title: Efficient graphene oxide coating improves cryo-EM sample preparation and data collection from tilted grids

abstract: Recent technical developments have made single particle cryo-EM a major structural biology technique, especially in the characterization of challenging samples that resist crystallization, can only be obtained in small amounts, or suffer from compositional or conformational heterogeneity. However, a number of hurdles that often challenge sample preparation still need to be overcome in order to increase the applicability and throughput of cryo-EM. These technical hurdles include obtaining enough particles per image, with close to random orientation, and without damage from interaction with the air-water interface. While coating EM grids with graphene oxide is a promising procedure for the improvement of sample preparation, it suffers from some technical problems that limit its applicability. We have modified the established drop cast method for adhering graphene oxide onto holey patterned grids to increase graphene coverage. Our method relies on the use of a polycationic polymer to coat the surface of the grid prior to graphene oxide application, thereby preventing the repulsion of the negatively charged graphene oxide sheets from the negatively charged grid surface. With this improved preparation method, we show that graphene oxide supports can increase the number of particles in the field of view by an order of magnitude with respect to open holes, while keeping them away from the damaging air-water interface. We also show how graphene oxide coated gold foil grids can be used to collect tilted cryo-EM data in order to overcome preferred orientation issues, without experiencing the large amount of drift observed with conventional amorphous carbon supports, thus allowing data collection that can lead to high-resolution reconstructions.


title: BCOR and BCORL1 mutations disrupt PRC1.1 repressive function in leukemia by unlinking the RING-PCGF1 enzymatic core from target genes

abstract: BCOR and its paralog BCORL1 encode subunits of the Polycomb repressive complex 1.1 (PRC1.1) and are recurrently mutated in myeloid malignancies. We show that leukemia-associated BCOR/BCORL1 mutations unlink the PRC1.1 RING-PCGF enzymatic core from the KDM2B-containing chromatin targeting auxiliary subcomplex, either by causing complete protein loss or expression of a C-terminally truncated protein lacking the PCGF Ub-like fold discriminator (PUFD) domain. By uncoupling PRC1.1 repressive function from target genes, BCOR/BCORL1 mutations activate aberrant cell signaling programs that confer acquired resistance to treatment. This study provides a mechanistic basis for Polycomb repressive dysfunction as a key oncogenic driver in myeloid malignancies and identifies a potential strategy for targeted therapy in BCOR-mutated cancer.

category: cancer biology


title: An unusually long 5'UTR of ATP dependent cold shock DEAD-box RNA helicase gene csdA negatively regulates its own expression in Escherichia coli

abstract: Cold-shock DEAD-box protein A (CsdA) is an ATP dependant cold shock DEAD-box RNA helicase. It is a major cold shock protein needed for the cold adaptation in Escherichia coli. Although the CsdA has been studied at the protein level, further studies are necessary to understand its mechanisms of gene regulations. In this regard, we have constructed a promoter less vector with the ORF of a GFP reporter and found that the promoter of the csdA gene resides far upstream (more than 800 bases) of its coding region. Furthermore, our in vivo deletion experiment has confirmed the existence of this extraordinarily long 5UTR. Our results show that it represses its own expression. In addition, the short peptide encoding (26 aa) yrbN gene resides within this 5UTR as an operon with 8 overlapping nucleotides with the csdA coding region. Besides, we observed that the csdA gene expression may also occur along with immediate upstream (180 nucleotides) nlpI gene both at 37{degrees}C and 15{degrees}C and from the pnp gene (1173 nucleotides upstream) only during cold. In conclusion, csdA gene has operon feature like prokaryotes, in contrast, it also contains an extraordinarily long 5UTR, found in eukaryotes.

category: molecular biology


title: Co-culture of type I and type II pneumocytes as a model of alveolar epithelium

abstract:
The epithelial tissues of the distal lung are continuously exposed to inhaled air, and are of research interest in studying respiratory exposure to both hazardous and therapeutic materials. Pharmaco-toxicological research depends on the development of sophisticated models of the alveolar epithelium, which better represent the different cell types present in the native lung and interactions between them.

We developed an air-liquid interface (ALI) model of the alveolar epithelium which incorporates cell lines representative of both type I (NCI-H441) and type II (hAELVi) epithelial cells. We compared morphology of single cells and the structure of cell layers of the two lines using light and electron microscopy. Working both in monotypic cultures and cocultures, we measured barrier function by trans-epithelial electrical resistance (TEER), and demonstrated that barrier properties can be maintained for 30 days. We created a mathematical model of TEER development over time based on these data in order to make inferences about the interactions occurring in these culture systems. We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation.

The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung. We described cavities which arise within stratified cell layers in NCI-H441 and cocultured cells, and present evidence that these cavities represent an aberrant apical surface. In summary, our results support the coculture of these two cell lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium.

category: cell biology


title: Characterization of RNA content in individual phase-separated coacervate microdroplets

abstract: Liquid-liquid phase separation or condensation is a form of macromolecular compartmentalization. Condensates formed by complex coacervation were hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of condensation in cells and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and how single condensates differ in their RNA composition. Therefore, we developed an approach to read the RNA content from single condensates using high-throughput sequencing. We find that RNAs which are enriched for specific sequence motifs efficiently accumulate in condensates. These motifs show high sequence similarity to short interspersed elements (SINEs). We observed similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

category: genomics


title: A comparative recombination analysis of human coronaviruses and implications for the SARS-CoV-2 pandemic

abstract: The SARS-CoV-2 pandemic prompts evaluation of recombination in human coronavirus (hCoV) evolution. We undertook recombination analyses of 158,118 public seasonal hCoV, SARS-CoV-1, SARS-CoV-2 and MERS-CoV genome sequences using the RDP4 software. We found moderate evidence for 8 SARS-CoV-2 recombination events, two of which involved the spike gene, and low evidence for one SARS-CoV-1 recombination event. Within MERS-CoV, 229E, OC43, NL63 and HKU1 datasets, we noted 7, 1, 9, 14, and 1 high-confidence recombination events, respectively. There was propensity for recombination breakpoints in structural genes, and recombination severely skewed the temporal structure of these data, especially for NL63 and OC43. Bayesian time-scaled analyses on recombinant-free data indicated the sampled diversity of seasonal CoVs emerged in the last 70 years, with 229E displaying continuous lineage replacements. These findings emphasize the importance of genomic based surveillance to detect recombination in SARS-CoV-2, particularly if recombination may lead to immune evasion.


title: Human nasal and lung tissues infected ex vivo with SARS-CoV-2 provide insights into differential tissue-specific and virus-specific innate immune responses in the upper and lower respiratory tract

abstract:
The nasal-mucosa constitutes the primary entry site for respiratory viruses including SARS-CoV-2. While the imbalanced innate immune response of end-stage COVID-19 has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local-mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with rapid increase in tissue-associated viral sub-genomic mRNA, and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon stimulated genes, cytokines and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tract, that are distinct to SARS-CoV-2. The studies shed light on the role of the nasal-mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19.

IMPORTANCEIn order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal-mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues, infected in parallel with SARS-CoV-2 and influenza virus, we have revealed distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal-mucosal infection model can be employed to assess the impact of viral evolutionary changes, and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

category: microbiology


title: SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection

abstract:
SARS-CoV-2 spillback from humans into domestic and wild animals has been well-documented. We compared variants of cell culture-expanded SARS-CoV-2 inoculum and virus recovered from four species following experimental exposure. Five nonsynonymous changes in nsp12, S, N and M genes were near fixation in the inoculum, but reverted to wild-type sequences in RNA recovered from dogs, cats and hamsters within 1-3 days post-exposure. Fourteen emergent variants were detected in viruses recovered from animals, including substitutions at spike positions H69, N501, and D614, which also vary in human lineages of concern. The rapidity of in vitro and in vivo SARS-CoV-2 selection reveals residues with functional significance during host-switching, illustrating the potential for spillback reservoir hosts to accelerate evolution, and demonstrating plasticity of viral adaptation in animal models.

One-Sentence SummarySARS-CoV-2 variants rapidly arise in non-human hosts, revealing viral evolution and potential risk for human reinfection.

category: evolutionary biology


title: Sialic acid-Dependent Binding and Viral Entry of SARS-CoV-2

abstract: Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 M) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral coreceptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

category: biochemistry


title: Comparative host interactomes of the SARS-CoV-2 nonstructural protein 3 and human coronavirus homologs

abstract: Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, non-structural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) dataset for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance nuclear protein important for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.


title: Comparative studies of the seven human coronavirus envelope proteins using topology prediction and molecular modelling to understand their pathogenicity

abstract: Human (h) coronaviruses (CoVs) 229E, NL63, OC43, and HKU1 are less virulent and cause mild, self-limiting respiratory tract infections, while SARS-CoV, MERS-CoV, and SARS-CoV-2, are more virulent and have caused severe outbreaks. The CoV envelope (E) protein, an important contributor to the pathogenesis of severe hCoVs infections, may provide insight into this disparate severity of the disease. Topology prediction programs and 3D modelling software was used to predict and visualize structural aspects of the hCoV E protein related to its functions. All seven hCoV E proteins largely adopted different topologies, with some distinction between the more virulent and less virulent ones. The 3D models refined this distinction, showing the PDZ-binding motif (PBM) of SARS-CoV, MERS-CoV, and SARS-CoV-2 to be more flexible than the PBM of hCoVs 229E, NL63, OC43, and HKU1. We speculate that the increased flexibility of the PBM may provide the more virulent hCoVs with a greater degree of freedom, which can allow them to bind to different host proteins and can contribute to a more severe form of the disease. This is the first paper to predict the topologies and model 3D structures of all seven hCoVs E proteins, providing novel insights for possible drug and/or vaccine development.

category: bioinformatics


title: Structural Analysis of Spike Protein Mutations in an Emergent SARS-CoV-2 Variant from the Philippines

abstract: A SARS-CoV-2 lineage designated as P.3 with multiple signature mutations in the Spike protein region was recently reported with cases from the Central Visayas Region of the Philippines. Whole genome sequencing revealed that the 33 samples under this lineage all contain the E484K, N501Y, and P681H Spike mutations previously found in variants of concern (VOC) such as the B.1.351, the P.1 and B.1.1.7 variants first reported in South Africa, Brazil, and the United Kingdom, respectively. The possible implications of the mutations found in the Spike protein of P.3 were analyzed for their potential effects on structure, stability, and molecular surface character. The analysis suggests that these mutations could significantly impact the possible interactions of the Spike protein with the ACE2 receptor and neutralizing antibodies, and warrants further clinical investigation. Some of the mutations affecting the N and C terminal domains may have effects on Spike monomer and trimer stability. This report provides insights on relevant targets for the design of future diagnostics, therapeutics and vaccines against the evolving SARS-CoV-2 variants in the Philippines.

category: biophysics


title: Structural Analysis of Spike Protein Mutations in the SARS-CoV-2 P.3 Variant


title: Differential effects on neuromuscular physiology between Sod loss-of-function mutation and paraquat-induced oxidative stress in Drosophila

abstract: Oxidative stress is thought to be a major contributor to aging processes. Here, we report differential effects on neurotransmission caused by loss-of-function mutations of Superoxide dismutase (Sod) and by paraquat (PQ) feeding in Drosophila. We demonstrated alterations in Sod mutants; the larval neuromuscular junction displayed supernumerary discharges and the adult giant-fiber escape pathway showed increased latency and poor response to repetitive high-frequency stimulation. Even though the concentrations used led to motor coordination defects and lethality, PQ feeding failed to reproduce such performance deficits in these larval and adult preparations, indicating mechanistic distinctions between genetic and pharmacological manipulation of oxidative stress.

category: neuroscience


title: Hillslope geodiversity shapes ammonia-oxidizing communities and other microbial regulators in a semi-arid shrubland

abstract:
The determinants and consequences of drought-related shrub mortality were studied for over a decade, as a model for desertification processes, in a semi-arid long-term ecological research station. Recent studies have shown that geodiversity is an important spatial predictor of plant viability under extreme drought conditions. Homogeneous hillslopes, with a deep soil profile and lack of stoniness, could not support shrubs under long term drought conditions due to low water storage in their soil. Conversely, heterogeneous hillslopes, with shallow soil profiles and high stoniness, supported shrub communities under similar conditions, due to the comparatively greater soil-water content. In the current study, we investigated the effect of hillslope geodiversity on the soil microbial diversity. Using DNA metabarcoding, we found small but consistent differences in the microbial community compositions of the homogeneous and heterogeneous hillslopes; more ammonia oxidizing and reducing-sugar degrading bacteria are found in the homogeneous hillslopes, possibly dwindling the ammonia supply to shrubs. Additionally, based on functional metagenomic reconstruction, we suggest that homogeneous hillslopes have lower superoxide and antibiotics production, leading to reduced protection against pathogens. In fungi, we observed an increase in possible pathogens, at the expense of lichen forming fungi. Lichens are considered to support soil-water by slowly releasing intercepted raindrops. In conclusion, we show that not only plant-diversity but also microbial-diversity is shaped by geodiversity, and that the community shift in homogeneous hillslopes may further promote shrub mortality in this drought-prone, water limited ecosystem.

HIGHLIGHTSO_LIHomogeneous hillslopes reduce soil water storage and increase aeration.
C_LIO_LIAmmonia oxidizers and reducing-sugar degraders dwindle ammonia supply for plants.
C_LIO_LIHomogenous hillslopes do not support moisture providing lichens.
C_LIO_LIReduced antibiotics and superoxide secretion capacitate pathogens.
C_LIO_LIGeodiversity facilitates microbial regulation during drought.
C_LI

GRAPHICAL ABSTRACT

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title: G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum

abstract: G-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified the formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.


title: Capsaicin activates TRPV1 in human Langerhans cells and inhibits mucosal HIV-1 transmission via secreted CGRP

abstract:
Upon its mucosal transmission, human immunodeficiency virus type 1 (HIV-1) rapidly targets resident antigen-presenting Langerhans cells (LCs) in genital epithelia, which subsequently trans-infect CD4+ T-cells. We previously described an inhibitory neuro-immune sensory mucosal crosstalk, whereby peripheral pain-sensing nociceptor neurons, innervating all mucosal epithelia and associating with LCs, secret the neuropeptide calcitonin gene-related peptide (CGRP) that strongly inhibits HIV-1 trans-infection. Moreover, we reported that LCs secret low levels of CGRP that are further increased by CGRP itself via an autocrine/paracrine mechanism. As nociceptors secret CGRP following activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), we investigated whether LCs also express functional TRPV1. We found that human LCs expressed TRPV1 mRNA and protein. TRPV1 in LCs was functional, as the TRPV1 agonists capsaicin (CP) and resiniferatoxin (RTX) induced Ca2+ influx in a dose-dependent manner. Treatment of LCs with CP and the TRPV1 agonist rutaecarpine (Rut) increased CGRP secretion, reaching concentrations close to its IC50 for inhibition of HIV-1 trans-infection. Accordingly, CP significantly inhibited HIV-1 trans-infection, which was abrogated by antagonists of both TRPV1 and the CGRP receptor. Finally, pre-treatment of inner foreskin tissue explants with CP markedly increased CGRP secretion, and upon subsequent polarized exposure to HIV-1, inhibited increase in LC-T-cell conjugate formation and T-cell infection. Together, our results reveal that alike nociceptors, LCs express functional TRPV1, whose activation induces CGRP secretion that inhibits mucosal HIV-1 transmission. Our studies could permit re-positioning of formulations containing TRPV1 agonists, already approved for pain relief, as novel topical microbicides against HIV-1.

Significance StatementUpon its sexual transmission, HIV-1 targets different types of mucosal immune cells, such as antigen-presenting Langerhans cells (LCs). In turn, LCs transfer HIV-1 to its principal cellular targets, namely CD4+ T-cells, in a process termed trans-infection. We previously discovered that the mucosal neuropeptide CGRP strongly inhibits trans-infection. CGRP is principally secreted from pain-sensing peripheral neurons termed nociceptors, once activated via their TRPV1 ion channel. Herein, we reveal that LCs also express functional TRPV1, whose activation induces secretion of CGRP that inhibits mucosal HIV-1 transmission. Accordingly, molecules activating TRPV1 and inducing CGRP secretion could be used to prevent mucosal HIV-1 transmission. This approach represents an original neuro-immune strategy to fight HIV-1.


title: Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

abstract:
Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus.

ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacteriums major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.


title: Full-length native CGRP neuropeptide and its stable analogue SAX, but not CGRP peptide fragments, inhibit mucosal HIV-1 transmission

abstract: The vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously reported that CGRP strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suprresing Langerhans cells (LCs)-mediated HIV-1 transfer to T-cells during trans-infection. To understand the requirements for CGRP receptor (CGRP-R) activation during inhibition of HIV-1 transmission, we here investigated the anti-HIV-1 activities of full-length native CGRP and its recently developed stable analogue SAX, as well as several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. We show that SAX significantly inhibits LCs-mediated HIV-1 trans-infection but with lower potency than CGRP, while all CGRP peptide fragments tested have no effect. In addition, CGRP readily enters the epithelial compartment of mucosal tissues and does not modify the distribution and density of mucosal immune cells. In-vivo, a single CGRP treatment in humanized mice, before vaginal challenge with high-dose HIV-1, restricts the increase in plasma viral load and maintains higher CD4+ T-cell counts. Together, our results call for the optimization and design of CGRP analogues and agonists, which could be harnessed for prevention of mucosal HIV-1 transmission.


title: Antimicrobials from a feline commensal bacterium inhibit skin infection by drug-resistant S. pseudintermedius.

abstract: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug-resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain (S. felis C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Competition experiments in mice showed that S. felis significantly reduced MRSP skin colonization and an antimicrobial extract from S. felis significantly reduced necrotic skin injury from MRSP infection. Fluorescence and electron microscopy revealed that S. felis antimicrobials disrupted bacterial but not eukaryotic cell membranes. LC/MS identified several S. felis phenol-soluble modulin beta (PSM{beta}) peptides that exhibited antimicrobial and anti-inflammatory activity. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections.


title: DNA methylation patterns in bacteria of the genus Ensifer during free living growth and during nitrogen-fixing symbiosis with Medicago spp.

abstract: Methylation of specific DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Using single-molecule real-time sequencing, six genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cells, methylation of GANTC motifs increased progressively from the ori to ter regions in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. In contrast, there was near full genome-wide GANTC methylation in the early stage of symbiotic differentiation. This was followed by a moderate decrease in the overall extent methylation and a progressive decrease in chromosomal GANTC methylation from the ori to ter regions in later stages of differentiation. We interpret these observations as evidence of dysregulated and constitutive CcrM activity during terminal differentiation, and we hypothesize that it is a driving factor for endoreduplication of terminally differentiated bacteroids.


title: DNA methylation in Ensifer species during free-living growth and during nitrogen-fixing symbiosis with Medicago spp.


title: Apparent growth paradox of Staphylococcus epidermidis in response to extremely low frequency electromagnetic radiation exposure

abstract: Staphylococcus epidermidis is a normal part of the human microbiome; however, it is an opportunistic pathogen and can cause infections when the delicate balance of this microbiome is disrupted. Furthermore, infections caused by this bacterium can be hard to treat as a result of antibiotic resistance and biofilm production. This experiment aimed to determine whether electromagnetic field radiation (ELF-EMF) could be a deterrent of bacterial growth, as an alternative treatment to antibiotics. A non-pathogenic strain of S. epidermidis was used for experimentation, which took place in a school laboratory setting. The experimental group was exposed to ELF-EMF, while the control group did not receive the ELF-EMF treatment. The number of bacterial colonies, represented as colony forming units (CFUs) and area of random colonies were calculated to determine the effect of this treatment. There was no dramatic difference of colony formation on days 0, 1, and 2 of the four day period of ELF-EMF exposure. However, colony formation for days 3 and 4 showed a significant difference between the control and the experimental groups, as the experimental group had a significantly higher CFU count than the control. The average CFU count for day 3 in the control group was 420.6 and 1,097.4 for the experimental group (p<0.0001, t=12.9803). On the final day of the experimentation (Day 4) the average CFU count for the control group was 424.6 and 896.4 for the experimental group (p<0.0001, t=5.8926). The area for five randomly chosen colonies from each petri dish was calculated on the fourth day of experimentation. The area for the experimental group was significantly lower than that control (p<0.0001, with t=6.8659). The average area for the control group was 1.3249 mm2 and a lower average of 0.6375mm2 for the experimental group. These results demonstrate that the ELF-EMF treatment had an inhibitory effect on the area growth of S. epidermidis, but not on the colony-forming ability of S. epidermidis. This suggests that ELF-EMF influences the means by which the bacterium S. epidermidis grows.


title: Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses

abstract: Although a variety of SARS-CoV-2 related coronaviruses have been identified, the evolutionary origins of this virus remain elusive. We describe a meta-transcriptomic study of 411 samples collected from 23 bat species in a small (~1100 hectare) region in Yunnan province, China, from May 2019 to November 2020. We identified coronavirus contigs in 40 of 100 sequencing libraries, including seven representing SARS-CoV-2-like contigs. From these data we obtained 24 full-length coronavirus genomes, including four novel SARS-CoV-2 related and three SARS-CoV related genomes. Of these viruses, RpYN06 exhibited 94.5% sequence identity to SARS-CoV-2 across the whole genome and was the closest relative of SARS-CoV-2 in the ORF1ab, ORF7a, ORF8, N, and ORF10 genes. The other three SARS-CoV-2 related coronaviruses were nearly identical in sequence and clustered closely with a virus previously identified in pangolins from Guangxi, China, although with a genetically distinct spike gene sequence. We also identified 17 alphacoronavirus genomes, including those closely related to swine acute diarrhea syndrome virus and porcine epidemic diarrhea virus. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species in Southeast Asia and southern China, with the largest contiguous hotspots extending from South Lao and Vietnam to southern China. Our study highlights both the remarkable diversity of bat viruses at the local scale and that relatives of SARS-CoV-2 and SARS-CoV circulate in wildlife species in a broad geographic region of Southeast Asia and southern China. These data will help guide surveillance efforts to determine the origins of SARS-CoV-2 and other pathogenic coronaviruses.


title: Vinculin controls endothelial cell junction dynamics during vascular lumen formation

abstract: Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors, which depend on dynamic cell-cell interactions. Remodeling of endothelial cell-cell junctions promote morphogenetic cellular events while preserving vascular integrity. Here, we have analyzed the dynamics of endothelial cell-cell junctions during lumen formation in angiogenic sprouts. By live-imaging of the formation of intersegmental blood vessels in zebrafish, we demonstrate that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Formation and maintenance of these junctional fingers are positively regulated by blood pressure whereas inhibition of blood flow prevents their formation. Using fluorescent reporters, we show that the tension-sensor Vinculin localizes to junctional fingers. Furthermore, loss of vinculin function, in vinculin a and -b double knockouts, prevents junctional finger formation in angiogenic sprouts, whereas endothelial expression of a vinculin transgene is sufficient to restore junctional fingers. Taken together, our findings suggest a mechanism in which lumen expansion during angiogenesis leads to an increase in junctional tension, which triggers recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells may employ force-dependent junctional remodeling to react to changes in external forces to protect cell-cell contacts and to maintain vascular integrity during sprouting angiogenesis.


title: Genome Size Estimation and Full-length Transcriptome of Sphingonotus tsinlingensis: Genetic Background for the Drought-Adapted Grasshopper

abstract: Sphingonotus Fieber, 1852 (Orthoptera: Acrididae) is a species-rich grasshopper genus with ~146 species. All species of this genus prefer dry environments, such as: desert, steppe, sand, and stony benchland. This genomic study aimed to understand the evolution and ecology of these grasshopper species. Here, the genome size of Sphingonotus tsinlingensis was estimated using flow cytometry and the first high-quality full-length transcriptome of this species is presented, which may serve as a reference genetic resource for the drought-adapted grasshopper species of Sphingonotus Fieber. The genome size of Sphingonotus tsinlingensis was ~12.8 Gb. Based on the 146.98 Gb Pacbio isoform sequencing data, 221.47 Mb full-length transcripts were assembled. Among these transcripts, 88,693 non-redundant isoforms were identified with an average length of 2,497 bp and an N50 value of 2,726 bp, which was much longer than the formal grasshopper transcriptome assemblies. A total of 48,502 protein coding sequences were determined, and 37,569 were annotated in public gene function databases. A total of 36,488 simple tandem repeats, 12,765 long non-coding RNAs, and 414 transcription factors were also identified. According to gene functions, 70 heat shock proteins and 61 P450 genes that may correspond to drought adaptation of S. tsinlingensis were identified. The genome of Sphingonotus tsinlingensis is an ultra-large and complex genome. Full-length transcriptome sequencing is an ideal strategy for genomic research. This is the first full-length transcriptome of the genus Sphingonotus. The assembly parameters were better than all known grasshopper transcriptomes. This full-length transcriptome may be used to understand its genetic background and the evolution and ecology of grasshoppers.

category: genetics


title: Trio-binned genomes of the woodrats Neotoma bryanti and N. lepida reveal novel gene islands and rapid copy number evolution of xenobiotic metabolizing cytochrome p450 genes

abstract: The genomic architecture underlying the origins and maintenance of biodiversity is an increasingly accessible feature of species, due in large part to third-generation sequencing and novel analytical toolsets. Woodrats of the genus Neotoma provide a unique opportunity to study how vertebrate herbivores respond to climate change, as two sister species (N. bryanti and N. lepida) independently achieved a major dietary feat - switching to the novel and toxic food source creosote bush (Larrea tridentata) - in the aftermath of a natural warming event. To better understand the genetic mechanisms underlying this ability, we employed a trio binning sequencing approach with a N. bryanti x N. lepida F1 hybrid, resulting in phased, chromosome-level, highly complete, haploid genome assemblies for each species from one individual. Using these new assemblies, we explored the genomic architecture of three cytochrome p450 subfamilies (2A, 2B, and 3A) that play key roles in the metabolism of naturally occurring toxic dietary compounds. We found that woodrats show expansions of all three p450 gene families, including the evolution of multiple novel gene islands within the 2B and 3A subfamilies. Our assemblies demonstrate that trio binning from an F1 hybrid rodent effectively recovers parental genomes from species that diverged more than a million years ago. Turnover and novelty in detoxification gene islands in herbivores is widespread within distinct p450 subfamilies, and may have provided the crucial substrate for dietary adaptation during environmental change.


title: A cross-cohort analysis of autosomal DNA methylation sex differences in the term placenta

abstract:
BackgroundHuman placental DNA methylation (DNAme) data is a valuable resource for studying sex differences during gestation, as DNAme profiles after delivery reflect the cumulative effects of gene expression patterns and exposures across gestation. Here, we present an analysis of sex differences in autosomal patterns of DNAme in the uncomplicated term placenta (n=343) using the Illumina 450K array.

ResultsUsing a false discovery rate < 0.05 and a mean sex difference in DNAme beta value of > 0.10, we identified 162 autosomal CpG sites that were differentially methylated by sex, and that replicated in an independent cohort of samples (n=293). Several of these differentially methylated CpG sites were part of larger correlated regions of differential DNAme, and many also exhibited sex-specific DNAme variability. Although global DNAme levels did not differ by sex, the majority of significantly differentially methylated CpGs were more highly methylated in male placentae, the opposite of what is seen in differential methylation analyses of somatic tissues. Interestingly, patterns of autosomal DNAme at these significantly differentially methylated CpGs organized placental samples along a continuum, rather than into discrete male and female clusters, and sample position along the continuum was significantly associated with maternal age and newborn birthweight standard deviation.

ConclusionsOur results provide a comprehensive analysis of sex differences in autosomal DNAme in the term human placenta. We report a list of high-confidence autosomal sex-associated differentially methylated CpGs, and identify several key features of these loci that suggest their relevance to sex differences observed in normative and complicated pregnancies.


title: Transgenic mice for in vivo epigenome editing with CRISPR-based systems

abstract: The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.


title: Genome-Wide Covariation in SARS-CoV-2

abstract: The SARS-CoV-2 virus causing the global pandemic is a coronavirus with a genome of about 30Kbase length [Song et al., 2019]. The design of vaccines and choice of therapies depends on the structure and mutational stability of encoded proteins in the open reading frames(ORFs) of this genome. In this study, we computed, using Expectation Reflection, the genome-wide covariation of the SARS-CoV-2 genome based on an alignment of {approx} 130000 SARS-CoV-2 complete genome sequences obtained from GISAID[Shu & McCauley, 2017]. We used this covariation to compute the Direct Information between pairs of positions across the whole genome, investigating potentially important relationships within the genome, both within each encoded protein and between encoded proteins. We then computed the covariation within each clade of the virus. The covariation detected recapitulates all clade determinants and each clade exhibits distinct covarying pairs.


title: Mixed fatty acid-phospholipid protocell networks

abstract: Self-assembled membranes composed of both fatty acids and phospholipids are both permeable for solutes and structurally stable, which was likely an advantageous combination for the development of primitive cells on the early Earth. Here we report on the solid surface-assisted formation of primitive mixed-surfactant membrane compartments, i.e. model protocells, from multilamellar lipid reservoirs composed of different ratios of fatty acids and phospholipids. Similar to the previously discovered enhancement of model protocell formation on solid substrates, we achieve spontaneous multi-step self-transformation of mixed surfactant reservoirs into closed surfactant containers, interconnected via nanotube networks. Some of the fatty acid containing compartments in the networks exhibit colony-like growth. We demonstrate that the compartments generated from fatty acid-containing phospholipid membranes feature increased permeability coefficients for molecules in the ambient solution, for fluorescein up to 7*10-6 cm/s and for RNA up to 3.5*10-6 cm/s. Our findings indicate that surface-assisted autonomous protocell formation and development, starting from mixed amphiphiles, is a plausible scenario for the early stages of the emergence of primitive cells.


title: AI-driven prediction of SARS-CoV-2 variant binding trends from atomistic simulations

abstract: We present a novel technique to predict binding affinity trends between two molecules from atomistic molecular dynamics simulations. The technique uses a neural network algorithm applied to a series of images encoding the distance between two molecules in time. We demonstrate that our algorithm is capable of separating with high accuracy non-hydrophobic mutations with low binding affinity from those with high binding affinity. Moreover, we show high accuracy in prediction using a small subset of the simulation, therefore requiring a much shorter simulation time. We apply our algorithm to the binding between several variants of the SARS-CoV-2 spike protein and the human receptor ACE2.


title: Integrin α5β1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity

abstract: Nanometer-scale properties of the extracellular matrix influence many biological processes, including cell motility. While much information is available for single cell migration, to date, no knowledge exists on how the nanoscale presentation of extracellular matrix receptors influences collective cell migration. In wound healing, basal keratinocytes collectively migrate on a fibronectin-rich provisional basement membrane to re-epithelialize the injured skin. Among other receptors, the fibronectin receptor integrin 5{beta}1 plays a pivotal role in this process. Using a highly specific integrin 5{beta}1 peptidomimetic combined with nanopatterned hydrogels, we show that keratinocyte sheets regulate their migration ability at an optimal integrin 5{beta}1 nanospacing. This efficiency relies on the effective propagation of stresses within the cell monolayer independent of substrate stiffness. For the first time, this work highlights the importance of extracellular matrix receptor nanoscale organization required for efficient tissue regeneration.


title: Allicin From Garlic Disrupts the Cytoskeleton in Plants

abstract:
Allicin is a defence substance produced by garlic cells upon injury. It is a thiosulfinate showing redox-activity and a broad range of antimicrobial and biocidal activity. It is known that allicin efficiently oxidizes thiol-groups and it has been described as a redox toxin. In order to learn more about the effect of allicin on plants we used pure synthetized allicin, and investigated cytoplasmic streaming in sterile filaments of Tradescantia fluminensis, organelle movement using transgenic Arabidopsis with organelle-specifics GFP-tags, and effects on actin and tubulin in the cytoskeleton using GFP-tagged lines. Auxin distribution in roots was investigated using PIN1:GFP, PIN3:GFP, DR5:GFP and DII-VENUS Arabidopsis reporter lines.

Allicin inhibited cytoplasmic streaming in T. fluminensis and organelle movement of peroxisomes and the Golgi apparatus in a concentration-dependent manner, inhibited root growth and destroyed the correct root tip distribution of auxin.

We speculate that the cytoskeleton can be a primary "receptor" for allicins oxidizing properties and as a consequence cytoskeleton-dependent cellular processes are disrupted.

category: plant biology


title: ANCHOR, a technical approach to monitor single-copy locus localization in planta

abstract: RESUMEGene expression is governed by several layers of regulation which in addition to genome organization, local chromatin structure, gene accessibility and the presence of transcription factors also includes gene positioning. Although basic mechanisms are expected to be conserved in Eukaryotes, surprisingly little information on the role of gene positioning is available in plant cells, mainly due to the lack of a highly resolutive approach. In this manuscript, we adapted the use of the ANCHOR system to perform real-time single-locus detection in planta. ANCHOR is a DNA-labelling tool derived from the partitioning system. We demonstrate its suitability to monitor a single-locus in planta and used this approach to track chromatin mobility during cell differentiation in Arabidopsis root epidermal cells. Finally, we discuss the potential of this approach to investigate the role of gene positioning during transcription and DNA repair in plants.


title: In vitro and in vivo interaction of caspofungin with isavuconazole against Candida auris planktonic cells and biofilms

abstract: The in vitro and in vivo efficacy of caspofungin was determined in combination with isavuconazole against Candida auris. Drug-drug interactions were assessed utilising the fractional inhibitory concentration indices (FICIs), the Bliss independence model and an immunocompromised mouse model. Median planktonic minimum inhibitory concentrations (pMICs) of 23 C. auris isolates were between 0.5 and 2 mg/L and between 0.015 and 4 mg/L for caspofungin and isavuconazole, respectively. Median pMICs for caspofungin and isavuconazole in combination showed 2-128-fold and 2-256-fold decreases, respectively. Caspofungin and isavuconazole showed synergism in 14 out of 23 planktonic isolates (FICI range 0.03-0.5; Bliss cumulative synergy volume range 0-4.83). Median sessile MICs (sMIC) of 14 biofilm-forming isolates were between 32 and >32 mg/L and between 0.5 and >2 mg/L for caspofungin and isavuconazole, respectively. Median sMICs for caspofungin and isavuconazole in combination showed 0-128-fold and 0-512-fold decreases, respectively. Caspofungin and isavuconazole showed synergistic interaction in 12 out of 14 sessile isolates (FICI range 0.023-0.5; