title: BglC of Streptomyces scabiei is a scopolin-hydrolyzing β-glucosidase interfering with the host defense mechanism

abstract: The beta-glucosidase BglC fulfills multiple functions in both primary metabolism and induction of pathogenicity of Streptomyces scabiei, the causative agent of the common scab disease of root and tuber crops. Indeed, this enzyme hydrolyzes cellobiose and cellotriose to directly feed glycolysis with glucose, but also modifies the intracellular concentration of these cello-oligosaccharides which are the virulence elicitors. The inactivation of bglC also led to unexpected phenotypes such as the constitutive overproduction of thaxtomin A, the main virulence determinant of S. scabiei. In this work we revealed a new target substrate of BglC, the phytoalexin scopolin. Removal of the glucose moiety of scopolin generates scopoletin, a potent inhibitor of thaxtomin A production. The hydrolysis of scopolin by BglC presents substrate inhibition kinetics which contrasts with the typical Michaelis-Menten saturation curve previously observed for the degradation of its natural substrate cellobiose. Our work therefore reveals that BglC targets both cello-oligosaccharide elicitors emanating from the hosts of S. scabiei, and the scopolin phytoalexin generated by the host defense mechanisms, thereby occupying a key position to fine-tune the production of the main virulence determinant thaxtomin A.


title: S100A8 and S100A9, biomarkers of SARS-Cov-2-infected patients, suppress HIV replication in primary macrophages

abstract: S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils and form a heterodimer complex. Recently, both proteins were identified as novel biomarkers of SARS-CoV-2 infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibits HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads in SARS-CoV2 co-infection.

category: microbiology


title: DOT1L bridges transcription and heterochromatin formation at pericentromeres

abstract: Repetitive DNA elements are packaged in heterochromatin, but many require bursts of transcription to initiate and maintain long-term silencing. The mechanisms by which these heterochromatic genome features are transcribed remain largely unknown. Here, we show that DOT1L, a conserved histone methyltransferase that modifies lysine 79 of histone H3 (H3K79), has a specialized role in transcription of major satellite repeats to maintain pericentromeric heterochromatin and genome stability. We discover that H3K79me3 is enriched at repetitive elements, that DOT1L loss specifically compromises pericentromeric satellite transcription, and that this function depends on interaction between DOT1L and the chromatin remodeler SMARCA5. Activation of pericentromeric repeats by DOT1L drives the first establishment of heterochromatin structures in cleavage-stage embryos and is required for preimplantation viability. Our findings uncover a vital instructive role for DOT1L as a bridge between transcriptional activation of heterochromatic repeats and maintenance of genome integrity, and illuminate global chromatin dynamics during early development.

category: molecular biology


title: Massively parallel identification of zipcodes in primary cortical neurons

abstract: Cells adopt highly polarized shapes and form distinct subcellular compartments largely due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called "zipcodes." Their recognition by RNA-binding proteins (RBPs) leads to the integration of the mRNAs into macromolecular complexes and their localization. While there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here, we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3UTRs. This approach combines a method of separating the principal subcellular compartments of neurons - cell bodies and neurites - with a massively parallel reporter assay. Our analysis identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons and suggests a strategy for detecting many more.


title: DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells

abstract: DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.


title: Population genomics of ancient and modern Trichuris trichiura

abstract: The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

category: genomics


title: Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa

abstract:
Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi-omics approach, followed-up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus.

A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted deep sequencing of candidate resistance genes and genomic loci detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNP was in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where CYP9K1 P450 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited a drastic reduction of genetic diversity in Uganda, in contrast to other locations, highlighting an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid mutation located between the meander and cysteine pocket of CYP9K1 was detected in all Ugandan mosquitoes.

This study sheds further light on the complex evolution of metabolic resistance in a major malaria vector, by adding further resistance genes and variants that can be used to design field applicable markers to better track this resistance Africa-wide.

Author SummaryMetabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used several DNA based approach to associate genomic differences between resistant and susceptible mosquitoes from several field and laboratory populations of the malaria vector Anopheles funestus. We followed-up our genomic analyses with functional validation of a candidate resistance gene in East Africa. This gene (CYP9K1) is a member of the cytochrome P450 gene-family that helps to metabolise, and thereby detoxify, pyrethroid insecticides. We show that this gene is a major driver of resistance to a specific sub-class of pyrethroid insecticides only, with moderate to no effects on other insecticides used against Anopheles funestus. We were able to link resistance in this gene to a mutation that changes the amino acid glycine to alanine that may impact how the protein-product of this gene binds to target insecticides. In addition to demonstrating the biochemical specificity of an evolutionary response, we have broadened the available pool of genes can be used to monitor the spread of insecticide resistance in this species.


title: Sequence Recognition by a Pseudorotational Cycle of Sugar Puckers: A Base Pair Switching Model via N-E Sugar Pucker Conversion for Homologous Recombination

abstract: A triplex DNA model bound to a helical filament of homologous recombination protein, such as Escherichia coli RecA, is presented. This model suggests that a function of the nucleoprotein filament could be at least partly attributed to the lowering of transition energy of duplex DNA structure having the E-type sugar puckers. Key events during homologous recombination such as sequence recognition, base pair switching and elongation/contraction of the helical pitch may correlate with the pseudo-rotation angle of sugar puckers along the N-, E-, S- and W-types. A conformational change of sugar puckers during the reaction is resolved into two motions by introducing a pair of parameters, 1 and {nu}3, which defines a swing orientation of bases and a torsional angle of phosphate backbones, respectively.

category: biochemistry


title: The Formation of a Stable Sliding Clamp Discriminates MSH2-MSH3 and MSH2-MSH6 Mismatch Interaction

abstract: MutS homologs (MSH) are highly conserved core components of DNA mismatch repair (MMR). Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Once on DNA the MSH sliding clamps provide a platform for the assembly of MMR strand-specific excision components beginning with the highly conserved MutL homologs (MLH/PMS). Previous studies with short mismatch-containing oligonucleotides revealed an MSH ATP hydrolysis (ATPase) cycle that included mismatch recognition, the formation of an ATP-bound sliding clamp and dissociation from the end of a mismatched DNA that ultimately recovers the mismatch binding conformation. We found that ATP-bound MSH complexes on blocked-end or very long DNA are extremely stable under a range of ionic conditions. These observations underpinned the development of a high-throughput fluorescence resonance energy transfer (FRET) system capable of clearly distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 activities that is suitable for chemical inhibitor screens.


title: Effects of Oncohistone Mutations and PTM Crosstalk on the N-terminal Acetylation Activities of NatD

abstract: Acetylation at the -N-terminus (N) is one of the most abundant modifications detected on histone H4 and H2A, which is catalyzed by N-terminal acetyltransferase D (NatD). NatD substrate recognition motif, N-terminal SGRGK, is enriched with frequent oncohistone mutations and post-translational modifications (PTMs). However, there is no information on how oncohistone mutations and other PTMs affect NatD-catalyzed acetylation. Herein, we determined how changes of local chemical environment on the N-terminal SGRGK sequence regulate NatD-catalyzed N acetylation on histone H4/H2A. Our studies indicate that all oncohistone mutations at SGRG suppressed the catalytic efficiency of NatD. Meanwhile, H4 serine 1 phosphorylation and arginine 3 methylation also negatively affect the NatD activity, but the lysine 5 acetylation has a marginal effect on NatD. This work reveals the impacts of oncohistone mutations on NatD activity and unravels the crosstalk between NatD and PTMs. To our knowledge, this is the first report on the potential regulatory mechanism of NatD, highlighting different revenues to interrogate the NatD-mediated pathway in the future.


title: The Mettl3 epitranscriptomic writer amplifies p53 stress responses

abstract: The p53 transcription factor, encoded by the most frequently mutated gene in human cancer, plays a critical role in tissue homeostasis in response to stress signals. The mechanisms through which p53 promotes downstream tumor suppressive gene expression programs remain, however, only superficially understood. Here, we used tandem affinity purification and mass spectrometry to reveal new components of the p53 response. This approach uncovered Mettl3, a component of the m6A RNA methyltransferase complex (MTC), as a p53-interacting protein. Analysis of Mettl3- deficient cells revealed that Mettl3 promotes p53 protein stabilization and target gene expression in response to DNA damage. Mettl3 acts in part by competing with the p53 negative regulator, Mdm2, for binding to the p53 transactivation domains to promote methyltransferase-independent stabilization of p53. In addition, Mettl3 relies on its catalytic activity to augment p53 responses, with p53 recruiting Mettl3 to p53 target genes to co-transcriptionally direct m6A modification of p53 pathway transcripts to enhance their expression. Mettl3 also promotes p53 activity downstream of oncogenic signals in vivo, in both allograft and autochthonous lung adenocarcinoma models, suggesting cooperative action of p53 and Mettl3 in tumor suppression. Accordingly, we found in diverse human cancers that mutations in MTC components perturb expression of p53 target genes and that MTC mutations are mutually exclusive with TP53 mutations, suggesting that the MTC enhances the p53 transcriptional program in human cancer. Together, these studies reveal a fundamental role for Mettl3 in amplifying p53 signaling through protein stabilization and epitranscriptome regulation.

category: cancer biology


title: TAL effectors with avirulence activity in African strains of Xanthomonas oryzae pv. oryzae

abstract:
BackgroundXanthomonas oryzae pv. oryzae causes bacterial leaf blight, a devastating disease of rice. Among the type-3 effectors secreted by Xanthomonas oryzae pv. oryzae to support pathogen virulence, the Transcription Activator-Like Effector (TALE) family plays a critical role. Some TALEs are major virulence factors that activate susceptibility (S) genes, overexpression of which contributes to disease development. Host incompatibility can result from TALE-induced expression of so-called executor (E) genes leading to a strong and rapid resistance response that blocks disease development. In that context, the TALE functions as an avirulence (Avr) factor. To date no such avirulence factors have been identified in African strains of Xanthomonas oryzae pv. oryzae.

