to both ears or to each ear separately. In the first case, the beat sensation results from hearing an amplitude-modulated signal (monaural beat). In the second case, it is generated by phase-sensitive neurons in the brain stem (binaural beat). We investigated the effects of monaural and binaural 5 Hz beat stimulation on neural activity and memory performance in neurosurgical patients performing an associative recognition task. Previously, we had reported that these beat stimulation conditions modulated memory performance in opposite directions. Here, we analyzed data from a patient subgroup, in which microwires were implanted in the amygdala, hippocampus, entorhinal cortex and parahippocampal cortex. We identified neurons responding with firing rate changes to binaural versus monaural 5 Hz beat stimulation. In these neurons, we correlated the differences in firing rates for binaural versus monaural beats to the memory-related differences for remembered versus forgotten items and associations. In the left hemisphere for these neurons, we detected statistically significant negative correlations between firing rate differences for binaural versus monaural beats and remembered versus forgotten items/associations. Importantly, such negative correlations were also observed between beat stimulation-related firing rate differences in the baseline window and memory-related firing rate differences in the poststimulus windows. In line with concepts of homeostatic plasticity, we interpret our findings as indicating that beat stimulation is linked to memory performance via shifting baseline firing levels.
Swirling Instability of the Microtubule Cytoskeleton
Authors: Stein David B. , De Canio Gabriele , Lauga Eric , Shelley Michael J. , Goldstein Raymond E.
Published: August 2020

In the cellular phenomena of cytoplasmic streaming, molecular motors carrying cargo along a network of microtubules entrain the surrounding fluid. The piconewton forces produced by individual motors are sufficient to deform long microtubules, as are the collective fluid flows generated by many moving motors. Studies of streaming during oocyte development in the fruit flyD. melanogasterhave shown a transition from a spatially-disordered cytoskeleton, supporting flows with only short-ranged correlations, to an ordered state with a cell-spanning vortical flow. To test the hypothesis that this transition is driven by fluid-structure interactions we study a discrete-filament model and a coarse-grained continuum theory for motors moving on a deformable cytoskeleton, both of which are shown to exhibit aswirling instabilityto spontaneous large-scale rotational motion, as observed.
Regulation of Vacuole Morphology by PIEZO Channels in Spreading Earth Moss
Authors: Radin Ivan , Richardson Ryan A. , Weiner Ethan R. , Bascom Carlisle S. , Bezanilla Magdalena , Haswell Elizabeth S.
Published: August 2020

The perception of mechanical force is a fundamental property of most, if not all cells. PIEZO channels are plasma membrane-embedded mechanosensitive calcium channels that play diverse and essential roles in mechanobiological processes in animals1,2. PIEZO channel homologs are found in plants3,4, but their role(s) in the green lineage are almost completely unknown. Plants and animals diverged approximately 1.5 billion years ago, independently evolved multicellularity, and have vastly different cellular mechanics5. Here, we investigate PIEZO channel function in the mossPhyscomitrium patens, a representative of one of the first land plant lineages.PpPIEZO1 andPpPIEZO2 were redundantly required for normal growth, size, and shape of tip-growing caulonema cells. Both were localized to vacuolar membranes and facilitated the release of calcium into the cytosol in response to hypoosmotic shock. Loss-of-function (ΔPppiezo1/2) and gain-of-function (PpPIEZO2-R2508Kand-R2508H) mutants revealed a role for moss PIEZO homologs in regulating vacuole morphology. Our work here shows that plant and animal PIEZO homologs have diverged in both subcellular localization and in function, likely co-opted to serve different needs in each lineage. The plant homologs of PIEZO channels thus provide a compelling lens through which to study plant mechanobiology and the evolution of mechanoperceptive strategies in multicellular eukaryotes.
Phylogenetic population structureand drug resistanceof Mycobacterium tuberculosis complex in the Volta Region of Ghana
Authors: Ameke Selassie , Asare Prince , Aboagye Sammy Yaw , Otchere Isaac Darko , Osei-Wusu Stephen , Yeboah-Manu Dorothy , Asante-Poku Adwoa
Published: August 2020

Context

Available molecular epidemiological data from recent studies suggest significant genetic variation between the different phylogenetic lineages ofMycobacterium tuberculosiscomplex (MTBC) and the MTBC lineages might have adapted to different human populations

Aim

This study sought to determine the phylogenetic population structure of clinical MTBC isolates from the Volta Region of Ghana.

Methods

The MTBC isolates obtained from collected sputum samples were characterized by standard methods. Non-tuberculous mycobacterial isolates were characterized by amplification of the heat shock protein 65 (hsp65) gene and sequencing. The drug susceptibility profiles of the MTBCs determined using GenoType MTBDRplus

Results

One hundred and seventeen (117, 93.6%) out of 125 mycobacterial positive isolates were characterized as members of the MTBC of whichM. tuberculosissensu stricto (MTBss) andM. africanum(Maf) were respectively 94 (80.3%) and 23 (19.7%). In all, 39 distinct spoligotype patterns were obtained; 26 for MTBss and 13 for Maf lineages. Spoligotyping identified 89 (76.04 %) Lineage 4, 16 (13.7 %) Lineage 5, 7 (6.0%) Lineage 6, 3 (2.6%) Lineage 2, 1(0.9%) Lineage 3 and 1 (0.9%) Lineage 1. Among the Lineage 4 isolates, 62/89 (69.7%) belonged to Cameroon sub-lineage, 13 (14.6%) Ghana, 8 (9.0%) Haarlem, 2 (2.2%) LAM, 1 (1.1%) Uganda I, 1 (1.1%) X and the remaining two were orphan. Significant localization of Maf was found within the Ho municipality (n=13, 29.5%) compared to the more cosmopolitan Ketu-South/Aflao (n=3, 8.3%) (p-value= 0.017). Eight (8) non-tuberculous mycobacteria were characterized asM. abscessus(7) andM. fortuitum(1)

Conclusion

We confirmed the importance ofM. africanumlineages as a cause of TB in the Volta region of Ghana.

Key Message

The phylogenetic population structure obtained agrees with previously described prevalence ofM. tuberculosiscomplex phylogenetic lineages from other regions of Ghana. It also confirms the stable prevalence ofM.africanum as an important human TB causing pathogen in Ghana.
Hypoxia-induced miR-92a regulates p53 signalling pathway and apoptosis by targeting calcium-sensing receptor in Genetically Improved Farmed Tilapia (Oreochromis niloticus)
Authors: Qiang Jun , He Jie , Tao Yi-Fan , Bao Jin-Wen , Zhu Jun-Hao , Xu Pao
Published: August 2020

miR-92a miRNAs are immune molecules that regulate apoptosis (programmed cell death) during the immune response. Apoptosis helps to maintain the dynamic balance in tissues of fish under hypoxia stress. The aim of this study was to explore the role and potential mechanisms of miR-92a in the liver of tilapia under hypoxia stress. We first confirmed thatCaSR(encoding a calcium-sensing receptor) is a target gene of miR-92a in genetically improved farmed tilapia (GIFT) using luciferase reporter gene assays. In GIFT under hypoxia stress, miR-92a was up-regulated andCaSRwas down-regulated in a time-dependent manner. Knocked-downCaSRexpression led to inhibited expression ofp53,TP53INP1andcaspase-3/8, reduced the proportion of apoptotic hepatocytes, and decreased the activity of calcium ions induced by hypoxia in hepatocytes. GIFT injected in the tail vein with an miR-92a agomir showed up-regulation of miR-92a and down-regulation ofCaSR,p53,TP53INP1, andcaspase-3/8genes in the liver, resulting in lower serum alanine aminotransferase and aspartate aminotransferase activities under hypoxia stress. These findings suggest that stimulation of miR-92a interferes with hypoxia-induced apoptosis in hepatocytes of GIFT by targetingCaSR, thereby alleviating liver damage. These results provide new insights into the adaptation mechanisms of GIFT to hypoxia stress.
Rapamycin as a potent and selective inhibitor of vascular endothelial growth factor receptor in breast carcinoma
Authors: Muhammad Sakri Muhammad Shahidan , Abdul Rahman Wan Faiziah Wan , Al-Astani Tengku Din Tengku Ahmad Damitri , Jaafar Hasnan , Gopalan Vinod
Published: August 2020

Angiogenesis is the process of new vascular formation, which is derived from various factors. For suppressing cancer cell growth, targeting angiogenesis is one of the therapeutic approaches. Vascular endothelial growth factor family receptors, including Flt-1, Flk-1, and Flt-4, have been found to play an essential role in regulating angiogenesis. In the present study, we evaluated the effects of rapamycin and platelet factor-4 toward breast carcinoma at the proteomic and genomic levels. A total of 60 N-Methyl-N-Nitrosourea-induced rat breast carcinomas were treated with rapamycin, platelet factor-4, and rapamycin+platelet factor-4. The tumors were subsequently subjected to immunohistological protein analysis and polymerase chain reaction gene analysis. Protein analysis was performed using a semi-quantitative scoring method, while the mRNA expression levels were analyzed based on the relative expression ratio. There was a significant difference in the protein and mRNA expression levels for the selected markers. In the rapamycin+platelet factor-4 treated group, the Flt-4 marker was downregulated, whereas there were no differences in the expression levels of other markers, such as Flt-1 and Flk-1. On the other hand, platelet factor-4 did not exhibit a superior angiogenic inhibiting ability in this study. Rapamycin is a potent anti-angiogenic drug; however, platelet factor-4 proved to be a less effective drug of anti-angiogenesis on rat breast carcinoma model.
A comparison of several media types and basic techniques used to assess outdoor airborne fungi in Melbourne, Australia
Authors: Black Wesley D
Published: August 2020

Despite the recent increase in interest in indoor air quality regarding mould, there is no single widely accepted standard media for the detection of airborne fungi, nor verification of many commonly used techniques. Commonly used media including malt-extract agar (MEA), Sabouraud dextrose agar (Sab, SDA, SabCG), potato dextrose agar (PDA) with and without antibiotics chloramphenicol & gentamycin (CG) were compared for their suitability in detecting a range of common airborne fungi by collecting 150 L outdoor air on a number of different days and seasons via an Anderson 400-hole sampler in suburban Melbourne, Australia. There was relatively little variation in mean numbers of colony forming units (CFU) and types of fungi recovered between MEA, PDA, SabCG media groups relative to variation within each group. There was a significant difference between SabCG, Dichloran-18% glycerol (DG18) and V8® Original juice agar media, however. Antibiotics reliably prevented the growth of bacteria that typically interfered with the growth and appearance of fungal colonies. There was no significant evidence for a growth enhancing factor from potato, mineral supplements or various vegetable juices. Differing glucose concentrations had modest effects, showing a vague ideal at 2%-4% with peptone. Sanitisation/sterilisation of the aluminium Andersen 400-hole sampler top-plate by flame is possible, but not strictly required nor advisable. The use of SabCG as a standard medium was generally supported.
Parental genome unification is highly erroneous in mammalian embryos
Authors: Cavazza Tommaso , Politi Antonio Z , Aldag Patrick , Baker Clara , Elder Kay , Blayney Martyn , Lucas-Hahn Andrea , Niemann Heiner , Schuh Melina
Published: August 2020

The vast majority of human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but the origin of this remains elusive. Using bovine embryos as a model for human embryos, we identify an error-prone mechanism of parental genome unification which often results in aneuploidy. Surprisingly, genome unification initiates hours before breakdown of the two pronuclei that encapsulate the parental genomes. While still within intact pronuclei, the parental genomes polarize towards each other, in a process driven by centrosomes, dynein, and microtubules. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other. Parental genome clustering often fails however, leading to massive chromosome segregation errors, incompatible with healthy embryo development. Nucleoli, which associate with chromatin, also cluster at the pronuclear interface in human zygotes. Defects in nucleolar clustering correlate with failure in human embryo development, suggesting a conserved mechanism.
Hydroxylation of antitubercular drug candidate, SQ109, by mycobacterial cytochrome P450
Authors: Bukhdruker Sergey , Varaksa Tatsiana , Grabovec Irina , Marin Egor , Shabunya Polina , Kadukova Maria , Grudinin Sergei , Kavaleuski Anton , Gusach Anastasiia , Gilep Andrei , Borshchevskiy Valentin , Strushkevich Natallia
Published: August 2020

Spreading of the multidrug-resistant (MDR) strains of the deadliest pathogenMycobacterium tuberculosis(Mtb) generates the need for new effective drugs. SQ109 showed activity against resistant Mtb and already advanced to Phase II/III clinical trials. Fast SQ109 degradation is attributed to the human liver Cytochrome P450s (CYPs). However, no information is available about interactions of the drug with Mtb CYPs. Here, we show that Mtb CYP124, previously assigned as a methyl-branched lipid monooxygenase, binds and hydroxylates SQ109in vitro. A 1.25 Å-resolution crystal structure of the CYP124–SQ109 complex unambiguously shows two conformations of the drug, both positioned for hydroxylation of the ω-methyl group in the trans position. The hydroxylated SQ109 presumably forms stabilizing H-bonds with its target, the Mycobacterial membrane protein Large 3 (MmpL3). We anticipate that Mtb CYPs could function as analogs of drug-metabolizing human CYPs affecting pharmacokinetics and pharmacodynamics of antitubercular (anti-TB) drugs.
Developing and Deploying a Scalable Computing Platform to Support MOOC Education in Clinical Data Science
Authors: Mayer David , Russell Seth , Wilson Melissa P. , Kahn Michael G. , Wiley Laura K.
Published: August 2020

One of the challenges of teaching applied data science courses is managing individual students’ local computing environment. This is especially challenging when teaching massively open online courses (MOOCs) where students come from across the globe and have a variety of access to and types of computing systems. There are additional challenges with using sensitive health information for clinical data science education. Here we describe the development and performance of a computing platform developed to support a series of MOOCs in clinical data science. This platform was designed to restrict and log all access to health datasets while also being scalable, accessible, secure, privacy preserving, and easy to access. Over the 19 months the platform has been live it has supported the computation of more than 2300 students from 101 countries.
Draft Genome Sequences of Nine Japanese Strains of the Kiwifruit Bacterial Canker Pathogen,Pseudomonas syringaepv. actinidiae Biovar 3
Authors: Fujikawa Takashi , Sawada Hiroyuki
Published: August 2020

Pseudomonas syringaepv. actinidiae is the pathogen that causes kiwifruit bacterial canker and is categorized into several groups (biovars). In Japan, biovar 3, known as the pandemic group, was first discovered in 2014. Here, we sequenced the genomes of nine Japanese biovar 3 strains.
A glycoengineered antigen exploiting a conserved proteinO-glycosylation pathway in theBurkholderiagenus for diagnosis of glanders infections
Authors: Wang Guanbo , Glaser Lena , Scott Nichollas E. , Mohamed Yasmine Fathy , Ingram Rebecca , Laroucau Karine , Valvano Miguel A.
Published: August 2020

We recently described a proteinO-glycosylation pathway conserved in all species of theBurkholderiagenus that results in synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders.Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that mainly infects horses, mules, donkeys and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination and eradication ofB. malleiinfections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotatedBurkholderiaglycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in theB. cenocepaciaK56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction ofB. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100 % specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected withB. pseudomallei, B. mallei, B. multivoransandB. cenocepacia. Our results indicate that proteinO-glycosylation inBurkholderiacan be exploited as a biomarker for diagnosis ofBurkholderia-associated infections.

IMPORTANCE

Glanders is a severe zoonotic disease caused by the Gram-negative bacteriumBurkholderia mallei, which affects horses, mules and donkeys, as well as humans.B. malleiis also considered a category B biothreat agent. Due to insufficient pathognomonic symptoms in the early stages of glanders, diagnosis can be difficult. Complement fixation is the most accurate and reliable serological test prescribed by the World Organization for Animal Health; however, this test has a considerable number of false-positive results. We have recently described a conserved proteinO-glycosylation pathway present in all species of theBurkholderiagenus; we also demonstrated thatBurkholderia-infected humans develop anti-glycan antibodies. Here, we exploited this system to construct and evaluate a synthetic glycoengineered protein antigen as a diagnostic tool for glanders. Our results show 100 % specificity in the detection of antibodies from infected horses, indicating that proteinO-glycosylation inBurkholderiacan be exploited as a biomarker for diagnosis ofBurkholderia-associated infections.