title: Essential oils of plants and their combinations as an alternative adulticides against Anopheles gambiae (Diptera: Culicidae) populations.

abstract: The persistence of malaria and the increasing of resistance of Anopheles gambiae species to chemicals remain major public health concerns in sub-Saharan Africa. Faced to these concerns, the search for alternative vector control strategies as use of essential oils need to be implemented. Here, the five essential oils (EOs) from Cymbopogon citratus, Cymbopogon nardus, Eucalyptus camaldulensis, Lippia multiflora, Ocimum americanum obtained by hydro distillation were tested according to WHO procedures on Anopheles gambiae "Kisumu" and field strains collected in "Vallee du Kou". Also, the binary combinations of C. nardus and O. americanum were examined. As results, among the EOs tested, L. multiflora was the most efficient regarding KDT50 and KDT95 and rate of morality values. Our current study showed that C8 (C.n 80% : O.a 20%) and C9 (C.n 90% : O.a 10%), were more toxic to Anopheles gambiae strain VK with the mortality rates reaching 80.7 and 100% at the 1% concentration, respectively. These two binary combinations shown a synergistic effect on the susceptible population. However, only C9 showed a synergistic effect on VK population. The bioactivity of the two EOs, Cymbopogon nardus and Ocimum americanum, was improved by the combinations at certain proportions and may constitute an alternative to pyrethroid resistance.


title: Genetic modification of the ant Lasius niger using CRISPR-Cas9 technology

abstract: CRISPR-Cas9 has become one of the most prominent gene editing tools available and it has been utilized in various organisms from bacteria to fungi, plants, and animals. In this study, we developed a CRISPR-Cas9 protocol for the black garden ant Lasius niger, a common and easily available study species for lab and field environments. To create indel mutations using CRISPR-Cas9 in L. niger, we targeted three different locations in a well-studied eye pigmentation gene cinnabar, generating several mutations that disrupt the ommochrome biosynthesis pathway and result in the lack of this pigment and therefore, abnormal eye coloration in adult workers. We also developed a protocol to collect L. niger eggs, inject them with CRISPR-Cas9 construct, and rear the eggs into mature adult workers with assistance of nursing workers. We demonstrated for the first time with L. niger that CRISPR-Cas9 is an excellent tool to create targeted mutations for this species. Our protocol can be referred to when developing similar studies for other species of ants and eusocial insects.

category: genetics


title: 20 years of misunderstanding: compensatory traits can explain U-shaped cost function of purely sexual traits

abstract: The cost of ornamentation is often measured experimentally to study the relative importance of sexual and viability selection for ornamentation, but these experiments can lead to a misleading conclusion when compensatory trait is ignored. A famous, classic experiment on the outermost tail feathers in the barn swallow Hirundo rustica, explains that the U-shaped aerodynamic performance of the outermost tail feathers would be the evolutionary outcome mainly through viability selection for optimal tail length, but this conclusion depends on the assumption that compensatory traits do not cause reduced performance. Using a simple model experiment, I demonstrated that ornamentation evolved purely though sexual selection can produce a U-shaped cost function under the presence of compensatory traits. Therefore, U-shaped cost function (and the low performance of individuals with reduced ornaments) cannot be used to infer the evolutionary force favoring ornamentation, due to a previously overlooked concept, "overcompensation", which can increase, rather than decrease, the apparent cost of ornamentation.

category: animal behavior and cognition


title: Atomic model of Vesicular Stomatitis Virus and Mechanism of Assembly

abstract: Like other negative-strand RNA viruses (NSVs) such as influenza and rabies, vesicular stomatitis virus (VSV) has a three-layered organization: a layer of matrix protein (M) resides between the membrane envelope, studded by glycoprotein (G), and the nucleocapsid, composed of the nucleocapsid protein (N) and the encapsidated genomic RNA. Lack of in situ atomic structures of these viral components has limited our understanding of the virion assembly mechanism. Here, by cryoEM and sub-particle reconstruction, we have determined the in situ structures of M and N inside VSV at 3.47 [A] resolution. In the virion, N and M have a stoichiometry of 1:2. The in situ structures of both N and M differ from their crystal structures in their N-terminal segments and oligomerization loops. N-RNA, N-N, and N-M-M interactions govern the formation of the capsid. A double layer of M contributes to packaging of the helical nucleocapsid: the inner M (IM) joins neighboring turns of the N helix, while the outer M (OM) contacts G and the membrane envelope. The pseudo-crystalline organization of G is further mapped by cryoET. The mechanism of VSV assembly is delineated by the network interactions of these viral components.

category: biochemistry


title: Structure of V-ATPase from citrus fruit

abstract: Vacuolar-type ATPases (V-ATPases) are rotary proton pumps involved in numerous essential cellular processes in all eukaryotes. Difficulty in obtaining preparations of purified V-ATPase from plants in sufficient quantities for structural analysis has hindered determining the 3D structure of the plant V-ATPase. We used the Legionella pneumophila effector SidK to affinity-purify the endogenous V-ATPase from lemon fruit. The preparation was sufficient to partially cover an electron microscopy specimen grid, allowing structure determination for the enzyme in two rotational states. The structure defines the ATP:H+ ratio of the enzyme, demonstrating that it can establish a maximum {Delta}pH of [~]3, which is insufficient to maintain the low pH observed in the vacuoles of juice sac cells in lemons and other citrus fruit. Compared to the yeast and mammalian enzymes, the membrane region of the plant V-ATPase lacks subunit f and possesses an unusual configuration of transmembrane  helices. Subunit H, which inhibits ATP hydrolysis in the isolated catalytic region of V-ATPase, adopts two different conformations in the intact complex, hinting at a role in modulating activity in the intact enzyme.


title: Engineering cell morphology by CRISPR interference in Acinetobacter baylyi ADP1

abstract: Microbial production of intracellular compounds can be engineered by, for example, redirecting the carbon flux towards products and increasing the cell size. Potential engineering strategies include exploiting clustered regularly interspaced short palindromic repeats interference (CRISPRi)-based tools for controlling gene expression. Here, we applied CRISPRi for engineering Acinetobacter baylyi ADP1, a model bacterium for synthesizing intracellular storage lipids, namely wax esters. We firstly established an inducible CRISPRi system for strain ADP1, which enables tightly controlled repression of target genes. We then targeted the glyoxylate shunt to redirect carbon flow towards wax esters. Secondly, we successfully employed CRISPRi for modifying cell morphology by repressing ftsZ, an essential gene required for cell division, in combination with targeted knock-outs to generate significantly enlarged filamentous or spherical cells, respectively. The engineered cells sustained increased wax ester production metrics, demonstrating the potential of cell morphology engineering in the production of intracellular lipids.

category: bioengineering


title: The Rosenbluth sampling Calculation of Hydrophobic-Polar Model

abstract: AO_SCPLOWBSTRACTC_SCPLOWLattice proteins are models resembling real proteins. They comprise an energy function and a set of conditions specifying the interaction between elements occupying adjacent lattice sites. In this paper we present an approach examining the behavior of chains of a large number of molecules. We investigate this by solving a restricted random walk problem on a cubic lattice and square lattice. More specifically, we apply the hydrophobic-polar model to examine the spatial characteristics of protein folds using the Monte Carlo method. This technique is the so-called Rosenbluth sampling method for solving restricted random walk problems. Specifically, by solving such walks we obtain plausible folds. In addition, this method can be extended to solve the hydrophobic-polar model. In this paper, we describe this method as an algorithm that calculates the energy spectrum for the hydrophobic-polar model, and the related formula for estimating the number of folds. Moreover, we estimate the number of folds for each sequence using hydrophobic-polar model energy estimation. On test sequences the predicted protein folds were obtained with a mismatch of one unit according to the energy. We also observe that the estimated number of folds depends only on the length and not on the type of sequence. This promising strategy can be extended to quantify other proteins in nature.

category: bioinformatics


title: Interpretable deep learning to uncover the molecular binding patterns determining TCR-epitope interactions

abstract:
BackgroundThe recognition of an epitope by a T-cell receptor (TCR) is crucial for eliminating pathogens and establishing immunological memory. Prediction of the binding of any TCR-epitope pair is still a challenging task, especially with novel epitopes, because the underlying patterns that drive the recognition are still largely unknown to both domain experts and machine learning models.

ResultsThe binding of a TCR and epitope sequence can only occur when amino acids from both sequences are in close contact with each other. We analyze the distance between interacting molecules of the TCR and epitope sequences and compare this to the amino acids that are important for TCR-epitope prediction models. Important residues are determined by using interpretable deep learning techniques or, more specifically, feature attribution extraction methods, on two state-of-the-art TCR-epitope prediction models: ImRex and TITAN. Highlighting feature attributions on the molecular complex reveals additional insights to the domain expert about why the prediction was made and can offer novel insights into the factors that determine TCR affinity on a molecular level. We also show which residues of the TCR and epitope sequences determine binding prediction for ImRex and TITAN and use those to explain model performance.

ConclusionsExtracting feature attributions is a useful way to verify your model and data for challenging problems where small hard-to-detect problems can accumulate to inaccurate results.


title: A de novo MS1 feature detector for the Bruker timsTOF Pro

abstract:
1Identification of peptides by analysis of data acquired by the two established methods for bottom-up proteomics, DDA and DIA, relies heavily on the fragment spectra. In DDA, peptide features detected in mass spectrometry data are identified by matching their fragment spectra with a peptide database. In DIA, a peptides fragment spectra are targeted for extraction and matched with observed spectra. Although fragment ion matching is a central aspect in most peptide identification strategies, the precursor ion in the MS1 data reveals important characteristics as well, including charge state, intensity, monoisotopic m/z, and apex in retention time. Most importantly, the precursors mass is essential in determining the potential chemical modification state of the underlying peptide sequence. In the timsTOF, with its additional dimension of collisional cross-section, the data representing the precursor ion also reveals the peptides peak in ion mobility. However, the availability of tools to survey precursor ions with a wide range of abundance in timsTOF data across the full mass range is very limited.

Here we present a de novo feature detector called three-dimensional intensity descent (3DID). 3DID can detect and extract peptide features down to a configurable intensity level, and finds many more features than several existing tools. 3DID is written in Python and is freely available with an open-source MIT license to facilitate experimentation and further improvement (DOI 10.5281/zenodo.6513126). The dataset used for validation of the algorithm is publicly available (ProteomeXchange identifier PXD030706).

2 Author SummaryIn the identification of peptides in mass spectrometry data, much attention has been given to the targeting and extraction of mass spectra produced by fragmentation of precursor ions. However, important information about the peptide is revealed by the data representing the precursor ion itself, such as the peptides charge state, mass-to-charge ratio, intensity, and retention time. The timsTOF produces the additional dimension of ion mobility, which provides richer information about the precursor. Although tools exist for the analysis of timsTOF data, they are hampered by limited dynamic range. In this work, we describe a de novo feature detector called 3DID that detects peptide features across the full mass range. Our detector can detect more peptides than existing tools across a broader range of abundance, which enables more comprehensive analysis of the data. We believe 3DID will make a valuable contribution to the proteomics toolbox.


title: Resolution of the curse of dimensionality in single-cell RNA sequencing data analysis

abstract: Single-cell RNA sequencing (scRNA-seq) can determine gene expression in numerous individual cells simultaneously, promoting progress in the biomedical sciences. However, scRNA-seq data are high-dimensional with substantial technical noise, including dropouts. During analysis of scRNA-seq data, such noise engenders a statistical problem known as the curse of dimensionality (COD). Based on high-dimensional statistics, we herein formulate a noise reduction method, RECODE (resolution of the curse of dimensionality), for high-dimensional data with random sampling noise. We show that RECODE consistently eliminates COD in relevant scRNA-seq data with unique molecular identifiers. RECODE does not involve dimension reduction and recovers expression values for all genes, including lowly expressed genes, realizing precise delineation of cell-fate transitions and identification of rare cells with all gene information. Compared to other representative imputation methods, RECODE employs different principles and exhibits superior overall performance in cell-clustering and single-cell level analysis. The RECODE algorithm is parameter-free, data-driven, deterministic, and high-speed, and notably, its applicability can be predicted based on the variance normalization performance. We propose RECODE as a general strategy for preprocessing noisy high-dimensional data.


title: Displacement and dissociation of oligonucleotidesduring DNA hairpin closure under strain

abstract: The simple hybridization kinetic of an oligonucleotide to its template is a fundamental step in many biological processes such as replication arrest, CRISPR recognition, DNA sequencing, DNA origami, etc. Although approaches exist that address special cases of this problem, there are no simple general prediction schemes. In this work, we have measured experimentally, with no fluorescent labelling, the displacement of an oligonucleotide from its substrate in two situations: one corresponding to oligonucleotide binding/unbinding on ssDNA and one where the oligonucleotide is displaced by the refolding of a dsDNA fork. In this second situation, the fork is expelling the oligonucleotide thus reducing significantly its residence time. To account for our data in these two situations, we have constructed a mathematical model, based on the known nearest neighbor dinucleotide free energies, and provided a good estimate of the residence times of different oligonucleotides (DNA, RNA, LNA) with various length in different experimental conditions (force, temperature, buffer conditions, presence of mismatches, etc.). This study provides a foundation for the dynamics of oligonucleotide displacement, a process of importance in a number of biological and bioengineering contexts.

category: biophysics


title: Mechanical communication-induced cell directional migration and branching connections mediated by calcium channels, integrin β1 and N-cadherin

abstract: Cell-cell mechanical communications at large spatial scale (above hundreds of micrometers) have been increasingly recognized in recent decade, which shows importance in tissue-level assembly and morphodynamics. The involved mechanosensing mechanism and resulted physiological functions are still to be fully understood. Recent work showed that traction force sensation in the matrix induces cell communications for self-assembly. Here, based on the experimental model of cell directional migration on Matrigel hydrogel containing 0.5 mg/ml type I collagen, we studied the mechano-responsive pathways for cell distant communications. Airway smooth muscle (ASM) cells assembled network structure on the hydrogel, whereas stayed isolated individually when cultured on glass without force transmission. Cell directional migration, or network assembly was significantly attenuated by inhibited actomyosin activity, or inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) calcium channel or SERCA pump on endoplasmic reticulum (ER) membrane, or L-type calcium channel on the plasma membrane. Inhibition of integrin {beta}1 with siRNA knockdown reduced cell directional migration and branching assembly, whereas inhibition of cell junctional N-cadherin with siRNA had little effect on distant attractions but blocked branching assembly. Our work demonstrated that the ER calcium channels and integrin are mechanosensing signals for cell mechanical communications regulated by actomyosin activity, while N-cadherin is responsible for traction force-induced cell stable connections in the assembly.


title: Distinct Mechanisms of Innate and Adaptive Immune Regulation Underlie Poor Oncologic Outcomes Associated with KRAS-TP53 Co-Alteration in Pancreatic Cancer

abstract: Co-occurrent KRAS and TP53 mutations define a majority of patients with pancreatic ductal adenocarcinoma (PDAC) and define its pro-metastatic proclivity. Here, we demonstrate that KRAS-TP53 co-alteration is associated with worse survival compared with either KRAS-alone or TP53-alone altered PDAC in 245 patients with metastatic disease treated at a tertiary referral cancer center, and validate this observation in two independent molecularly annotated datasets. Compared with non-TP53 mutated KRAS-altered tumors, KRAS-TP53 co-alteration engenders disproportionately innate immune-enriched and CD8+ T-cell-excluded immune signatures. Leveraging in silico, in vitro, and in vivo models of human and murine PDAC, we discover a novel intersection between KRAS-TP53 co-altered transcriptomes, TP63-defined squamous trans-differentiation, and myeloid-cell migration into the tumor microenvironment. Comparison of single-cell transcriptomes between KRAS-TP53 co-altered and KRAS-altered/TP53WT tumors revealed cancer cell-autonomous transcriptional programs that orchestrate innate immune trafficking and function. Moreover, we uncover granulocyte-derived inflammasome activation and TNF signaling as putative paracrine mediators of innate immunoregulatory transcriptional programs in KRAS-TP53 co-altered PDAC. Immune subtyping of KRAS-TP53 co-altered PDAC reveals conflation of intratumor heterogeneity with progenitor-like stemness properties. Coalescing these distinct molecular characteristics into a KRAS-TP53 co-altered "immunoregulatory program" predicts chemoresistance in metastatic PDAC patients enrolled in the COMPASS trial, as well as worse overall survival.

category: cancer biology


title: An Integrated 3D bioprinted disease in a dish lung cancer model to simultaneously study drug efficacy, toxicity and metabolism

abstract:
Discovery and development of new drugs is a long, expensive and high risk proposition. Millions of dollars spent and decade plus years of time taken to discover a new drug have haunted pharma industry for many years.

In part, the reliance on animal models to make go or no go decisions for selecting drugs for human trials has been a problem because animal biology does not capture human disease in entirety. In recognition of this, the last decade has seen the emergence of more human like tools being developed in the hope of better prediction of human outcomes.

Towards that end we have developed a 3D bioprinted disease in a dish lung cancer model which uses human cells and includes ability to measure drug efficacy, toxicity and metabolism simultaneously. For drug profiling studies in our disease in a dish model we 3D bioprinted intestinal cells, layered below which were liver cells and finally underneath were target lung cancer cells. The idea was to simulate the path taken by an oral drug which encounters the gut, followed by liver and target organs. We demonstrate here that a 3D bioprinted disease model composed of human derived cells is able to concurrently measure in vitro drug efficacy, toxicity and metabolism. Such humanized models will help make early go or no go decisions on the potential of a drug to enter human trials.

category: pharmacology and toxicology


title: Modelling somatic mutation accumulation and expansion in a long-lived tree with hierarchical modular architecture

abstract: In a long-lived organism with a modular architecture, such as trees, somatic mutations accumulate throughout the long lifespan and result in genetic mosaicism in each module within the same individual. In recent years, next-generation sequencing technology has provided a snapshot of such intra-organismal genetic variability. However, the dynamic processes underlying the accumulation and expansion of somatic mutations during the growth remain poorly understood. In this study, we constructed a model to describe these processes in a form that can be applied to a real sequenced tree. Given that the proliferation dynamics of meristematic cells vary across plant species, multiple possible processes for elongation and branching were comprehensively expressed in our model. Using published data from a poplar tree, we compared the prediction of the models with the observation and explained the cell lineage dynamics underlying somatic mutations accumulation that were not evident from the snapshot of the sequenced data. We showed that the somatic genetic drift during growth decreased the intra-meristem mosaicism, resulting in mutation distribution strictly reflecting tree topology. Our modelling framework can help interpret and replenish the new empirical findings of genetic mosaicism in long-lived trees.

category: plant biology


title: Neurocomputational evidence that conflicting prosocial motives guide distributive justice

abstract: In the history of humanity, most conflicts within and between societies have originated from perceived inequality in resource distribution. How humans achieve and maintain distributive justice has therefore been an intensely studied issue. However, most research on the corresponding psychological processes has focused on inequality aversion and has been largely agnostic of other motives that may either align or oppose this behavioral tendency. Here we provide behavioral, computational, and neuroimaging evidence that distribution decisions are guided by three distinct motives - inequality aversion, harm aversion, and rank reversal aversion - that interact and in fact can also deter individuals from pursuing equality. At the neural level, we show that these three motives are encoded by separate neural systems, compete for representation in various brain areas processing equality and harm signals, and are integrated in the striatum, which functions as a crucial hub for translating the motives to behavior. Our findings provide a comprehensive framework for understanding the cognitive and biological processes by which multiple prosocial motives are coordinated in the brain to guide redistribution behaviors. This framework enhances our understanding of the brain mechanisms underlying equality-related behavior, suggests possible neural origins of individual differences in social preferences, and provides a new pathway to understand the cognitive and neural basis of clinical disorders with impaired social functions.

category: neuroscience


title: Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification

abstract: Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 12 homeobox genes in 19 distinct neuron classes that are mostly unrelated by location, function and lineage history. 10 of these 19 neuron classes had no homeobox gene function ascribed to them before, while in the other 9 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analysis, 112 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancient feature of animal nervous systems.


title: Inhibition of c-Jun in AgRP neurons mediates chronic stress-induced anxiety-like behaviors and colitis susceptibility

abstract: Psychiatric disorders, such as anxiety, are frequently associated with inflammatory bowel diseases (IBD), however, the neural mechanisms are unknown. Here, we showed that hypothalamic agouti-related protein (AgRP) neuronal activity was suppressed under chronic restraint stress (CRS), a condition known to induce anxiety-like behaviors and increase colitis susceptibility. Consistently, chemogenic activation (inhibition) of AgRP neurons reversed (mimicked) CRS-induced anxiety-like behaviors and colitis susceptibility. Furthermore, CRS inhibited AgRP neuronal activity by suppressing the expression of c-Jun. As expected, overexpression of c-Jun in these neurons protected against the CRS-induced these effects and knockdown of c-Jun in AgRP neurons (c-Jun{Delta}AgRP) promoted anxiety-like behaviors and colitis. Moreover, relieving the anxiety with cyamemazine (an anxiolytic drug) alleviated colitis susceptibility in c-Jun{Delta}AgRP mice. Finally, according to a proteomic analysis, the levels of the secreted protein thrombospondin 1 (THBS1) were negatively associated with the increased anxiety-like behaviors and colitis susceptibility, supplementing recombinant THBS1 rescued colitis in c-Jun{Delta}AgRP mice. Taken together, these results reveal a critical role of hypothalamic AgRP neuron-derived c-Jun in orchestrating chronic stress-induced anxiety-like behaviors and colitis susceptibility. These results provide a new perspective for understanding the neuronal mechanisms and potential therapeutic target for the comorbidity of psychiatric disorders, such as anxiety, and IBD.


title: Choosing memory retrieval strategies: a critical role for inhibition in the dentate gyrus

abstract:
Remembering the location of food is essential for survival. Rodents and humans employ mainly hippocampus-dependent spatial strategies, but when being stressed they shift to striatum-mediated stimulus-based strategies. To investigate underlying brain circuits, we tested mice with a heightened stress susceptibility due to a lack of the GABA-synthetizing enzyme GAD65 (GAD65-/- mice) in a dual solution task. Here, GAD65-/- mice preferred to locate a food reward in an open field via a proximal cue, while their wildtype littermates preferred a spatial strategy. The analysis of cFos co-activation across brain regions and of stress-induced mRNA expression changes of GAD65 pointed towards the hippocampal dorsal dentate gyrus (dDG) as a central structure for mediating stress effects on strategy choices via GAD65. Reducing the GAD65 expression locally in the dDG by a shRNA mediated knock down was sufficient to replicate the phenotype of the global GAD65 knock out and to increase dDG excitability. Using DREADD vectors to specifically interfere with dDG circuit activity during dual solution retrieval but not learning confirmed that the dDG modulates strategy choices and that a balanced excitability of this structure is necessary to establish spatial strategy preference.

These data highlight the dDG as a critical hub for choosing between spatial and non-spatial foraging strategies. The inhibitory/excitatory balance in the DG appears as a crucial factor for determining strategy selection in stress-susceptible mice.

HighlightsO_LIStress reduces spatial memory preferences for locating rewards in an open field
C_LIO_LIGAD65 deficient mice show reduced preferences for spatial memory strategy
C_LIO_LIDorsal dentate gyrus knock down of GAD65 is sufficient to reduce spatial strategies
C_LIO_LIExcitability in the dorsal dentate gyrus modulates retrieval strategy choices
C_LI

Graphical abstract

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title: CSF circulation and dispersion yield rapid clearancefrom intracranial compartments

abstract: In this paper we used a computational model to estimate the clearance of tracer driven by circulation of cere-brospinal fluid (CSF) produced in the choroid plexus (CP) located within the lateral ventricles. CSF was assumed to exit the subarachnoid space (SAS) via different outflow routes such as the parasagittal dura, cribriform plate and/or meningeal lymphatics. We also modelled a reverse case where fluid was produced within the spinal canal and absorbed in the CP in line with observation on certain iNPH patients. No directional interstitial fluid flow was assumed within the brain parenchyma. Tracers were injected into the foramen magnum. The models demonstrate that convection in the SAS yield rapid clearance from both the SAS and the brain interstitial fluid (ISF) and can speed up intracranial clearance from years, as would be the case for purely diffusive flow, to days.


title: PfHDAC1 is an essential regulator of parasite asexual growth with its altered genomic occupancy and activity associated with artemisinin drug resistance in Plasmodium falciparum

abstract: Plasmodium falciparum is a deadly protozoan parasite and the causative agent of malaria, which accounts for close to 200 million cases and 400,000 deaths every year. It has been identified to possess a tightly regulated gene expression profile that is integrally linked to its timely development during the intraerythrocytic stage. Epigenetic modifiers of the histone acetylation code have been identified as key regulators of the parasites transcriptome. In this study, we characterize the solitary class I histone deacetylase PfHDAC1 and demonstrate that phosphorylation is required for its catalytic activity. PfHDAC1 binds to and regulates parasite genes responsible for housekeeping and stress-responsive functions. We show that PfHDAC1 activity in parasites is crucial for normal intraerythrocytic development and that its cellular abundance is correlated with parasitemia progression. We further show that PfHDAC1 has differential abundance and genomic occupancy in artemisinin drug-resistant vs sensitive parasites and that inhibition of its deacetylase activity can modulate the sensitivity of parasites to the drug. We also identify that artemisinin exposure can interfere with PfHDAC1 phosphorylation and its genomic occupancy. Collectively, our results demonstrate PfHDAC1 to be an important regulator of basic biological functions in parasites while also deterministic of responses to environmental stresses such as antimalarial drugs.

category: microbiology


title: Minimal functional domains of the core polarity regulator Dlg

abstract:
The compartmentalized domains of polarized epithelial cells arise from mutually antagonistic actions between the apical Par complex and the basolateral Scrib module. In Drosophila, the Scrib module proteins Scribble (Scrib) and Discs-large (Dlg) are required to limit Lgl phosphorylation at the basolateral cortex, but how Scrib and Dlg could carry out such a  protection activity is not clear. We tested Protein Phosphatase 1 (PP1) as a potential mediator of this activity but demonstrate that a significant component of Scrib and Dlg regulation of Lgl is PP1-independent and found no evidence for a Scrib-Dlg-PP1 protein complex. However, the Dlg SH3 domain plays a role in Lgl protection and, in combination with the N-terminal region of the Dlg HOOK domain, in recruitment of Scrib to the membrane. We identify a  minimal Dlg comprised of the SH3 and HOOK domains that is both necessary and sufficient for Scrib localization and epithelial polarity function in vivo.

Summary StatementA minimal SH3-HOOK fragment of Dlg is sufficient to support epithelial polarity through mechanisms independent of the PP1 phosphatase.

category: developmental biology


title: Consistency in defence and competitiveness trade-off in a planktonic predator-prey system

abstract:
O_LIPredator-prey interactions play a central role in community dynamics and thus energy and matter transfers in food webs. Intraspecific variation in traits and particularly in trait combinations involved in trade-offs can alter predator-prey interactions but the underlying mechanisms governing these interactions are still unclear. Quantifying the relevant traits forming trade-off relationships and how these traits determine prey and predator fitness remains a major challenge, even for a single species. Here, we measured multiple traits related to defensive and competitive abilities to investigate the intraspecific trade-off between defence and competitiveness in 6 different strains of the green alga Chlamydomonas reinhardtii exposed to predation by the rotifer Brachionus calyciflorus and examine the consistency of the trait relationships and its consequence for the predator.
C_LIO_LIWe found significant differences in defence and competitiveness traits that were used to categorized prey strains as defended against predation and poor competitors, undefended against predation and good competitors, or intermediate in both traits. Furthermore, we found that the different morphological and trophic traits related to defence and competitiveness of prey strains were negatively correlated. The position of prey strains in trait space were consistent independent of the defence and competitiveness trait considered. As we compared trait differences between prey strains coming from environments where selection has favoured one trait or the other, these negative correlations strongly suggested the presence of a trade-off between defence and competitiveness.
C_LIO_LIOur study represents the first empirical evidence of the consistency in the expression of a defence-competitiveness trade-off at the intraspecific level. Assessing the relation between relevant traits and trade-offs and understanding how it translates into fitness of prey and predator allows improving general theory on the outcomes of predator-prey interactions and ecosystem processes.
C_LI

category: ecology


title: MITE infestation of germline accommodated by genome editing in Blepharisma

abstract: During a sophisticated developmental process, ciliates excise numerous internally eliminated sequences (IESs) from a germline genome copy, producing a functional somatic genome. Most IESs ultimately originate from transposons but homology is obscured by sequence decay. To obtain more representative perspectives on ciliate genome editing, we assembled forty thousand IESs of Blepharisma stoltei, from a much earlier-diverging lineage than existing models. Short IESs (< 115 bp) were largely non-repetitive, with a pronounced ~10 bp length periodicity, whereas longer IESs (max 7 kbp) were non-periodic and contained abundant interspersed repeats. Contrary to current models, the Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8000 copies) was a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We propose MITEs as an important and eventually self-limiting IES source. Rather than defending germline genomes against mobile elements, we argue that transposase domestication actually facilitates junk DNA accumulation.

category: genomics


title: Associations of human femoral condyle cartilage structure and composition with viscoelastic and constituent-specific material properties at different stages of osteoarthritis

abstract: The relationships between structure and function in human knee femoral cartilage are not well-known at different stages of osteoarthritis. Thus, we characterized the depth-dependent composition and structure of normal and osteoarthritic human femoral condyle cartilage (n = 47) and related them to their viscoelastic and constituent-specific mechanical properties. We observed that, in superficial cartilage, the collagen network disorganization and proteoglycan loss were associated with the smaller initial fibril network modulus (collagen pretension). Furthermore, the proteoglycan loss was associated with the greater strain-dependent fibril network modulus (a measure of nonlinear mechanical behavior). The proteoglycan loss was also associated with greater cartilage viscosity at a low loading frequency (0.005 Hz), while the disorganization of the collagen network was associated with greater cartilage viscosity at a high loading frequency (1 Hz). Our results suggest that proteoglycan degradation and collagen disorganization reduce the pretension of the collagen network while proteoglycan degradation also increases the nonlinear mechanical response of the collagen network. Further, the results also highlight that proteoglycan degradation and collagen disorganization increase the viscosity of cartilage, but their contribution to increased viscosity occurs in completely different loading frequencies.


title: Translational approach to increase phosphate accumulation in two plant species through perturbance of inositol pyrophosphates

abstract:
Inorganic phosphate (Pi), while indispensable for all biological organisms and a major agricultural macronutrient, is an increasingly limited and nonrenewable resource. Recent studies demonstrate the importance of inositol pyrophosphates (PP-InsPs) in plant Pi signaling and homeostasis, however the extent to which PP-InsPs impact plant development is not well understood. We report that transgenic expression of the Saccharomyces cerevisiae enzyme Diadenosine and Diphosphoinositol Polyphosphate Phosphohydrolase (DDP1) in Arabidopsis thaliana and Thlaspi arvense (pennycress) provide a unique translational utility for Pi phytoremediation as well as unique germplasm and insight on the long-term impacts of reduced PP-InsPs. Transgenic DDP1 expression in Arabidopsis decreased PP-InsPs, impacted growth and development, and increased Pi accumulation leading to Pi toxicity. Analysis of Pi Starvation Response (PSR) marker genes indicated that the PSR is activated in DDP1 expressing plants. We assessed translational utility through transformation of pennycress, a spring annual cover crop with emerging importance as a biofuel crop, with a DDP1 transgene. Pennycress plants expressing DDP1 showed similar altered Pi accumulation phenotypes, suggesting that these plants could potentially serve to remove Pi from Pi-rich soils. Our study addresses the long-term impacts of PP-InsP reduction on plant growth, as well as establishing a starting material for a unique Pi reclaiming cover crop.

SIGNIFICANCE STATEMENTA major challenge to food security is the phosphorus (P) crisis. A global P shortage is imminent based on the misuse of current resources and will be further aggravated by climate change and a lack of policy addressing sustainability. Our work addresses this crisis by investigating the sustained impact of altering inositol pyrophosphates to manipulate plant P accumulation, a strategy that could be used to remediate nutrient-polluted environments.


title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

abstract: NK cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human hematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2R{beta} expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations.

category: immunology


title: Integrated viral elements unveil the dual lifestyle of Tetraselmis spp. polinton-like viruses

abstract: In this study, we aimed at exploring horizontal gene transfer between viruses and Chlorodendraceae green algae (Chlorophyta) using available genomic and transcriptomic sequences for 20 algal strains. We identified a significant number of genes sharing a higher sequence similarity with viral homologues, thus signaling their possible involvement in HGTs with viruses. Further characterization showed that many of these genes were clustered in DNA regions of several tens to hundreds of kilobases in size, originally belonging to viruses related to known Tetraselmis spp. viruses (TetV and TsV). In contrast, the remaining candidate HGT genes were randomly dispersed in the algal genomes, more frequently transcribed and belonged to large multigene families. The presence of homologs in Viridiplantae suggested that these latter were more likely of algal rather than viral origin. We found a remarkable diversity Polinton-like virus elements inserted in Tetraselmis genomes, all of which were most similar to the Tetrasemis striata virus TsV. The genes of PLV-like elements are transcriptionally inactive with the notable exception of the homologue of the TVSG_00024 gene of TsV whose function is unknown. We suggest that this gene may be involved in a sentinel process to trigger virus reactivation and excision in response to an environmental stimulus. Altogether, these results provide evidence that TsV-related viruses have a dual lifestyle, alternating between a free viral phase (i.e., virion) and a phase integrated into host genomes.


title: Dauer quiescence as well as continuity of the life cycle after dauer-exit in Caenorhabditis elegans are dependent on the endoribonuclease activity of XRN-2

abstract: Caenorhabditis elegans embarks on a quiescent dauer state upon exposure to unfavourable conditions and can sustain for a very long period without food, but it returns to continuous life cycle upon arrival of suitable conditions. Thus, dauer state plays a critical role in its adaptive fitness and survival. ATP-independent endoribonuclease activity of XRN-2 has been implicated in dauer microRNA metabolism, perturbation of which causes their collapse within a very short span of time. Here, we present a detailed comparative analyses of dauer transcriptomes from a conditional mutant strain for the endoribonuclease activity of XRN-2, maintained under control and experimental conditions. We observed disruption of microRNA homeostasis in experimental dauers resulted in deregulation of a large number of mRNA targets. Our bioinformatic analyses, supported by morphological, physiological, and behavioral evidence further demonstrate critical changes in metabolism leading to a state unsupportive of dauer maintenance, alongside potential defects in multiple neuronal activities, which might have caused an overall disruption of dauer plasticity. We also explore a possible role of this endoribonuclease activity towards the maintenance of chromatin architecture and transposon expression that in turn might affect the transcriptional program critically required for the maintenance of non-aging, long-lived dauers. Finally, we also demonstrate that perturbation of the endoribonuclease activity during the dauer state exerts drastic adverse effects on the continuity of life cycle after dauer-exit. They not only fail to recapitulate the wild type events of germline development and embryogenesis, but also present traits of very old worms and formation of tumor-like structures in the proximal gonad.

category: molecular biology


title: Loss of mitochondrial Chchd10 or Chchd2 in zebrafish leads to an ALS-like phenotype and Complex I deficiency independent of the mt-ISR

abstract: Mutations in CHCHD10 and CHCHD2, coding for two paralogous mitochondrial proteins, have been identified in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTD), and Parkinsons disease (PD). Here we investigated the biological roles of these proteins during vertebrate development using knockout (KO) models in zebrafish. We demonstrate that loss of either or both proteins leads to a motor impairment, reduced survival, and compromised neuromuscular junction (NMJ) integrity in larval zebrafish. Compensation by Chchd10 was observed in the chchd2-/- model, but not by Chchd2 in the chchd10 -/- model. The assembly of mitochondrial respiratory chain Complex I was impaired in chchd10 -/- and chchd2 -/- zebrafish larvae, but unexpectedly not in the double chchd10 -/- & chchd2 -/- model, suggesting that reduced mitochondrial Complex I cannot be solely responsible for the observed phenotypes, which are generally more severe in the double KO. Activation of the mitochondrial integrated stress response (mt-ISR) was only observed in the double KO model, possibly implicating this pathway in the recovery of the Complex I defect, and suggesting that Complex I assembly defect in our single KO is independent of the mt-ISR. Our results demonstrate that both proteins are required for normal vertebrate development, but their precise molecular function in the mitochondrial biology of motor neurons remains to be discovered.


title: Microbiome signatures in Acropora cervicornis are associated with genotypic resistance to elevated nutrients and heat stress

abstract: The staghorn coral, Acropora cervicornis, was once abundant in the Caribbean, but now is listed as critically endangered. To recover A. cervicornis populations, restoration efforts have focused on preserving genetic diversity and increasing coral cover. However, identifying stress-resistant corals can help to increase restoration success, by allocating genotypes to reefs where they are more likely to survive. We assessed the performance (growth, survivorship, and photochemical efficiency) and characterized the microbiome (prokaryotes) of six A. cervicornis genotypes that were maintained at control temperatures (~26 {degrees}C) and either ambient nutrients or elevated nutrients (elevated NH4, and elevated NH4 + PO4) for > 2 months. We then compared how these parameters changed when the corals were exposed to heat-stress (3-weeks at ~31.5 {degrees}C). We found that exposure to elevated nutrients reduced A. cervicornis performance under control temperatures and heat stress. However, there was a wide range of variation among genotypes, with three genotypes maintaining relatively higher survivorship and growth rates when exposed to nutrients alone, and nutrients followed by heat stress. Heat stress alone changed the microbial composition among genotypes more than elevated nutrients alone, but heat stress also interacted with nutrient pre-exposure to affect microbial communities. The relative abundance of Midichloriaceae and Spirochaetaceae varied by coral genotype and a high abundance of these bacterial taxa was a positive predictor of coral survivorship rate, suggesting a microbial signature that could aid in identifying resistant A. cervicornis genotypes. Our findings suggest there is significant variation among genotypes in the response of A. cervicornis to elevated nutrients and temperatures. Resistant genotypes may be identifiable via their microbiomes and prioritized for outplanting at sites characterized by high levels of nutrient pollution. Large-scale microbiome screening may help expedite targeted outplanting and could be tested and extended to facilitate the identification of genotypes with other resistance characteristics.


title: Heterologously secreted MbxA from Moraxella bovis induces a membrane blebbing response of the human host cell

abstract: Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live- cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.


title: SARS-CoV-2 Main Protease: a Kinetic Approach

abstract: In this article, I present a new model of the interaction of the main protease (Mpro) from SARS-CoV-2 virus with its substrate. The reaction scheme used to describe this mechanism is an extension of the well-known Michaelis-Menten model proposed in 1913 by Leonor Michaelis and Maud Menten [1]. The model I present here takes into account that one Mpro enzyme monomer interacts with another Mpro monomer in the presence of the substrate, leading to the formation of an enzyme dimer bound to one substrate molecule. Indeed, this dimer is formed by the sequentially binding of one Mpro enzyme monomer to one molecule of substrate, followed by another Mpro enzyme monomer binding to this Mpro-substrate complex. This reaction mechanism is also known in the literature as substrate-induced dimerization [3]. Starting from this new reaction scheme established for this catalytic mechanism, I derived a mathematical expression describing the catalytic rate of the active Mpro enzyme dimer as a function of the substrate concentration [S]. The plot corresponding to this substrate-induced dimerization reaction shows a function f ([S]) that is not monotonic, i.e. not strictly increasing or decreasing, but with a second derivative initially negative and then becoming positive after having passed the Vmax point. This is typically a type of curve showing a phenomenon like the one of substrate inhibition (for instance, inhibition by excess-substrate [7]). The graphical representation of this process shows an interesting behaviour: from zero M/s, the reaction rate increases progressively, similar to the kind of curve described by the Michaelis-Menten model. However, after having reached its maximum catalytic rate, Vmax, the reaction rate decreases progressively as we continue to increase the substrate concentration. I propose an explanation to this interesting behavior. At the moment where Vcat is maximum, we can assume that, in theory, every single substrate molecule in solution is bound to two enzyme monomers (i.e. to one active dimer). The catalytic rate is thus theoretically maximized. At the time where the reaction rate begins to decrease, we observe a new phenomenon that appears: the enzyme monomers begin to be "diluted" in the solution containing the excess substrate. The dimers begin to dissociate and to bind increasingly to the substrate as inactive monomers instead of active dimers. Hence, it is more and more unlikely for the enzyme monomers to sequentially bind twice to the same substrate molecule (here, [E] << [S]). Thus, at this stage, the substrate-induced dimerization occurs less often. At the limit, when the substrate is in high excess, there is virtually no more dimerization which occurs. This is one example of excess-substrate inhibition. Furthermore, after having established this fact, I wanted to see if this catalytic behavior was also observed in vitro. Therefore, I conducted an experiment where I measured the catalytic rate of the Mpro dimer for different substrate concentrations. The properties of my substrate construct were such, that I could determine the catalytic rate of the enzyme dimer by directly measuring the spectrophotometric absorbance of the cleaved substrate at{lambda} = 405 nm. The results show explicitly -- within a margin of error -- that the overall shape of the experimental curve looks like the one of the theoretical curve. I thus conclude that the biochemical behavior of the Mpro in vitro follows a new path when it is in contact with its substrate: an excess substrate concentration decreases the activity of the enzyme by the phenomenon of a type of excess-substrate inhibition. This finding could open a new door in the discovery of drugs directed against the Mpro enzyme of the SARS-CoV-2 virus, acting on the inhibition by excess-substrate of the Mpro enzyme, this protein being a key component in the metabolism of the virus. Furthermore, I have established that the maximum of the fitted curve, Vmax, depends only on [E]T and not on [S]. [Formula] exhibits the same dependence pattern. Therefore, if I keep [E]T close to zero, the catalytic rate of the enzyme will also be greatly reduced, which can be understood intuitively. Finally, if we dilute the enzyme sufficiently in the host cell by injecting a suitably high concentration of the octapeptide substrate AVLQSGFR (an inhibitor of the original substrate), this artificial substrate will bind to the "intermediate" dimer from the polypeptide and prevent the precursor Mpro from auto-cleaving and dimerizing due to the "distorted key" effect of the octapeptide on the "intermediate" dimer. The precursor peptide Mpro will auto-cleave to a lesser extent than in the absence of the artificial octapeptide and thus the concentration of the total enzyme [E]T will be lowered in the cell. It would therefore be possible to control the virulence of the virus by adjusting the concentration of the artificial inhibitory octapeptide. However, this is only speculation and has yet to be verified in practice.


title: Quantitative analysis of neuroligin-3 expression in the enteric nervous system of the Neuroligin-3R451C mouse model of autism

abstract: Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction but its cellular expression in the GI tract remains to be characterised. Localisation of NLGN3 protein is challenging in intestinal tissue due to the lack of target-specific antibodies. Here, we combined RNAScope in situ hybridization for Nlgn3 mRNA and immunofluorescence for markers of all mouse enteric neurons, cholinergic submucosal neurons, non-cholinergic submucosal neurons, nitregic and calretinin-containing myenteric neurons as well as glial cells. We also developed a quantitative 3-dimensional image analysis method to measure Nlgn3 mRNA cellular expression levels in enteric neurons and glia. We show that Nlgn3 mRNA is expressed in most submucosal and myenteric neurons as well as in enteric glia. The R451C mutation reduces Nlgn3 mRNA expression levels in cholinergic, nitrergic and calretinin enteric neuronal subpopulations but does not affect Nlgn3 mRNA expression in VIPergic submucosal neurons. In summary, we show that the autism-associated R451C mutation in Nlgn3 reduces Nlgn3 mRNA expression in the mouse enteric nervous system (ENS). These findings could shed light on the pathophysiology of GI dysfunction in ASD.


title: BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection

abstract: The recently emerged SARS-CoV-2 Omicron sublineages BA.2.12.1, BA.2.13, BA.4 and BA.5 all contain L452 mutations and show potential higher transmissibility over BA.21. The new variants receptor binding and immune evasion capability require immediate investigation, especially on the role of L452 substitutions. Herein, coupled with structural comparisons, we show that BA.2 sublineages, including BA.2.12.1 and BA.2.13, exhibit increased ACE2-binding affinities compared to BA.1; while BA.4/BA.5 displays the weakest receptor-binding activity due to F486V and R493Q reversion. Importantly, compared to BA.2, BA.2.12.1 and BA.4/BA.5 exhibit stronger neutralization evasion against the plasma of 3-dose vaccinees and, most strikingly, of vaccinated BA.1 convalescents. To delineate the underlying evasion mechanism, we determined the escaping mutation profiles2, epitope distribution3 and Omicron sublineage neutralization efficacy of 1640 RBD-directed neutralizing antibodies (NAbs), including 614 isolated from BA.1 convalescents. Interestingly, post-vaccination BA.1 infection mainly recalls wildtype (WT) induced humoral memory and elicits antibodies that neutralize both WT and BA.1. These cross-reactive NAbs are significantly enriched on non-ACE2-competing epitopes; and surprisingly, the majority are undermined by R346 and L452 substitutions, namely R346K (BA.1.1), L452M (BA.2.13), L452Q (BA.2.12.1) and L452R (BA.4/BA.5), suggesting that R346K and L452 mutations appeared under the immune pressure induced by Omicron convalescents. Nevertheless, BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1 but do not respond to WT SARS-CoV-2 due to the high susceptibility to N501, N440, K417 and E484. However, these NAbs are largely escaped by BA.2 sublineages and BA.4/BA.5 due to D405N and F486V, exhibiting poor neutralization breadths. As for therapeutic NAbs, LY-CoV1404 (Bebtelovimab4) and COV2-2130 (Cilgavimab5) can still effectively neutralize BA.2.12.1 and BA.4/BA.5, while the S371F, D405N and R408S mutations carried by BA.2/BA.4/BA.5 sublineages would undermine most broad sarbecovirus NAbs. Together, our results indicate that Omicron can evolve mutations to specifically evade humoral immunity elicited by BA.1 infection. The continuous evolution of Omicron poses great challenges to SARS-CoV-2 herd immunity and suggests that BA.1-derived vaccine boosters may not be ideal for achieving broad-spectrum protection.


title: Comparative computational structural genomics highlights divergent evolution of fungal effectors

abstract: Elucidating the evolution of pathogen effector molecules is critical to understand infection mechanisms of fungal phytopathogens and ensure crop security. However, rapid diversification that diminishes sequence similarities between homologous effectors has largely concealed the roots of effector evolution. We predicted the folds of 26,653 secreted proteins from 21 species with AlphaFold and performed structure-guided comparative analyses on two aspects of effector evolution: uniquely expanded effector families and common folds present across the fungal species. Extreme expansion of nearly lineage-specific effector families was found only in several obligate biotrophs, Blumeria graminis and Puccinia graminis. The highly expanded effector families were the source of conserved sequence motifs, such as the Y/F/WxC motif. We identified additional lineage-specific effector families that include known (a)virulence factors, such as AvrSr35, AvrSr50 and Tin2. These families represented new classes of sequence-unrelated structurally similar effectors. Structural comparisons revealed that the expanded structural folds further diversify through domain duplications and fusion with disordered stretches. Sub-and neo-functionalized structurally similar effectors reconverge on regulation, expanding the functional pools of effectors in the pathogen infection cycle. We also found evidence that many effector families could have originated from ancestral folds that have been conserved across fungi. Collectively, our study highlights diverse mechanisms of effector evolution and supports divergent evolution of effectors that leads to emergence of pathogen virulence from ancestral proteins.


title: The highly conserved stem-loop II motif is important for the lifecycle of astroviruses but dispensable for SARS-CoV-2

abstract: The stem-loop II motif (s2m) is an RNA element present in viruses from divergent viral families, including astroviruses and coronaviruses, but its functional significance is unknown. We created deletions or substitutions of the s2m in astrovirus VA1 (VA1), classic human astrovirus 1 (HAstV1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For VA1, recombinant virus could not be rescued upon partial deletion of the s2m or substitutions of G-C base pairs. Compensatory substitutions that restored the G-C base-pair enabled recovery of VA1. For HAstV1, a partial deletion of the s2m resulted in decreased viral titers compared to wild-type virus, and reduced activity in a replicon system. In contrast, deletion or mutation of the SARS-CoV-2 s2m had no effect on the ability to rescue the virus, growth in vitro, or growth in Syrian hamsters. Our study demonstrates the importance of the s2m is virus-dependent.


title: Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation

abstract:
Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus- infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the highly pathogenic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2 (eIF2) phosphorylation and SG formation induced by exogenous stress (e.g. sodium arsenite treatment). Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV-2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2 phosphorylation, and the Nsp1 of SARS- CoV2 alone was sufficient to cause decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear retention of the SG- nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in infection compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication.

Author SummaryHost cells possess many mechanisms that can detect viral infections and trigger defense programs to suppress viral replication and spread. One of such antiviral mechanisms is the formation of stress granules - large aggregates of RNA and proteins that sequester viral components and cellular factors needed by the virus to replicate. Because of this threat, viruses evolved specific mechanisms that prevent stress granule formation. Understanding these mechanisms can reveal potential targets for therapies that would disable viral inhibition of stress granules and render cells resistant to infection. In this study we analyzed inhibition of stress granules by two human coronaviruses: the common cold coronavirus OC43 and the pandemic SARS-CoV2. We have demonstrated that these viruses employ at least two proteins - nucleocapsid protein (N) and the non-structural protein 1 (Nsp1) to suppress stress granules. These proteins act through distinct complementary mechanisms to ensure successful virus replication. Because both OC43 and SARS-CoV2 each dedicate more than one gene product to inhibit stress granule formation, our work suggests that viral disarming of stress granule responses is central for a productive infection.


title: Relationships of Worldwide Onobrychis species: from seed & pod morphology and molecular perspective

abstract:
The present study aimed to investigate the phylogeny of 31 Onobrychis species with the help of seed and fruit macro & micro-morphological traits and ISSR molecular markers. The species were found to be highly variable in their seed and fruit morphological traits. Also, a new type of fruit (O. crista galli type), in addition to the three predefined types of fruit, was introduced. From molecular perspective, a set of 22 ISSR primers were shown to have a good efficiency for genetic discrimination among the species. Bayesian model based STRUCTURE analysis grouped the genotypes into 4 genetic clusters, and the high value of Fst calculated for these clusters indicated a high level of genetic differentiation among them. On the other hand, based on the Maximum Likelihood phylogenetic tree, our results are in agreement with the subgenus classification (Onobrychis and Sisyrosema), while contrary to Sisyrosema, the Onobrychis subgenus (the Lophobrychis and Onobrychis sections) appears not to be monophyletic. Furthermore, in contrast to the traditional taxonomic classification, species belonging to the Lophobrychis section do not form a coherent group, indicating that this section could be considered as a heterogeneous unit and perhaps could be merge into the Onobrychis section.

HighlightThis paper discusses a worldwide phylogenic analysis and population structures of Onobrychis genus. The results of this paper provide a comprehensive answer to complexity of subgenus and section discrimination.

category: evolutionary biology


title: Visualizing the Coordination of APE1 and DNA Polymerase β During Base Excision Repair

abstract: Base Excision Repair (BER) is carried out by a series of DNA repair proteins that function in a step-by-step process to identify, remove, and replace DNA damage. As DNA damage is processed during BER, the DNA transitions through various intermediate states, called BER intermediates, which if left exposed can develop into double-strand DNA breaks and trigger programmed cell death signaling. Previous studies have proposed that in order to minimize exposure of the BER intermediates, each protein may remain bound to its product prior to the next protein binding. Thus, a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme may form between each step of the BER pathway. The transfer of BER intermediates between enzymes, known as BER coordination, has yet to be directly visualized and the mechanistic details of the process remain unclear. Here, we utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase {beta} (Pol {beta}). When preformed complexes comprised of APE1 and the incised AP-site product were subsequently bound by Pol {beta}, the Pol {beta} enzyme dissociated shortly after binding in a majority of the observations. In the events where Pol {beta} binding was followed by APE1 dissociation (i.e., DNA hand-off), Pol {beta} had remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that, in the absence of other BER factors, transfer of the BER intermediate from APE1 to Pol {beta} during BER is dependent on the dissociation kinetics of APE1 and the duration that Pol {beta} remains bound near the APE1-5 nick complex. These findings provide insight into how APE1 and Pol {beta} coordinate the transfer of DNA within the BER pathway.


title: Improving the RNA velocity approach using long-read single cell sequencing

abstract: The concept of RNA velocity has been recently developed that allowed to look at the otherwise static single-cell RNA sequencing data in a dynamic way, which permitted inferences about cell fates. However, the more precise parameters, such as the number of exons/introns, can also be determined using long-read methods. Comparing the numbers of exons and introns allows including more genes for downstream velocity analysis and resolves the precise cell fate. The recently developed concept of "RNA velocity" concerns with dynamic changes in mRNA expression and complements single-cell RNA sequencing (scRNA-seq) data, which are static snapshots of a certain cell state taken at a given time point1. RNA velocity measures the change in mRNA abundance by differentiating the newly transcribed unspliced pre-mRNAs from mature spliced mRNAs. The rapidly developing long-read sequencing technology lends itself for RNA velocity analysis of scRNA-seq data, which was previously performed primarily using second-generation sequencing.


title: Mechanically primed cells transfer memory to fibrous matrices for persistent invasion

abstract:
In disease and development, cells sense and migrate across mechanically dissimilar environments. We investigated whether mechanical memory of past environments empowers cells to navigate new, three-dimensional environments. Here, we show that cells primed by stiff matrices apply higher forces, compared to soft-primed cells, to accumulate and align collagen fibers towards sustained invasion. This priming advantage persists in dense or stiffened collagen. Through an energy-minimization model, we elucidate how memory-laden cells overcome mechanosensing of softer or challenging environments via a cell-matrix transfer of memory. Consistent with model predictions, depletion of -catenin and YAP hamper coordinated forces and cellular memory required for collagen remodeling before invasion. We release tension in collagen fibers via laser ablation and disable fiber remodeling by lysyl-oxidase inhibition; both of which disrupt cell-to-matrix transfer of memory and reduce invasion. These results have implications for cancer, fibrosis, and aging, where potential matrix memory may generate prolonged cellular response.

One-Sentence SummaryCell invasion across mechanically dissimilar environments is mediated by force-based storage and extraction of cell and matrix memory.


title: Expression of YAP1-MAML2 and constitutively active YAP1 drive the formation of meningioma-like tumors in mice that resemble NF2-mutant meningiomas

abstract: YAP1 is a transcriptional co-activator and oncogene under the control of the Hippo Signaling Pathway. Functional inactivation of Hippo Pathway tumor suppressors, including NF2, are frequent events in human cancers. Meningiomas are the most common primary brain tumors, and a large percentage exhibit heterozygous loss of chromosome 22 (harboring the NF2 gene) and functional inactivation of the remaining NF2 gene copy, implicating oncogenic YAP activity in the pathobiology of almost half of these tumors. An alternate type of activating YAP1 mutation are YAP1 gene fusions that have been identified in several cancer subtypes. Recently, fusions between YAP1 and MAML2 have been identified in a subset of pediatric NF2-wild type meningiomas. Here, we show that the expression profile of human YAP1-MAML2-positive pediatric meningiomas resembles that of the common NF2-mutant meningiomas based on global and YAP-related gene expression signatures. We then use the RCAS/tv-a system for postnatal gene transfer and show that the intracranial expression of YAP1-MAML2 in neonatal mice results in the formation of meningioma-like tumors that exert a similar gene expression pattern as seen in human YAP1 fusion-positive and NF2-mutant meningiomas and regulate classical YAP1 target genes. We demonstrate that YAP1-MAML2 exerts oncogenic YAP activity that is resistant to inhibitory Hippo pathway signaling and relies on the interaction with TEAD transcription factors. Pharmacological disruption of this interaction is sufficient to inhibit the viability of YAP1-MAML2-expressing mouse tumors ex vivo. Finally, we show that constitutively active YAP1 (S127/397A-YAP1) is also sufficient to cause of similar meningioma-like tumors suggesting that the YAP component of the gene fusion is the critical driver of these tumors. In summary, our results implicate YAP1-MAML2 as a sufficient oncogenic driver in YAP1-MAML2 fusion-positive meningiomas, which mimic NF2-mutant meningiomas, and highlight TEAD-dependent YAP activity as a potential therapeutic target in these tumors.


title: Ultradeep characterisation of translational sequence determinants refutes rare-codon hypothesis and unveils quadruplet base pairing of initiator tRNA and transcript

abstract: Translation is a key determinant of gene expression and an important biotechnological engineering target. In bacteria, 5-untranslated region (5-UTR) and coding sequence (CDS) are well-known mRNA parts controlling translation and thus cellular protein levels. However, the complex interaction of 5-UTR and CDS has so far only been studied for few sequences leading to non-generalisable and partly contradictory conclusions. Herein, we systematically assess the dynamic translation from over 1.2 million 5-UTR-CDS pairs in Escherichia coli to investigate their collective effect using a new method for ultradeep sequence-function mapping. This allows us to disentangle and precisely quantify effects of known and hypothetical sequence determinants of translation. We find that 5-UTR and CDS individually account for 53% and 20% of variance in translation, respectively, and show conclusively that, contrary to a common hypothesis, tRNA abundance does not explain expression changes between CDSs with different synonymous codons. Moreover, the obtained large-scale data clearly point to a base-pairing interaction between initiator tRNA and mRNA beyond the anticodon-codon interaction, an effect that is often masked for individual sequences and therefore inaccessible to low-throughput approaches. Our study highlights the indispensability of ultradeep sequence-function mapping to accurately determine the contribution of parts and phenomena involved in gene regulation.

category: synthetic biology


title: Syntaxin Habc is required to open Unc18 to template SNARE assembly

abstract: SNARE and Unc18 proteins form the core of the membrane fusion complex at synapses. The fusion machinery is evolutionarily ancient and mediates constitutive fusion in yeast. We demonstrate that the SNARE and Unc18 machinery in the nematode C. elegans can be replaced by yeast proteins and still carry out synaptic transmission. However, substitutions of individual components from yeast disrupts fusion. To understand the functional interactions within the core machinery we adopted an  interspecies complementation approach using yeast. Synaptic transmission could be restored in chimeras when two key interfaces were present: a novel Habc-Unc18 contact site and an Unc18-SNARE motif contact site. An open form of Unc18 could bypass the requirement for the Habc-Unc18 interface. Together, these data suggest that the Habc domain of syntaxin is required for Unc18 to adopt an open conformation; open Unc18 then templates SNARE complex formation.


title: Effects of the investigational drug sodium phenylbutyrate-TUDCA (AMX0035) on the transcriptional and metabolic landscape of sporadic ALS fibroblasts

abstract: ALS is a rapidly progressive, fatal disorder caused by motor neuron degeneration, for which there is a great unmet therapeutic need. AMX0035, a combination of sodium phenylbutyrate (PB) and taurursodiol (TUDCA, Turso), has shown promising results in early ALS clinical trials, but its mechanisms of action remain to be elucidated. To obtain an unbiased landscape of AMX0035 effects we investigated the transcriptomic and metabolomic profiles of primary skin fibroblasts from sporadic ALS patients and healthy controls treated with PB, TUDCA, or PB-TUDCA combination (Combo). Combo changed many more genes and metabolites than either PB or TUDCA individually. Most changes were unique to Combo and affected the expression of genes involved in ALS-relevant pathways, such as nucleocytoplasmic transport, unfolded protein response, mitochondrial function, RNA metabolism, and innate immunity. Weighted gene coexpression network analysis showed that significant correlations between ALS gene expression modules and clinical parameters were abolished by Combo. This study is the first to explore the molecular effects of Combo in ALS patient-derived cells. It shows that Combo has a greater and distinct impact compared to the individual compounds and provides clues to drug targets and mechanisms of actions, which may underlie the benefits of this investigational drug combination.


title: Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy

abstract:
Parkinsons disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of -Synuclein (-Syn) protein. Currently, no treatment can slow nor halt neurodegeneration. Multiplications and mutations of the -Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress -Syn replicate several features of PD. Decreasing total -Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with a  synucleinopathy. We previously performed a genetic screen for modifiers of -Syn levels, and found CDK14, a kinase of largely unknown function as a regulator of -Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we decreased Cdk14 in two mouse models of synucleinopathy. We found that reduction of Cdk14 mitigated neuropathological and neurobehavioral sequelae associated with -Syn overexpression. We further validated these findings in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable ex vivo. We found that in both mouse and human neurons, CDK14 inhibition decreases total and pathologically aggregated -Syn. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

One Sentence SummaryLoss of CDK14 mitigates -Synuclein pathology in Parkinsons disease (PD) mouse models and in human neurons, highlighting CDK14 as a druggable target for the treatment of PD.


title: An Epitope-Focused Trypanosome-Derived Vaccine Platform Elicits High-Affinity Antibodies and Immunity Against Fentanyl Effects

abstract: Poorly antigenic small molecules pose challenges for the production of clinically efficacious antibodies. To address this, we have developed an immunization platform derived from the antigenic surface coat of the African trypanosome. Through sortase-based conjugation of antigens to the trypanosome surface coat protein variant surface glycoprotein (VSG), we created the VAST (VSG-immunogen Array by Sortase Tagging). We used VAST to elicit protective immunity to the effects fentanyl. Immunizing mice with fentanyl-VAST generated serological memory and protection from fentanyl challenge. Employing single-cell RNAseq, we then developed a streamlined method that synergizes with the VAST to identify immunization-elicited memory B cells and the antibodies they encode. All antibodies selected by this method displayed picomolar affinities for fentanyl. Passive immunization protected animals from fentanyl, while crystallography revealed that the mAbs bind fentanyl in an unusually deep pocket suited to small molecules, demonstrating the ability of the VAST to elicit high-quality therapeutic antibodies.


title: Two Competing Guilds as a Core Microbiome Signature for Chronic Diseases

abstract: Summary ParagraphGut microbiota may work as an essential organ and its members interact closely with each other and form a higher-level organization called guilds. How such guild-level structure supports the gut microbiota to stably provide essential health-relevant functions to the host remains elusive. With high quality metagenome-assembled genomes as network nodes, here we identified a core microbiome signature made up of two robust competing guilds that together correlate with a wide range of host health conditions. Genomes in these two guilds kept their ecological relationship unchanged despite experiencing profound abundance changes during a 3-month high fiber intervention and 1-year follow-up in patients with type 2 diabetes. The genomes of one guild harbored more genes for plant polysaccharide degradation and butyrate production, while the other guild had more genes for virulence or antibiotic resistance. A Random Forest regression model showed that the abundance distributions of these genomes were associated with 41 out of 43 bio-clinical parameters in the study cohort. With these genomes as reference, Random Forest modeling successfully classified case and control of 8 chronic diseases in 12 independent metagenomic datasets from 1,816 participants across ethnicity and geography. This core microbiome signature may facilitate ecological management of chronic diseases.


title: In situ structural analysis reveals membrane shape transitions during autophagosome formation

abstract: Autophagosomes are unique organelles which form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves a series of membrane transformations with distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (ET) and novel data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. By mapping individual structures onto a timeline based on geometric features, we uncover dynamic changes in membrane shape and curvature. Moreover, we reveal the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles such as the vacuole and the ER. Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores towards closure without a templating cargo.

category: cell biology


title: Leishmania allelic selection during experimental sand fly infection correlates with mutational signatures of oxidative DNA damage

abstract: Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. Survival of these parasites in their vertebrate and invertebrate hosts relies on their capacity to differentiate into distinct stages that are the result of a co-evolutionary process. These stages show in addition important phenotypic shifts that often impacts infection, affecting for example parasite pathogenicity, tissue tropism, or drug susceptibility. Despite their clinical relevance, the evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here we use Leishmania donovani as a trypanosomatid model pathogen to shed first light on parasite evolutionary adaptation during experimental sand fly infection. Applying a comparative genomics approach on hamster- isolated amastigotes and derived promastigotes before (input) and after (output) infection of Phlebotomus orientalis revealed a strong bottleneck effect on the parasite population as judged by principal component and phylogenetic analyses of input and output parasite DNA sequences. Despite random genetic drift caused by the bottleneck effect, our analyses revealed various genomic signals that seem under positive selection given their convergence between independent biological replicates. While no significant fluctuations in gene copy number were revealed between input and output parasites, convergent selection was observed for karyotype, haplotype and allelic changes during sand fly infection. Our analyses further uncovered signature mutations of oxidative DNA damage in the output parasite genomes, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a new model of Leishmania genomic adaptation during sand fly infection, where oxidative DNA damage and DNA repair processes drive haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei and Trypanosoma cruzi.


title: Pleiotrophin drives a pro-metastatic immune niche within the breast tumor microenvironment

abstract: Metastatic cancer cells adapt to thrive in secondary organs. To investigate metastatic adaptation, we performed transcriptomic analysis of metastatic and non-metastatic murine breast cancer cells. We found that pleiotrophin (PTN), a neurotrophic cytokine, is a metastasis-associated factor that is expressed highly by aggressive breast cancers. Moreover, elevated PTN in plasma correlated significantly with metastasis and reduced survival of breast cancer patients. Mechanistically, we find that PTN activates NF-kB in cancer cells leading to altered cytokine production, subsequent neutrophil recruitment and an immune suppressive microenvironment. Consequently, inhibition of PTN, pharmacologically or genetically, reduces the accumulation of tumor associated neutrophils and reverts local immune suppression resulting in increased T cell activation and attenuated metastasis. Furthermore, inhibition of PTN significantly enhanced the efficacy of immune checkpoint blockade + chemotherapy in reducing metastatic burden in mice. These findings establish PTN as a previously unrecognized driver of a pro-metastatic immune niche and thus represents a promising therapeutic target for the treatment of metastatic breast cancer.


title: Context-dependent effects explain divergent prognostic roles of Tregs in cancer

abstract: Assessing cancer prognosis is a challenging task, given the heterogeneity of the disease. Multiple features (clinical, environmental, genetic) have been used for such assessments. The tumor immune microenvironment (TIME) is a key feature, and describing the impact of its many components on cancer prognosis is an active field of research. The complexity of the tumor microenvironment context makes it difficult to use the human TIME to assess prognosis, as demonstrated by the example of regulatory T cells (Tregs). The effect of Tregs on prognosis is ambiguous, with different studies considering them to be negative, positive or neutral. We focused on five different cancer types (breast, colorectal, gastric, lung and ovarian). We clarified the definition of Tregs and their utility for assessing cancer prognosis by taking the context into account via the following parameters: the Treg subset, the anatomical location of these cells, and the neighboring cells. With a meta-analysis on these three parameters, we were able to clarify the prognostic role of Tregs. We found that CD45RO+ Tregs had a reproducible negative effect on prognosis across cancer types, and we gained insight into the contributions of the anatomical location of Tregs and of their neighboring cells on their prognostic value. Our results suggest that Tregs play a similar prognostic role in all cancer types. We also established guidelines for improving the design of future studies addressing the pathophysiological role of Tregs in cancer.


title: A Phytophthora nucleolar effector, Pi23226, targets to host ribosome biogenesis for necrotrophic cell death

abstract: Pathogen effectors target diverse subcellular organelles to manipulate the plant immune system. Although nucleolus has been emerged as a stress marker, and several effectors are localized in the nucleolus, the roles of nucleolar-targeted effectors remain elusive. In this study, we showed Phytophthora infestans infection of Nicotiana benthamiana results in nucleolar inflation during the transition from biotrophic to necrotrophic phase. Multiple P. infestans effectors were localized in the nucleolus: Pi23226 induced cell death in Nicotiana benthamiana and nucleolar inflation similar to that observed in the necrotrophic stage of infection, whereas its homolog Pi23015 and a deletion mutant (Pi23226{Delta}C) did not induce cell death or affect nucleolar size. RNA immunoprecipitation and iCLIP-seq analysis indicated that Pi23226 bound to the 3'-end of 25S rRNA precursors, resulting in the accumulation of unprocessed 27S pre-rRNAs. The nucleolar stress marker NAC082 was strongly upregulated under Pi23226-expressing conditions. Pi23226 subsequently inhibited global protein translation in host cells by interacting with ribosomes. Pi23226 enhanced P. infestans pathogenicity, indicating that Pi23226-induced ribosome malfunction and cell death was beneficial for pathogenesis in the host. Our results provide evidence for the molecular mechanism underlying RNA-binding effector activity in host ribosome biogenesis, and lead to new insights into the nucleolar action of effectors in pathogenesis.


title: Evaluating and mitigating clinical samples matrix effects on TX-TL cell-free performance

abstract: Cell-free biosensors are promising tools for medical diagnostics, yet their performance can be affected by matrix effects arising from the sample itself or from external components. Here we systematically evaluate the performance and robustness of cell-free systems in serum, plasma, urine, and saliva using two reporter systems, sGFP and luciferase. In all cases, clinical samples have a strong inhibitory effect. Of different inhibitors, only the RNase inhibitor mitigated matrix effects. However, we found that the recovery potential of RNase inhibitor was partially muted by interference from glycerol contained in the commercial buffer. We solved this issue by designing a strain producing an RNase inhibitor protein requiring no additional step in extract preparation. Furthermore, our new extract yielded higher reporter levels than previous conditions and tempered interpatient variability associated with matrix effects. This systematic evaluation and improvements of cell-free system robustness unified across many types of clinical samples is a significant step towards developing cell-free diagnostics for a wide range of conditions.


title: Selection of Single Domain Anti-Transferrin Receptor Antibodies for Blood-Brain Barrier Transcytosis Using a Neurotensin Based Assay and Histological Assessment of Target Engagement in a Mouse Model of Alzheimer's Disease-Related Amyloid-Beta Pathology

abstract:
BackgroundThe blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti- mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents.

MethodsAnti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (A{beta}) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related A{beta} plaque pathology.

ResultsAn anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol/kg body weight, ranging from undetectable for nonbinding mutants to -6{degrees}C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest hypothermia effect was fused to the anti-A{beta} VHH dimer and labeled with Alexa647; the dye- labeled VHH fusion construct still bound both mTfR and A{beta} plaques. However, after intravenous injection at 600 nmol/kg body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex.

ConclusionsThere was a surprising dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with A{beta}-plaques in cortex. Alternative methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents.


title: A resource for generating and manipulating human microglial states in vitro

abstract: Microglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimers disease (AD). In response to CNS stimuli, these cells adopt distinct transcriptional and functional subtypes known as states. However, an understanding of the function of these states has been elusive, especially in human microglia, due to lack of tools to model and manipulate this cell-type. Here, we provide a platform for modeling human microglia transcriptional states in vitro. Using single-cell RNA sequencing, we found that exposure of human stem-cell differentiated microglia (iMGLs) to brain-related challenges generated extensive transcriptional diversity which mapped to gene signatures identified in human brain microglia. We identified two in vitro transcriptional clusters that were analogous to human and mouse disease-associated microglia (DAMs), a state enriched in neurodegenerative disease contexts. To facilitate scalable functional analyses, we established a lentiviral approach enabling broad and highly efficient genetic transduction of microglia in vitro. Using this new technology, we demonstrated that MITF (Melanocyte Inducing Transcription Factor), an AD-enriched transcription factor in microglia, drives both a disease-associated transcriptional signature and a highly phagocytic state. Finally, we confirmed these results across iMGLs differentiated from multiple iPSC lines demonstrating the broad utility of this platform. Together, these tools provide a comprehensive resource that enables the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.


title: Inner hair cell synapse density influences auditory processing

abstract: It has been proposed that inner hair cell (IHC) synaptopathy, i.e., loss of synapses between spiral ganglion neurons and IHCs, leads to hidden hearing loss (HHL), i.e. poor performance on challenging hearing tasks despite a normal audiogram. However, this has only been tested in animals after exposure to noise or ototoxic drugs, which can cause deficits beyond synaptopathy. Furthermore, the impact of supernumerary synapses on auditory processing has not been evaluated. Here, we studied mice in which IHC synapse counts have been increased or decreased by altering neurotrophin 3 (Ntf3) expression in IHC supporting cells. As we previously showed, postnatal Ntf3 knockdown or overexpression reduces or enhances suprathreshold ABR amplitude and IHC synapse density, respectively, without changing cochlear thresholds. Here, we show that IHC synapse density does not influence the acoustic startle reflex or prepulse inhibition, measures of reflexive responses to sound and sensory-motor gating. In contrast, gap-prepulse inhibition, a behavioral test for auditory temporal processing, was increased or reduced according to Ntf3 expression levels. These results indicate that cochlear synaptopathy causes temporal processing deficits, which may be a key contributor to the hearing impairments that define HHL. Furthermore, the improvement in temporal acuity achieved by increasing Ntf3 expression and synapse density suggests a therapeutic strategy for improving hearing in noise for individuals with synaptopathy of various etiologies.