ResultsWith respect to the importance of TALEs in the Rice-Xoo pathosystem, we aimed at identifying those that may act as Avr factor within African Xoo. We screened 86 rice accessions, and identified 12 that were resistant to two African strains while being susceptible to a well-studied Asian strain. In a gain of function approach based on the introduction of each of the nine tal genes of the avirulent African strain MAI1 into the virulent Asian strain PXO99A, four were found to trigger resistance on specific rice accessions. Loss-of-function mutational analysis further demonstrated the avr activity of two of them, talD and talI, on the rice varieties IR64 and CT13432 respectively. Further analysis of TalI demonstrated the requirement of its activation domain for triggering resistance in CT13432. Resistance in 9 of the 12 rice accessions that were resistant against African Xoo specifically, including CT13432, could be suppressed or largely suppressed by trans-expression of the truncTALE tal2h, similarly to resistance conferred by the Xa1 gene which recognizes TALEs generally independently of their activation domain.

ConclusionWe identified and characterized TalD and TalI as two African Xoo TALEs with avirulence activity on IR64 and CT13432 respectively. Resistance of CT13432 against African Xoo results from the combination of two mechanisms, one relying on the TalI-mediated induction of an unknown executor gene and the other on an Xa1-like gene or allele.

category: plant biology


title: A novel Pan-viral prophylaxis strategy using vaccine adjuvant CAF09b protects against influenza virus infection

abstract: The SARS-CoV-2 pandemic caused a massive health and societal crisis, although the fast development of effective vaccines reduced some of the impact. To prepare for future pandemics, a pan-viral prophylaxis could be used to control the initial virus outbreak in the period prior to vaccine approval. The liposomal vaccine adjuvant CAF(R)09b contains the TLR3 agonist polyinosinic:polycytidylic acid, which induces a type I interferon (IFN-I) response and an antiviral state in the affected tissues. When testing CAF09b as a potential pan-viral prophylaxis, we observed that intranasal administration of CAF09b to mice resulted in an influx of innate immune cells into the nose and lungs and upregulation of IFN-I related gene expression. When CAF09b was administered prior to challenge with mouse-adapted influenza A/Puerto Rico/8/1934 virus, it protected from severe disease, although virus was still detectable in the lungs. However, when CAF09b was administered after influenza challenge, the mice had a similar disease course to controls. In conclusion, CAF09b may be a suitable candidate as a pan-viral prophylactic treatment for epidemic viruses, but must be administered prior to virus exposure to be effective.

category: immunology


title: Commensal bacteria promote type I interferon signaling to maintain immune tolerance

abstract: Type I interferons (IFN) play essential roles in numerous physiological processes, acting as central coordinators in the host response against pathogens. Upon sensing of microbial ligands, host cells rapidly activate the type I IFN response to prime innate and adaptive immune responses. Recent studies suggest tonic IFN are maintained by commensal microbes and critical in mounting an effective immune response to viral pathogens. Further, emerging developments have extended an immunoregulatory role of type I IFN in the maintenance of immune homeostasis. Yet whether immunomodulatory bacteria from the gut microbiota operate through IFN signaling to promote immune tolerance remains largely unanswered. Here we show that commensal microbes are necessary to maintain type I IFN responses in intestinal tissues. Specifically, Bacteroides fragilis induced type I IFN response in dendritic cells (DCs) and this pathway is necessary for the induction of IL-10-producing Foxp3+ regulatory T cells (Tregs). In addition, we show upregulation of type I IFN related genes in Tregs from mesenteric lymph nodes and colonic lamina propria of mice colonized with B. fragilis. Our findings demonstrate type I interferon signaling plays an important role in microbiota-mediated immune tolerance in the gut.


title: Engineering transcription factor BmoR mutants for constructing multifunctional alcohol biosensors

abstract: Native transcription factor-based biosensors (TFBs) have the potential for in situ detection of value-added chemicals or byproducts. However, their industrial application is limited by their ligand promiscuity, low sensitivity, and narrow detection range. Alcohols exhibit similar structures, and no reported TFB can distinguish a specific alcohol from its analogs. Here, we engineered an alcohol-regulated transcription factor, BmoR, and obtained various mutants with remarkable properties. For example, the generated signal-molecule-specific BmoRs could distinguish the constitutional isomers n-butanol and isobutanol, with insensitivity up to an ethanol concentration of 800 mM (36.9 g/L). Linear detection of 0-60 mM of a specific higher alcohol could be achieved in the presence of up to 500 mM (23.0 g/L) ethanol as background noise. Notably, two mutants with raised outputs and over 1.0 x 107-fold higher sensitivity, and one mutant with an increased upper detection limit (14.8 g/L n-butanol or isobutanol) were screened out. Using BmoR as an example, this study systematically explored the ultimate detection limit of a TFB towards its small-molecule ligands, paving the way for in situ detection in the biofuel and wine industries.


title: Cooperative Effects of RIG-I-like Receptor Signaling and IRF1 on DNA Damage-Induced Cell Death

abstract: Properly responding to DNA damage is vital for eukaryotic cells, including the induction of DNA repair, growth arrest and, as a last resort to prevent neoplastic transformation, cell death. Besides being crucial for ensuring homeostasis, the same pathways and mechanisms are at the basis of chemoradiotherapy in cancer treatment, which involves therapeutic induction of DNA damage by chemical or physical (radiological) measures. Apart from typical DNA damage response mediators, the relevance of cell-intrinsic antiviral signaling pathways in response to DNA breaks has recently emerged. Originally known for combatting viruses via expression of antiviral factors including IFNs and establishing of an antiviral state, RIG-I-like receptors (RLRs) were found to be critical for adequate induction of cell death upon the introduction of DNA double-strand breaks. We here show that presence of IRF3 is crucial in this process, most likely through direct activation of pro-apoptotic factors rather than transcriptional induction of canonical downstream components, such as IFNs. Investigating genes reported to be involved in both DNA damage response and antiviral signaling, we demonstrate that IRF1 is an obligatory factor for DNA damage-induced cell death. Interestingly, its regulation does not require activation of RLR signaling, but rather sensing of DNA double strand breaks by ATM and ATR. Hence, even though independently regulated, both RLR signaling and IRF1 are essential for proper induction/execution of intrinsic apoptosis. Our results not only support more broadly developing IRF1 as a biomarker predictive for the effectiveness of chemoradiotherapy, but also suggest investigating a combined pharmacological stimulation of RLR and IRF1 signaling as a potential adjuvant regimen in tumor therapy.

category: cell biology


title: Haemocytes are critical for Drosophila melanogaster post-embryonic development, independent of control of the microbiota

abstract:
Proven roles for haemocytes (blood cells) have expanded beyond the control of infections in Drosophila. Despite this, the critical role of haemocytes in post-embryonic development has long been thought to be limited to control of microorganisms during metamorphosis. This has previously been shown by rescue of adult development in haemocyte-ablation models under germ-free conditions. Here we show that haemocytes have a critical role in post-embryonic development beyond their ability to control the microbiota. Using a newly generated, strong haemocyte-specific driver line for the GAL4/UAS system, we show that specific ablation of haemocytes is pupal lethal, even under axenic conditions. Genetic rescue experiments prove that this is a haemocyte-specific phenomena. RNA-seq data suggests that dysregulation of the midgut is a critical consequence of haemocyte ablation. We believe this novel role of haemocytes during metamorphosis is a major finding for the field. This is an exciting new Drosophila model to study the precise mechanisms in which haemocytes regulate tissue development, findings from which could have far reaching implications beyond invertebrate biology.

Summary StatementHaemocyte-ablation in Drosophila melanogaster with a strong haemocyte-specific driver causes pupal lethality

category: developmental biology


title: Maternal SMCHD1 controls both imprinted Xist expression and imprinted X chromosome inactivation

abstract: Embryonic development is dependent on the maternal supply of proteins through the oocyte, including factors setting up the adequate epigenetic patterning of the zygotic genome. We previously reported that one such factor is the epigenetic repressor SMCHD1, whose maternal supply controls autosomal imprinted expression in mouse preimplantation embryos and mid-gestation placenta. In mouse preimplantation embryos, X chromosome inactivation is also an imprinted process. Combining genomics and imaging, we show that maternal SMCHD1 is required not only for the imprinted expression of Xist in preimplantation embryos, but also for the efficient silencing of the inactive X in both the preimplantation embryo and mid-gestation placenta. These results expand the role of SMCHD1 in enforcing the silencing of Polycomb targets. The inability of zygotic SMCHD1 to fully restore imprinted X inactivation further points to maternal SMCHD1s role in setting up the appropriate chromatin environment during preimplantation development, a critical window of epigenetic remodelling.


title: The HIV-1 Integrase C-Terminal domain induces TAR RNA structural changes promoting Tat binding.

abstract: Recent evidence indicated that HIV-1 Integrase (IN) binds genomic viral RNA (gRNA) playing a critical role in viral particle morphogenesis and gRNA stability in host cells. Combining biophysical and biochemical approaches we show that the C-terminal flexible 18-residues tail of IN acts as a sensor of the peculiar apical structure of trans-activation response element RNA (TAR), directly interacting with its hexaloop. We highlighted how the whole IN C-terminal domain, once bound to TAR, can change its structure assisting the binding of Tat, the HIV trans-activator protein, which finally displaces IN from TAR. Our results are consistent with the emerging role of IN in early stage of proviral transcription and suggest new steps of HIV-1 life cycle that can be considered as therapeutic targets.


title: Temperature-dependent local conformations and conformational distributions of cyanine dimer labeled single-stranded -- double-stranded DNA junctions by 2D fluorescence spectroscopy

abstract: DNA replication, and the related processes of genome expression, require binding, assembly, and function of protein complexes at and near single-stranded (ss) - double-stranded (ds) DNA junctions. These central protein-DNA interactions are likely influenced by thermally induced conformational fluctuations of the DNA scaffold across an unknown distribution of functionally relevant states to provide regulatory proteins access to properly conformed DNA binding sites. Thus, characterizing the nature of conformational fluctuations and the associated structural disorder at ss-dsDNA junctions is likely critical for understanding the molecular mechanisms of these central biological processes. Here we describe spectroscopic studies of model ss-dsDNA fork constructs that contain dimers of  internally labeled cyanine (iCy3) chromophore probes that have been rigidly inserted within the sugar-phosphate backbones of the DNA strands. Our combined analyses of absorbance, circular dichroism (CD) and two-dimensional fluorescence spectroscopy (2DFS) permit us to characterize the local conformational parameters and conformational distributions. We find that the DNA sugar-phosphate backbones undergo abrupt successive changes in their local conformations - initially from a right-handed and ordered DNA state to a disordered splayed-open structure and then to a disordered left-handed conformation - as the dimer probes are moved across the ss-dsDNA junction. Our results suggest that the sugar-phosphate backbones at and near ss-dsDNA junctions adopt specific position-dependent local conformations and exhibit varying extents of conformational disorder that deviate widely from the Watson-Crick structure. We suggest that some of these conformations are likely to function as secondary-structure motifs for interaction with protein complexes that bind to and assemble at these sites.

category: biophysics


title: Differential effects of Wnt-β-catenin signaling in Purkinje cells and Bergmann glia in SCA1

abstract:
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease characterized by progressive ataxia and degeneration of specific neuronal populations, including Purkinje cells (PCs) in the cerebellum. Previous studies have demonstrated a critical role for various evolutionarily conserved signaling pathways in cerebellar patterning, such as the Wnt-{beta}-catenin pathway; however, the roles of these pathways in adult cerebellar function and cerebellar neurodegeneration are largely unknown. In this study, we found that Wnt-{beta}-catenin activity was progressively enhanced in multiple cell types in the adult SCA1 mouse cerebellum, and that activation of this signaling occurs in an ataxin-1 polyglutamine (polyQ) expansion-dependent manner. Genetic manipulation of the Wnt-{beta}-catenin signaling pathway in specific cerebellar cell populations revealed that activation of Wnt-{beta}-catenin signaling in PCs alone was not sufficient to induce SCA1-like phenotypes, while its activation in astrocytes including Bergmann glia (BG) resulted in gliosis and disrupted BG localization, which was replicated in SCA1 mouse models. Our studies identify a novel mechanism in which polyQ-expanded ataxin-1 positively regulates Wnt-{beta}-catenin signaling, and demonstrate that different cell types have distinct responses to the enhanced Wnt-{beta}-catenin signaling in the SCA1 cerebellum, underscoring an important role of BG in SCA1 pathogenesis.

Significance statementThe mechanisms underlying the degeneration of specific cellular populations in various neurodegenerative disorders remain unknown. Here, we show that the polyQ expansion of ataxin-1 activates the Wnt-{beta}-catenin signaling pathway in various cell types, including Purkinje cells and Bergmann glia, in the cerebellum of SCA1 mouse models. We used conditional mouse genetics to activate and silence this pathway in different cell types and found elevated activity of this signaling pathway impacted Bergmann glia and Purkinje cell populations differently. This study highlights the important role of Wnt-{beta}-catenin signaling pathway in glial cell types for SCA1 pathogenesis.

category: neuroscience


title: BRCA1 and ELK-1 regulate Neural Progenitor Cell Fate in the Optic Tectum in response toVisual Experience in Xenopus laevis tadpoles

abstract: In developing Xenopus tadpoles, the optic tectum begins to receive patterned visual input while visuomotor circuits are still undergoing neurogenesis and circuit assembly. This visual input regulates neural progenitor cell fate decisions such that maintaining tadpoles in the dark increases proliferation, expanding the progenitor pool, while visual stimulation promotes neuronal differentiation. To identify regulators of activity-dependent neural progenitor cell fate, we used RNA-Seq to profile the transcriptomes of proliferating neural progenitor cells and newly-differentiated immature neurons. Out of 1,130 differentially expressed (DE) transcripts, we identified six DE transcription factors which are predicted to regulate the majority of the other DE transcripts. Here we focused on Breast cancer 1 (BRCA1) and the ETS-family transcription factor, ELK-1. BRCA1 is known for its role in cancers, but relatively little is known about its potential role in regulating neural progenitor cell fate. ELK-1 is a multifunctional transcription factor which regulates immediate early gene expression. We investigated the effect of BRCA1 and ELK-1 on activity-regulated neurogenesis in the tadpole visual system using in vivo time-lapse imaging to monitor the fate of turbo-GFP-expressing SOX2+ neural progenitor cells in the optic tectum. Our longitudinal in vivo imaging analysis shows that knockdown of either BRCA1 or ELK-1 altered the fates of neural progenitor cells, and furthermore that the effects of visual experience on neurogenesis depend on BRCA1 expression, while the effects of visual experience on neuronal differentiation depend on ELK-1 expression. These studies provide insight into the potential mechanisms by which neural activity affects neural progenitor cell fate.


title: Diversification of multipotential postmitotic mouse retinal ganglion cell precursors into discrete types

abstract: The genesis of broad neuronal classes from multipotential neural progenitor cells has been extensively studied, but less is known about the diversification of a single neuronal class into multiple types. We used single-cell RNA-seq to study how newly-born (postmitotic) mouse retinal ganglion cell (RGC) precursors diversify into ~45 discrete types. Computational analysis provides evidence that RGC type identity is not specified at mitotic exit, but acquired by gradual, asynchronous fate restriction of postmitotic multipotential precursors. Some types are not identifiable until a week after they are generated. Immature RGCs may be specified to project ipsilaterally or contralaterally to the rest of the brain before their type identity has been determined. Optimal transport inference identifies groups of RGC precursors with largely non-overlapping fates, distinguished by selectively expressed transcription factors that could act as fate determinants. Our study provides a framework for investigating the molecular diversification of discrete types within a neuronal class.


title: Defining Cardiac Cell Populations and Relative Cellular Composition of the Early Fetal Human Heart

abstract: The human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Reliable identification of fetal cardiac cell types using protein markers is important for understanding cardiac development and delineating the cellular composition of the human heart during early development, which remains largely unknown. The aim of this study was to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8-12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and Troponin I (cTnI)), fibroblasts (DDR2, Thy1, Vimentin), endothelial cells (CD31) and smooth muscle cells (-SMA) were assessed. Flow cytometry revealed two distinct populations of cTnI expressing cells based on fluorescence intensity: cTnIHigh and cTnILow. MHC positive cardiomyocytes were cTnIHigh, whereas MHC negative non-myocyte cells were cTnILow. cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin was confirmed to be enriched in cultured fibroblast populations and flow cytometry revealed VimHigh and VimLow cell populations in the fetal heart. MHC positive cardiomyocytes were VimLow whilst CD31 positive endothelial cells were VimHigh. Based on the markers investigated, we estimate fetal human cardiomyocyte populations comprise 75-80% of total cardiac cells and exhibit the following marker profile: -MHC+/cTnIHigh/VimLow. For the non-cardiomyocyte population, we estimate they comprise 20-25% of total cardiac cells and exhibit the following marker profile: -MHC-/cTnILow/VimHigh. Our study suggests the marker profiles and proportions of fetal cardiac populations are distinct from that of the adult heart.


title: Influences of rare copy number variation on human complex traits

abstract: The human genome contains hundreds of thousands of regions exhibiting copy number variation (CNV). However, the phenotypic effects of most such polymorphisms are unknown because only larger CNVs (spanning tens of kilobases) have been ascertainable from the SNP-array data generated by large biobanks. We developed a new computational approach that leverages abundant haplotype-sharing in biobank cohorts to more sensitively detect CNVs co-inherited within extended SNP haplotypes. Applied to UK Biobank, this approach achieved 6-fold increased CNV detection sensitivity compared to previous analyses, accounting for approximately half of all rare gene inactivation events produced by genomic structural variation. This extensive CNV call set enabled the most comprehensive analysis to date of associations between CNVs and 56 quantitative traits, identifying 269 independent associations (P < 5 x 10-8) - involving 97 loci - that rigorous statistical fine-mapping analyses indicated were likely to be causally driven by CNVs. Putative target genes were identifiable for nearly half of the loci, enabling new insights into dosage-sensitivity of these genes and implicating several novel gene-trait relationships. CNVs at several loci created extended allelic series including deletions or duplications of distal enhancers that associated with much stronger phenotypic effects than SNPs within these regulatory elements. These results demonstrate the ability of haplotype-informed analysis to empower structural variant detection and provide insights into the genetic basis of human complex traits.

category: genetics


title: Mining transcriptomic data to identify  Saccharomyces cerevisiae  signatures related to improved and repressed ethanol production under fermentation

abstract: Saccharomyces cerevisiae is known for its outstanding ability to produce ethanol in industry. Identifying the dynamic of gene expression in S. cerevisiae in response to fermentation is required for the establishment of any ethanol production improvement program. The goal of this study was to identify the discriminative genes between improved and repressed ethanol production as well as clarifying the molecular responses to this process through mining the transcriptomic data. Through 11 machine learning based algorithms from RapidMiner employed on available microarray datasets related to yeast fermentation performance under Mg2+ and Cu2+ supplementation, 172 probe sets were identified by at least 5 AWAs. Some have been identified as being involved in carbohydrate metabolism, oxidative phosphorylation, and ethanol fermentation. Principal component analysis (PCA) and heatmap clustering were also validated the top-ranked selective probe sets. According to decision tree models, 17 roots with 100% performance were identified. OLI1 and CYC3 were identified as the roots with the best performance, demonstrated by the most weighting algorithms and linked to top two significant enriched pathways including porphyrin biosynthesis and oxidative phosphorylation. ADH5 and PDA1 are also recognized as differential top-ranked genes that contribute to ethanol production. According to the regulatory clustering analysis, Tup1 has a significant effect on the top-ranked target genes CYC3 and ADH5 genes. This study provides a basic understanding of the S. cerevisiae cell molecular mechanism and responses to two different medium conditions (Mg2+ and Cu2+) during the fermentation process.

category: bioinformatics


title: The amount range of membrane cholesterol required for robust cell adhesion and proliferation in serum-free condition.

abstract: Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines. However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture. In this study, we develop a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbeccos Modified Eagles Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated well (called as "DA-X condition") showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.


title: Probing behavior of the corn leafhopper Dalbulus maidis on susceptible and resistant maize hybrids

abstract: The corn leafhopper Dalbulus maidis is the main vector of the pathogens that cause corn stunt, a major disease of maize in the Americas. As host resistance is an efficient tool to control diseases, the findings of a previous report showed that some corn hybrids are resistant to D. maidis. In this work, we assessed the probing behavior of D. maidis on susceptible and resistant corn hybrids using EPG (Electrical Penetration Graph) technology. Fifteen-day-old females were monitored for 20 hours, with access to hybrids DK390, DK670, DK79-10, and DK72-10. Hybrids DK390 and DK72-10 showed resistance to D. maidis in phloem, since insects feeding on these hybrids presented more salivation events in phloem without subsequent ingestion, which are seen as failed attempts to ingest. A reduction of the total duration of phloem ingestion was observed, and accordingly of the time spent by insects with access to these hybrids on xylem ingestion. The hybrid DK390 also had mesophyll resistance, seen as less probing time and a higher number of probes of short duration. These findings support and are consistent with previous research, providing useful information to characterize maize hybrids resistant to D. maidis, and so to corn stunt.


title: Temporal pattern recognition in the human brain: a dual simultaneous processing

abstract: Pattern recognition is a major scientific topic. Strikingly, while machine learning algorithms are constantly refined, the human brain emerges as an ancestral biological example of such complex procedure. However, how it transforms sequences of single objects into meaningful temporal patterns remains elusive. Using magnetoencephalography (MEG) and magnetic resonance imaging (MRI), we discovered and mathematically modelled an inedited dual simultaneous processing responsible for pattern recognition in the brain. Indeed, while the objects of the temporal pattern were independently elaborated by a local, rapid brain processing, their combination into a meaningful superordinate pattern depended on a concurrent global, slower processing involving a widespread network of sequentially active brain areas. Expanding the established knowledge of neural information flow from low- to high-order brain areas, we revealed a novel brain mechanism based on simultaneous activity in different frequency bands within the same brain regions, highlighting its crucial role underlying complex cognitive functions.


title: Unravelling the role of thyroid hormones in seasonal neuroplasticity in European Starlings (Sturnus vulgaris)

abstract: 1Thyroid hormones clearly play a role in the seasonal regulation of reproduction, but any role they might play in song behavior and the associated seasonal neuroplasticity in songbirds remains to be elucidated. To pursue this question, we first established seasonal patterns in the expression of thyroid hormone regulating genes in male European starlings employing in situ hybridization methods. Thyroid hormone transporter LAT1 expression in the song nucleus HVC was elevated during the photosensitive phase, pointing towards an active role of thyroid hormones during this window of possible neuroplasticity. In contrast DIO3 expression was high in HVC during the photostimulated phase, limiting the possible effect of thyroid hormones to maintain song stability during the breeding season. Next, we studied the effect of hypothyroidism on song behavior and neuroplasticity using in vivo MRI. Hypothyroidism inhibited the photostimulation-induced increase in testosterone, confirming the role of thyroid hormones in activating the hypothalamic-pituitary-gonadal (HPG) axis. Surprisingly, apart from the myelination of several tracts during the photostimulated phase, most neuroplasticity related to song production was unaffected by hypothyroidism. Remarkably, T3 plasma concentrations were negatively correlated to the microstructural changes in several song control nuclei. Potentially, a global reduction of circulating thyroid hormones during the photosensitive period is necessary to lift the brake imposed by the photorefractory period, whereas local fine-tuning of thyroid hormone concentrations through LAT1 could activate underlying neuroplasticity mechanisms. Given the complexity of thyroid hormone effects, this study is a steppingstone to disentangle the influence of thyroid hormones on seasonal neuroplasticity.


title: Thalamus gates active dendritic computations in cortex during sensory processing

abstract: Perception is causally linked to a calcium-dependent spiking mechanism that is built into the distal dendrites of layer 5 pyramidal tract neurons - the major output cell type of the cerebral cortex. It is yet unclear which circuits activate this cellular mechanism upon sensory stimulation. Here we found that the same thalamocortical axons that relay sensory signals to layer 4 also densely target the dendritic domain by which pyramidal tract neurons initiate calcium spikes. Distal dendritic inputs, which normally appear greatly attenuated at the cell body, thereby generate bursts of action potentials in cortical output during sensory processing. Our findings indicate that thalamus gates an active dendritic mechanism to facilitate the combination of sensory signals with top-down information streams into cortical output. Thus, in addition to being the central hub for sensory signals, thalamus is also likely to ensure that the signals it relays to cortex are perceived by the animal.


title: Survey evaluation of dog owners feeding habits in a household setting and comparison of FDA hygiene protocols on dog bowl bacterial contamination as evaluated by total aerobic cell counts.

abstract: In-home pet food handling and food dish hygiene practices can have adverse health impacts for both humans and pets. Safe food and dish handling guidelines are not easily evidenced for pet owners. The study was designed to investigate dog owners feeding habits and evaluate the impact of the Food and Drug Association (FDA) hygiene protocols on dog food dish contamination. Procedures and surveys were approved by NCSU-IACUC and -IRB. Pet feeding and food dish hygiene data were collected from 417 dog owner surveys and 68 food dish swabs. Total aerobic cell counts (TAC) were performed on 68 dishes and randomly assigned into Group A (FDA pet food handling and dish hygiene guidelines), Group B (FDA pet and human food handling and dish hygiene guidelines), or Group C (no guidelines). Hygiene protocols were instituted in-home for 1 week, followed by a second TAC and follow-up survey. Survey from dog owners-households indicated: 4.7% were aware of FDA pet food handling and dish hygiene guidelines; 36% have individuals [≤] 13 years old and/or immunocompromised; 43% store dog food 0-5 feet from human food; 34% wash their hands after feeding; and 33% prepare their dog food on human food preparation surfaces. The hygiene protocols followed by Groups A and B resulted in significant decreases in food dish TAC (P<0.001; 1.40; p=0.026; 0.604, respectively), as compared to Group C (p=0.373). Hot water (>160F) washing decreased TAC (p=0.005) over cold/lukewarm water. In the follow-up survey, 8% of Group A and B respondents reported likely to adhere to protocols long-term. This study suggests a need for pet food handling and dish hygiene guideline education to minimize bacterial contamination of dishes, especially in high-risk households.


title: Impact of various preservation and storage methods on the viability of mycoplasma field strains isolated in Mali

abstract: The survival of five mycoplasma strains was studied in different storage media (Mycoplasma complet media without cryopreservative agent, Mycoplasma complete media with addition of horse serum, Mycoplasma complete media with addition of glycerol and lyophilized cultures without stabilizer) under different temperatures (+37{degrees}C, +4{degrees}C, -20{degrees}C, -85{degrees}C) during 24 months. Five Mycoplasma strains, Mycoplasma mycoides subsp mycoides (Mmm), Mycoplasma bovis (Mb), Mycoplasma agalactiae (Ma), Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) were isolated from various parts of the country. The initial titers of the strains determined by the agar plate count before storage were 42.4x107 UFC/ml (8.6 log UFC/ml) for Mmm strain; 32.4x108 UFC/ml (9.51 log UFC/ml) for M.bovis strain; 12.4x109 UFC/ml (10.09 log UFC/ml) for Ma strain; 2.4x109 UFC/ml (9.38 log UFC/ml) for Mg and 2.8x109 UFC/ml (9.45 log UFC/ml) for Ms strain. After 3 weeks of storage, no viable mycoplasmas were detected in all the conservation media at +37{degrees}C and after 3 months of storage at +4{degrees}C except for the lyophilized cultures in which an average viability rate of 17.81% was observed. Overall, the mycoplasma strains remained viable at freezing temperatures after 24 months regardless of the storage medium, but with decreasing titers, which was noticeable with mycoplasma complete media, and mycoplasma media with horse serum. Conversely, at -20{degrees}C the average viability rates after 24 months of storage were 84.36% (with glycerol) and 90.04% (lyophilized cultures). At -85{degrees}C after 24 months of storage, this was 87.98% (with glycerol) and 91.44% (lyophilized cultures). These findings suggest that, in the absence of the lysophylisation process, the addition of glycerol may be recommended for long-term storage of frozen mycoplasma isolates.


title: A holistic insight of mycobacteriophage induced changes in mycobacterial cells

abstract: Mycobacteriophages are phages that interact with mycobacteria resulting in their killing. Although lysis is the major mechanism by which mycobacteriophages cause cell death, other mechanisms may also be involved. The present study was initiated with the objective of investigating the changes that take place at the cellular level following the infection of mycobacterial cells by phage D29. To investigate this issue, we took recourse to performing immunofluorescence and electron microscopic studies. Transmission electron microscopic examination revealed the adsorption of phages on to the surface of mycobacteria, following which penetration of the tail through the thick mycoloic acid layer was seen. At later time points discrete populations of cells at different stages of lysis were observed, which comprised of completely lysed cells, in which the cells were fragmented and those at the early onset stage exhibited formation of membrane pores through which the phages and intracellular contents were released. SEM results also indicated that phages may come out through the entire surface of the cell, or alternatively through gaps in the surface. In some of the images we observed structures that apparently resembled membrane blebs which are normally encountered when cells undergo programmed cell death (PCD). In addition, we observed significant increase in DNA fragmentation as well as membrane depolarization, which are also indicative of occurrence of PCD. As several bacterial PCD pathways are mediated by the toxin-antitoxin (TA) modules, the expression profile of all the TA systems was examined before and after phage infection. Apart from specifically addressing the issue of PCD in mycobacteriophage infected cells, this investigation has led to the development of facile tools necessary for investigating mycobacteriophage-mycobacteria interactions by means of microscopic methods.


title: Structural basis for long-chain isoprenoids synthesis by cis-prenyltransferases

abstract: Isoprenoids are the largest group of natural products, found in all living organisms and play an essential role in numerous cellular processes. These compounds are synthesized by prenyltransferases, catalyzing the condensation reaction between an allylic diphosphate primer and a variable number of isopentenyl diphosphate (C5) units. This superfamily of enzymes can be subdivided into trans- or cis-prenyltransferases according to the stereoisomerism of the product. The cis branch can be further classified according to product length. While the active site volume was suggested to determine the final length in enzymes synthesizing short- and medium-chain products (up to C60), long-chain enzymes (up to C120) and rubber synthases (>C10,000) fail to conform to this paradigm. Here, to resolve the structural basis for long-chain isoprenoid synthesis, we focused on the human cis-prenyltransferase complex (hcis-PT). This enzyme, peripheral to the endoplasmic reticulum membrane, produces the precursor for dolichol phosphate, a membrane residing glycosyl carrier. In line with its crucial role in the cellular protein glycosylation machinery, disease-causing mutations in hcis-PT were shown to result in a wide spectrum of clinical phenotypes. The crystallographic structures of hcis-PT in four different substrate/product-bound conformations revealed an outlet enabling product elongation into the bulk solvent. Moreover, hydrogen-deuterium exchange mass spectrometry analysis in solution showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Finally, using a fluorescent substrate analog and a fluorescently-labeled lipid nanodiscs, we show that product elongation and membrane association are closely correlated. Together, our results support directional product synthesis in long-chain enzymes and rubber synthases, with a distinct substrate inlet and product outlet, allowing direct membrane insertion of the elongating isoprenoid during catalysis. This mechanism uncouples active site volume from product length and circumvents the need to expulse hydrophobic product into a polar environment prior to membrane insertion.


title: Conserved Structure Modules within the lncRNA SChLAP1 Mediate Protein Recognition Implicated in Aggressive Prostate Cancer

abstract: The lncRNA Second Chromosome Locus Associated with Prostate 1 (SChLAP1) was previously identified as a predictive biomarker and driver of aggressive prostate cancer. Recent work suggests that SChLAP1 may bind the SWI/SNF chromatin remodeling complex to promote prostate cancer metastasis, though the exact role of SWI/SNF recognition is debated. To date, there are no detailed biochemical studies of apo SChLAP1 or the SChLAP1:SWI/SNF complex. Herein, we report the first secondary structure model of SChLAP1 utilizing SHAPE-MaP both in vitro and in cellulo. Comparison of the in vitro and in cellulo data via {Delta}SHAPE identified putative protein binding sites within SChLAP1, specifically to evolutionarily conserved exons of the transcript. We also demonstrate that global SChLAP1 secondary structure is sensitive to both purification method and magnesium concentration. Further, we identified a 3-fragment of SChLAP1 (SChLAP1Frag) that harbors multiple potential protein binding sites and presents a robustly folded secondary structure, supporting a functional role for this region. This work lays the foundation for future efforts in selective targeting and disruption of the SChLAP1:protein interface and the development of new therapeutic avenues in prostate cancer treatment.


title: SiglecFhigh neutrophils in lung tumor tissues suppress local CD8 T cell responses and limit the efficacy of anti PD-L1 antibodies

abstract:
BackgroundTumor infiltrating neutrophils generally correlates to worst prognosis and refractoriness to immunotherapy yet the complexity and significance of diverse subsets resident in tumor tissues has just begun to emerge. In lung tumors, a network of neutrophils states with graded protumorigenic properties is conserved between mouse and humans and include a subset of mature, long lived cells expressing the sialic-acid-binding protein SiglecF (SiglecFhigh neu). The mechanism of recruitment of SiglecFhigh neu into tumor tissues and the impact on local anti-tumor T cell responses and interference with immunotherapy is still elusive.

MethodsWe used an immunogenic model of KrasG12D Tp53 null adenocarcinoma of the lung to screen for factors inducing the recruitment of SiglecFhigh neu, followed by gene editing to delete selected candidates. We analyzed frequencies and effector functions of endogenous CD8 T cell responses in controls and SiglecFhigh neu depleted tumors by flow cytometry and functional assays. Tissues fluorescence and confocal imaging of lung sections was used to explore the relative distribution of neu and CD8 T cells. To establish the impact of SiglecFhigh neu on anti-tumoral immune responses we treated cohort of animals with anti-PD-L1 antibodies to evaluate tumor growth in control conditions and under therapy.

ResultsWe found that tumor tissues express high levels of CXCL5, mapping to cancer cells. Upon deletion of chemokine expression by gene editing, the recruitment of SiglecFhigh neu was almost entirely abrogated. In tumors depleted of SiglecFhigh neu, the density of tumor specific endogenous CD8 T cells was 3-fold higher than in controls and showed significantly enhanced activation and effector functions. Importantly, checkpoint blockade with anti PD-L1 antibodies was ineffective in control tumors but showed a significant benefit in SiglecFhigh neu depleted tumors.

ConclusionThis study demonstrates that SiglecFhigh neu differentiating in lung tumor tissues inhibit local CD8 T cell responses and interfere with the success of checkpoint blockade. These data suggest that blocking selectively tissue resident neu may promote better responses to immunotherapy.


title: PI4P and BLOC-1 remodel endosomal membranes into tubules

abstract: Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers play major roles in the flux of membrane lipids and proteins to acceptor organelles. However, how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. By exploiting imaging approaches at different scales on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability and functions of recycling endosomal tubules. Endosomal PI4P production by type II PI4-kinases is needed to form nascent curved tubules through binding of BLOC-1 that stabilize and elongate them. Membrane remodeling by the PI4P/ BLOC-1 module functions not only in the recycling of endosomal cargoes, but also in the lifecycles of intracellular pathogens such as Chlamydia bacteria and influenza virus. This study demonstrates how a phospholipid and a protein complex coordinate as a minimal machinery to remodel cellular membranes into functional tubes.


title: Expression of brain-derived neurotrophic factor in basolateral amygdala inputs to lateral septum is necessary for mice to identify socially novel individuals

abstract: The lateral septum (LS) is a GABAergic region in the basal forebrain that is implicated in sociability. However, the neural circuits and cell signaling pathways that converge on the LS to mediate social behaviors arent well understood. Multiple lines of evidence suggest that brain-derived neurotrophic factor (BDNF) signaling through its receptor TrkB plays important roles in social behavior. While BDNF is not locally produced in LS, we demonstrate that nearly all GABAergic neurons in LS express TrkB. Local knock-down of TrkB expression from LS neurons decreased sociability and reduced recruitment of social novelty-induced neural activity. Since BDNF is not synthesized in LS, we evaluated which inputs to the LS could serve as potential BDNF sources for controlling sociability. By selectively ablating inputs to LS, we demonstrated that inputs from the basolateral amygdala (BLA), but not ventral CA1 (vCA1), regulate sociability. Moreover, depleting BDNF selectively in BLA-LS projection neurons phenocopied the decreased sociability observed following either local LS TrkB knockdown or ablation of BLA-LS inputs. These data support the hypothesis that BLA-LS projection neurons could serve as a critical source of BDNF for activating TrkB signaling in LS neurons to control sociability.


title: The basolateral amygdala to lateral septum circuit is critical for regulating sociability in mice


title: Non-motile subpopulations of Pseudomonas aeruginosa repress flagellar motility in motile cells through a type IV pili- and Pel-dependent mechanism

abstract:
The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host-colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that under swarming conditions, swarming motility can be repressed by cells that are non-motile due to the absence of a flagellum or flagellar rotation. Non-swarming cells, due to mutations that prevent either flagellum biosynthesis or rotation, present at 5% of the total population suppressed swarming of wild-type cells under the conditions tested in this study. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. In contrast, flagellated cells required only type IV pili, but not Pel production, in order for swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and non-motile cells may enhance the formation of sessile communities including those involving multiple genotypes, phenotypically-diverse cells, and perhaps other species.

ImportanceOur study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of the larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and non-motile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.


title: The uptake of avermectins in Caenorhabditis elegans is dependent on Intra-Flagellar Transport and other protein trafficking pathways

abstract:
Parasitic nematodes are globally important and place a heavy disease burden on infected humans, crops and livestock, while commonly administered anthelmintics used for treatment are being rendered ineffective by increasing levels of resistance. Although the modes of action and resistance mechanisms caused by detoxification and target site insensitivity for these compounds is well documented, the mechanisms for uptake, which can also cause resistance, are still poorly defined. It has recently been shown in the model nematode Caenorhabditis elegans that the avermectins or macrocyclic lactones such as ivermectin and moxidectin gain entry though the sensory cilia of the amphid neurons. This study interrogated the molecular mechanisms involved in the uptake of avermectins using a combination of forward genetics and targeted resistance screening approaches along with visualising a BODIPY labelled ivermectin analog and confirmed the importance of intraflagellar transport in this process. This approach also identified the protein trafficking pathways used by the downstream effectors and the components of the ciliary basal body that are required for effector entry into these non-motile structures. Mutations in many of the genes under investigation also resulted in resistance to the unrelated anthelmintic drugs albendazole and levamisole, giving insights into the potential mechanisms of multidrug resistance observed in field isolates of the parasitic nematodes that are a scourge of ruminant livestock. In total 50 novel C. elegans anthelmintic survival associated genes were identified in this study, three of which (daf-6, rab-35 and inx-19) are associated with broad spectrum cross resistance. When combined with previously known resistance genes, there are now 53 resistance associated genes which are directly involved in amphid, cilia and IFT function.

Author SummaryNematodes represent significant pathogens of man and domestic animals and control relies heavily on limited classes of Anthelminitic drugs. Single and multi-drug resistance is a growing problem however mechanisms of anthelmintic drug resistance and drug uptake by nematodes remain to be clearly elucidated. In Caenorhabditis elegans there has been an association between amphid and dye filling defects with resistance to avermectins however the effector and causal mechanisms remain elusive. This study uses a combination of fluorescently labelled anthelmintics and anthelmintic resistance screens to probe the uptake mechanisms for these drugs. The role of the sensory amphids in the uptake of avermectins was confirmed. The avermectins enter the distal segment of the cilia using an effector which is delivered by the UNC-119 and UNC-33/UNC-44 transport systems to the base of the cilia, followed by distal appendage dependent entry and transport along the cilia by the intraflagellar transport pathway. Of the genes investigated, three (daf-6, rab-35 and inx-19) were linked to cross resistance against all the anthelmintics tested (Ivermectin, Moxidectin, Albendazole and Levamisole). This study gives further insight into how important classes of anthelmintics enter nematodes and highlights the potential for this process to give rise to anthelmintic resistance.


title: A pathogenic fungus uses volatiles to entice male flies into fatal matings with infected female cadavers

abstract: To ensure dispersal, many parasites and pathogens behaviourally manipulate infected hosts. Other pathogens and certain insect-pollinated flowers use sexual mimicry and release deceptive mating signals. However, it is unusual for pathogens to rely on both behavioural host manipulation and sexual mimicry. Here, we show that the host-specific and behaviourally manipulating pathogenic fungus, Entomophthora muscae, generates a chemical blend of volatile sesquiterpenes and alters the level of natural host cuticular hydrocarbons in dead infected female house fly (Musca domestica) cadavers. Healthy male house flies respond to the fungal compounds and are enticed into mating with dead female cadavers. This is advantageous for the fungus as close proximity between host individuals leads to an increased probability of infection. The fungus-emitted volatiles thus represent the evolution of an extended phenotypic trait that exploit male flies willingness to mate and benefit the fungus by altering the behavioural phenotype of uninfected healthy male host flies.


title: Generation and characterization of Ccdc28b mutant mice links the Bardet-Biedl associated gene with social behavioral phenotypes

abstract:
CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). After confirming the depletion of Ccdc28b we performed a phenotypic characterization showing that Ccdc28b mut animals present a mild phenotype: i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors suggestive of autism spectrum disorder (ASD). This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Importantly however, our data suggests a possible causal link between CCDC28B and ASD-like phenotypes that exceeds the context of BBS: filtering for rare deleterious variants, we found CCDC28B mutations in eight probands from the Simmons Simplex Collection cohort. Furthermore, a phenotypic analysis showed that CCDC28B mutation carriers present lower BMI and mild communication defects compared to a randomly selected sample of SSC probands. Thus, our results suggest that mutations in CCDC28B lead to mild autism-like features in mice and humans. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and ASD-like phenotypes, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of ASD.

AUTHOR SUMMARYBardet-Biedl syndrome (BBS) is caused by mutations in any of 21 genes known to date. In some families, BBS can be inherited as an oligogenic trait whereby mutations in more than one BBS gene collaborate in the presentation of the syndrome. In addition, CCDC28B was identified as a modifier of BBS, associated with a more severe presentation of the syndrome. Different mechanisms, all relying on functional redundancy, have been proposed to explain this genetic interaction and the characterization of different BBS proteins supported this possibility as they were shown to play roles in the same cellular organelle, the primary cilium.

Similarly, CCDC28B also participates in cilia biology regulating the length of the organelle: knockdown of CCDC28B in cells results in cilia shortening and depletion in zebrafish also results in early embryonic phenotypes characteristic of other cilia mutants. Here, we sought to generate a mouse Ccdc28b mutant to determine whether it would be sufficient to cause a ciliopathy phenotype, and to generate a reagent critical to further dissect its modifying role in the context of BBS. Overall, Ccdc28b mutant mice presented a mild phenotype, a finding fully compatible with a modifier rather than a causal BBS gene. In addition, we found that Ccdc28b mutants showed a clear autism-like behavior, and autism is indeed a feature of several BBS patients. Importantly, we identified multiple individuals with autism from the Simmos Simplex Collection to carry disruptive mutations in CCDC28B suggesting that this gene is causally associated with autism independently of BBS.


title: Defining cellular complexity in human autosomal dominant polycystic kidney disease by multimodal single cell analysis

abstract: Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease and is characterized by the formation and progressive expansion of kidney cysts. Most ADPKD cases arise from mutations in either the PKD1 or PKD2 gene but the precise downstream signaling pathways driving cyst growth are not well understood, and relatively few studies investigate human cystic kidney due to sample scarcity. In order to better understand the cell types and states driving human ADPKD progression, we analyzed eight ADPKD and five healthy human kidney samples, generating a single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. The integrated datasets identified 11 primary cell clusters including most epithelial cell types as well as large endothelial and fibroblast cell clusters. Proximal tubular cells from ADPKD kidneys expressed a failed repair transcriptomic signature characterized by profibrotic and proinflammatory transcripts. We identified the G protein-coupled receptor GPRC5A as specifically upregulated in cyst lining cells derived from collecting duct. The principal cell subpopulation enriched for GPRC5A expression also exhibited increased transcription factor binding motif availability for NF-{kappa}B, TEAD, CREB and retinoic acid receptor families and we identified and validated a distal enhancer regulating GPRC5A expression containing these transcription factor binding motifs. This study establishes the single cell transcriptomic and epigenomic landscape of ADPKD, revealing previously unrecognized cellular heterogeneity.


title: Mining of thousands of prokaryotic genomes reveals high abundance of prophage signals

abstract: Phages and prophages are one of the principal modulators of microbial populations. However, much of their diversity is still poorly understood. Here, we extracted 33,624 prophages from 13,713 complete prokaryotic genomes in order to explore the prophage diversity and their relationships with their host. Our results reveal that prophages were present in 75% of the genomes studied. In addition, Enterobacterales were significantly enriched in prophages. We also found that pathogens are a significant reservoir of prophages. Finally, we determined that the prophage relatedness and the range of genomic hosts were delimited by the evolutionary relationships of their hosts. On a broader level, we got insights into the prophage population, identified in thousands of publicly available prokaryotic genomes, by comparing the prophage distribution and relatedness between them and their hosts.


title: Mitochondrial fission process 1 (MTFP1) controls bioenergetic efficiency and prevents inflammatory cardiomyopathy and heart failure in mice

abstract: Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in adult-onset dilated cardiomyopathy (DCM) characterized by sterile inflammation and cardiac fibrosis that progressed to heart failure and middle-aged death. Failing hearts from cardiomyocyte-restricted knockout mice displayed a general decline in mitochondrial gene expression and oxidative phosphorylation (OXPHOS) activity. Pre-DCM, we observed no defects in mitochondrial morphology, content, gene expression, OXPHOS assembly nor phosphorylation dependent respiration. However, knockout cardiac mitochondria displayed reduced membrane potential and increased non-phosphorylation dependent respiration, which could be rescued by pharmacological inhibition of the adenine nucleotide translocase ANT. Primary cardiomyocytes from pre-symptomatic knockout mice exhibited normal excitation-contraction coupling but increased sensitivity to programmed cell death (PCD), which was accompanied by an opening of the mitochondrial permeability transition pore (mPTP). Intriguingly, mouse embryonic fibroblasts deleted for Mtfp1 recapitulated PCD sensitivity and mPTP opening, both of which could be rescued by pharmacological or genetic inhibition of the mPTP regulator Cyclophilin D. Collectively, our data demonstrate that contrary to previous in vitro studies, the loss of the MTFP1 promotes mitochondrial uncoupling and increases cell death sensitivity, causally mediating pathogenic cardiac remodeling.

category: physiology


title: Cytometric Analysis of Diverse Glaucophyte Species Reveals Distinctive Signals Useful for Fluorescence-Based Detection and Sorting

abstract: Glaucophytes, red algae and viridiplants (green algae and land plants) are formally united in the supergroup Archaeplastida. Although diverse molecular and genomic evidence suggest the common origin of the three Archaeplastida lineages, the lack of a robust glaucophyte knowledgebase has limited comprehensive evaluations of competing hypotheses. Glaucophytes are rare and apparently confined to freshwater habitats. However, the distribution and diversity of these algae have not been thoroughly explored owing to challenges with detecting and isolating novel specimens. Here we examined the cytometric signatures of representative species of the genera Cyanophora, Cyanoptyche, Glaucocystis and Gloeochaete for a distinctive signal that would aid identification. Most glaucophytes analyzed presented a relatively high red fluorescence signal due to the presence of the blue phycobiliproteins C-phycocyanin and allophycocyanin. Cell-size differences and the concurrent presence of the red phycobiliprotein phycoerythrin in other algal lineages, such as red algae and cryptophytes, allowed us to distinguish glaucophytes from other photosynthetic cells containing blue phycobiliproteins. Our results indicate that the peculiar autofluorescence signal of glaucophytes will facilitate further identification and isolation on novel specimens of this scarce but important algal group.


title: Can intraspecific variation in a herbivorous mite alter responses to drought-stressed host plant? A common garden experiment in the context of climate change

abstract: Drought associated with climate change can stress plants, altering their interactions with phytophagous arthropods. Dryness not only impacts cultivated plants but also their parasites, which in some cases are favoured by drought. Herbivorous arthropods feeding on drought-stressed plants typically produce bigger offspring and develop faster. However, how much responses to abiotic factors differ among populations of a species remains poorly documented. Here, we evaluate variability among populations of a major agricultural pest, the two spotted spider mite, Tetranychus urticae, in response to drought-stress. We compare key life-history parameters of twelve populations that originate from climates ranging from wet and cool Atlantic locations to medium to dry hot Mediterranean locations. We evaluated how plant drought stress affects four life history traits: development time, fecundity, sex-ratio and emigration rate in an experiment comparing well-watered and drought-stressed bean plants. Mites feeding on drought-stressed plants developed faster and attempted to leave leaves less often, and young females were more fecund. The mites from wet temperate climates exhibited greater plasticity between the two water regimes than mites originating from dryer and hot climates. Thus, climate in the area of origin influences mite response to drought.

category: zoology


title: Herbivore life histories are altered by drought stress in their hosts plants


title: Cytotoxic CD4+ T cells driven by T-cell intrinsic IL-18R/MyD88 signaling predominantly infiltrate Trypanosoma cruzi-infected hearts

abstract: Increasing attention has been directed to cytotoxic CD4+ T cells (CD4CTLs) in different pathologies, both in humans and mice. The impact of CD4CTLs in immunity and the mechanisms controlling their generation, however, remain poorly understood. Here, for the first time, we showed that CD4CTLs abundantly differentiate during mouse infection with an intracellular parasite. CD4CTLs appear in the spleen in parallel to Th1 cells, display pathogen-derived peptide-specific cytotoxicity against antigen-presenting cells and express immunoregulatory and/or exhaustion markers. We demonstrated that CD4CTL absolute numbers and activity are severely reduced in both Myd88-/- and Il18ra-/- mice. Of note, the infection of mixed-bone marrow chimeras revealed that WT, but not Myd88-/-, cells transcribe the CD4CTL gene signature and that Il18ra-/-CD4+ phenocopy Myd88-/-CD4+ T cells. Moreover, the adoptive transfer of WT CD4+GzB+ T cells to susceptible Il18ra-/- mice increased their survival. Importantly, cells expressing the CD4CTL phenotype predominate among CD4+ T cells infiltrating the infected cardiac tissue, are increased in the circulation of Chagas patients and their frequency correlates with severe cardiomyopathy. Our findings describe CD4CTLs as a major player in immune response to a relevant human pathogen and disclose T-cell intrinsic IL-18R/MyD88 signaling as a key pathway controlling the magnitude of the CD4CTL response.


title: Escherichia coli ribosomal protein S1 enhances the kinetics of ribosome biogenesis and RNA decay

abstract: Escherichia coli ribosomal protein S1 is essential for translation initiation of mRNAs and for cellular viability. Two oligonucleotide binding (OB)-fold domains located in the C-terminus of S1 are dispensable for growth, but their deletion causes a cold-shock phenotype, loss of motility and deregulation of RNA mediated stress responses. Surprisingly, the expression of the small regulatory RNA RyhB and one of its repressed target mRNA, sodB, are enhanced in the mutant strain lacking the two OB domains. Using in vivo and in vitro approaches, we show that RyhB retains its capacity to repress translation of target mRNAs in the mutant strain but becomes deficient in triggering rapid turnover of those transcripts. In addition, the mutant is defective in of the final step of the RNase E-dependent maturation of the 16S rRNA. This work unveils an unexpected function of S1 in facilitating ribosome biogenesis and RyhB-dependent mRNA decay mediated by the RNA degradosome. Through its RNA chaperone activity, S1 participates to the coupling between ribosome biogenesis, translation, and RNA decay.


title: Moran Process Version of the Tug-of-War Model: Complex Behavior Revealed by Mathematical Analysis and Simulation Studies

abstract: AO_SCPLOWBSTRACTC_SCPLOWIn a series of publications McFarland and co-authors introduced the tug-of-war model of evolution of cancer cell populations. The model is explaining the joint effect of rare advantageous and frequent slightly deleterious mutations, which may be identifiable with driver and passenger mutations in cancer. In this paper, we put the Tug-of-War model in the framework of a denumerable-type Moran process and use mathematics and simulations to understand its behavior. The model is associated with a time-continuous Markov Chain (MC), with a generator that can be split into a sum of the drift and selection process part and of the mutation process part. Operator semigroup theory is then employed to prove that the MC does not explode, as well as to characterize a strong-drift limit version of the MC which displays "instant fixation" effect, which was an assumption in the original McFarlands model. Mathematical results are fully confirmed by simulations of the complete and limit versions. They also visualize complex stochastic transients and genealogies of clones arising in the model.

category: evolutionary biology


title: Unlocking the functional potential of polyploid yeasts

abstract: Breeding and domestication have generated widely exploited crops, animals and microbes. However, many Saccharomyces cerevisiae industrial strains have complex polyploid genomes and are sterile, preventing genetic improvement strategies based on breeding. Here, we present a novel strain improvement approach based on the budding yeasts property to promote genetic recombination when meiosis is interrupted and cells return-to-mitotic-growth (RTG). We demonstrated that two unrelated sterile industrial strains with complex triploid and tetraploid genomes were RTG-competent and developed a visual screening for easy and high-throughput identification of recombined RTG clones based on colony phenotypes. Sequencing of the evolved clones revealed unprecedented levels of RTG-induced genome-wide recombination. We generated and extensively phenotyped a RTG library and identified clones with superior biotechnological traits. Thus, we propose the RTG-framework as a fully non-GMO workflow to rapidly improve industrial yeasts that can be easily brought to the market.


title: fcfdr: an R package to leverage continuous and binary functional genomic data in GWAS

abstract:
SummaryGWAS discovery is limited in power to detect associations that exceed the stringent genome-wide significance threshold, but this limitation can be alleviated by leveraging relevant auxiliary data. Frameworks utilising the conditional false discovery rate (cFDR) can be used to leverage continuous auxiliary data (including GWAS and functional genomic data) with GWAS test statistics and have been shown to increase power for GWAS discovery whilst controlling the FDR. Here, we describe an extension to the cFDR framework for binary auxiliary data (such as whether SNPs reside in regions of the genome with specific activity states) and introduce an all-encompassing R package to implement the cFDR approach, fcfdr, demonstrating its utility in an application to type 1 diabetes.

Availability and implementationThe fcfdr R package is freely available at: https://github.com/annahutch/fcfdr. Scripts and data to reproduce the analysis in this paper are freely available at: https://annahutch.github.io/fcfdr/articles/t1d_app.html


title: A Null Allele of the Pol IV Second Subunit is Viable in Oryza sativa

abstract: All eukaryotes possess three DNA-dependent RNA polymerases, Pols I-III, while land plants possess two additional polymerases, Pol IV and Pol V. Derived through duplication of Pol II subunits, Pol IV produces 24-nt siRNAs that interact with Pol V transcripts to target de novo DNA methylation and silence transcription of transposons. Members of the grass family encode additional duplicated subunits of Pol IV and V, raising questions regarding the function of each paralog. In this study, we identify a null allele of the putative Pol IV second subunit, NRPD2, and demonstrate that NRPD2 is the sole subunit functioning with NRPD1 in small RNA production and CHH methylation in leaves. Homozygous nrpd2 mutants have neither gametophytic defects, nor embryo lethality, although adult plants are dwarf and sterile.


title: Actin filaments act as a size-dependent diffusion barrier around centrosomes

abstract:
The centrosome, a non-membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that the centrosome may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we found that centrosomes were highly accessible to soluble molecules with a Stokes radius of [≤] 5.1 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size-dependent diffusion barrier at centrosomes. The permeability of barriers was tightly regulated by branched actin filaments outside of centrosomes. Such barrier gated the microtubule nucleation. We propose that actin filaments spatiotemporally constrain the distribution of molecules at centrosomes in a size-dependent manner.

Significance StatementCentrosome maintains its microenvironment without membrane. Whether the dense protein complexes outside centrosomes including pericentriolar matrix, microtubules, and branched actin filaments provide physical obstruction is unclear yet. We here established a series of new tools for quantitative evaluation of the diffusion rates of varisized soluble proteins in different sub-compartments of centrosomes. Our results demonstrated that branched actin filaments, but not pericentriolar matrix or microtubules, around centrosome have acted as a size-dependent diffusion barrier and physically constrain centrosome microtubule nucleation.


title: Potassium-chelating drug sodium polystyrene sulfonate enhances lysosomal function and suppresses proteotoxicity

abstract:
Lysosomes are crucial for degradation and recycling of damaged proteins and cellular components. Therapeutic strategies enhancing lysosomal function are a promising approach for aging and age-related neurodegenerative diseases. Here, we show that an FDA approved drug sodium polystyrene sulfonate (SPS), used to reduce high blood potassium in humans, enhances lysosomal function both in C.elegans and in human neuronal cells. Enhanced lysosomal function following SPS treatment is accompanied by the suppression of proteotoxicity caused by expression of the neurotoxic peptides A{beta} and TAU. Additionaly, treatment with SPS imparts health benefits as it significantly increases lifespan in C.elegans. Overall our work supports the potential use of SPS as a prospective geroprotective intervention.

HIGHLIGHTSO_LISodium polystyrene sulfonate improves pH-dependent processing of lysosomal cargo, enhances proteotoxic stress resistance and extends lifespan in C. elegans
C_LIO_LISodium polystyrene sulfonate boosts lysosomal function in human neuronal cells and reduces level of aggregation-associated phosphorylated-TAU
C_LI


title: Contribution of cytoplasm viscoelastic properties to mitotic spindle positioning

abstract:
Cells are filled with macromolecules and polymer networks that set scale-dependent viscous and elastic properties to the cytoplasm. Although the role of these parameters in molecular diffusion, reaction kinetics and cellular biochemistry is being increasingly recognized, their contributions to the motion and positioning of larger organelles, such as mitotic spindles for cell division remain unknown. Here, using magnetic tweezers to displace and rotate mitotic spindles in living embryos, we uncovered that the cytoplasm can impart viscoelastic reactive forces that move spindles, or passive objects with similar size, back to their original position. These forces are independent of cytoskeletal force generators, yet reach hundreds of piconewtons and scale with cytoplasm crowding. Spindle motion shears and fluidizes the cytoplasm, dissipating elastic energy and limiting spindle recoils with functional implications for asymmetric and oriented divisions. These findings suggest that bulk cytoplasm material properties may constitute important control elements for the regulation of division positioning and cellular organization.

Significance StatementThe regulation of mitotic spindle positioning is a key process for tissue architecture, embryo development and stem cells. To date, most models have assumed that spindles are positioned by forces exerted by polar cytoskeleton networks, like microtubule asters or acto-myosin bundles. Here, using in situ magnetic tweezers to apply calibrated forces and torques to mitotic spindles in live dividing sea urchin cells, we found that the viscoelastic properties of the cytoplasm medium in which spindles are embedded can hold spindles in place, and move them back if their original position is perturbed. These viscoelastic forces are large and may significantly participate in the force balance that position and orient mitotic spindles in many cell types.


title: Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

abstract: Elevations in plasma phosphate concentrations (hyperphosphatemia) occur in chronic kidney disease (CKD), in certain genetic disorders, and following the intake of a phosphate-rich diet. Whether hyperphosphatemia and/or associated changes in metabolic regulators, including elevations of fibroblast growth factor 23 (FGF23) directly contribute to specific complications of CKD is uncertain. Here we report that similar to patients with CKD, mice with adenine-induced CKD develop inflammation, anemia and skeletal muscle wasting. These complications are also observed in mice fed high phosphate diet even without CKD. Ablation of pathologic FGF23- FGFR4 signaling did not protect mice on an increased phosphate diet or mice with adenine- induced CKD from these sequelae. However, low phosphate diet ameliorated anemia and skeletal muscle wasting in a genetic mouse model of CKD. Our mechanistic in vitro studies indicate that phosphate elevations induce inflammatory signaling and increase hepcidin expression in hepatocytes, a potential causative link between hyperphosphatemia, anemia and skeletal muscle dysfunction. Our study suggests that high phosphate intake, as caused by the consumption of processed food, may have harmful effects irrespective of pre-existing kidney injury, supporting not only the clinical utility of treating hyperphosphatemia in CKD patients but also arguing for limiting phosphate intake in healthy individuals.


title: Thioflavin T indicates membrane potential in mammalian cells and can affect it in a blue light dependent manner.

abstract: The fluorescent benzothiazole Thioflavin T (ThT) has a high binding affinity to protein aggregates and is used as a marker for the study of this process, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilisation of ThT in research and diagnosis. Here, we show that ThT localises into the mitochondria of HeLa cells in a membrane-potential dependent manner. Specifically, ThT colocalised in cells with a well-established mitochondrial membrane-potential indicator Tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light ({lambda}=405 nm) exposure, but neither factor alone, caused depolarisation of mitochondrial membrane potential. This depolarisation effect was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its light-dependent binding in mitochondria. These results show that ThT can act as a membrane potential dye in mammalian cells, when used at low concentrations and with low blue-light exposure, while it causes dissipation of the mitochondrial membrane potential at higher concentrations and in the presence of blue light excitation. This conclusion motivates a re-evaluation of ThTs use at micromolar range in live-cell analyses, while indicating that this dye can enable future studies on the potential connections between membrane potential dynamics and protein aggregation.


title: Microbial stowaways - waterbirds as dispersal vectors of aquatic pro- and microeukaryotic communities

abstract:
AimWaterbirds are important dispersal vectors of multicellular organisms such as macrophytes, aquatic macroinvertebrates, and zooplankton. However, no study to date has focused on their potential role in dispersing aquatic microbial communities (i.a., bacteria, algae, protozoa). Here, we explicitly studied passive transport (endozoochory) of prokaryotes and unicellular microeukaryotes by waterbirds based on DNA metabarcoding approaches. By directly comparing the dispersed set of organisms to the source pool of a natural metacommunity, we aimed for a realistic estimate of the overall importance of waterbird zoochory for natural microbial communities.

LocationShallow saline temporary ponds (soda pans) in the cross-border region of Austria and Hungary.

TaxonProkaryotes and unicellular microeukaryotes.

MethodsIn 2017 and 2018, water samples from 25 natural aquatic habitats along with fresh droppings of the dominant greylag goose (Anser anser) and four other waterbird species were collected in a habitat network of temporary ponds. Their prokaryotic and microeukaryotic communities were identified via 16S and 18S rRNA gene amplicon sequencing. Sequence reads were analysed using mothur. After quality filtering of the reads, pro- and microeukaryotic amplicon sequencing variant (ASV) compositions were compared between the aquatic and dropping samples, across years and waterbird species.

ResultsWe found that 28% of the dominant aquatic prokaryotic and 19% of the microeukaryotic ASVs were transported by A. anser. ASV richness in A. anser droppings was lower, but compositional variation was higher compared to the aquatic communities, probably resulting from stochastic pick-up of microbes from multiple aquatic habitats. We furthermore found that the composition of prokaryotic ASVs in bird droppings were different among the two years and reflected the actual aquatic communities. The dispersed set of microbes were largely similar among the different waterbird species except for the planktivore filter-feeder northern shoveler (Spatula clypeata) which was outstanding by dispersing a more species-rich subset of microeukaryotes than shorebirds or geese.

Main conclusionsBy using a combined amplicon-sequencing approach to characterize microorganisms in waterbird droppings and in the associated environment, our study provides strong evidence for endozoochory of natural aquatic microorganism communities. These results imply that waterbirds may be crucial in maintaining ecological connectivity between discrete aquatic habitats at the level of microbial communities.

category: ecology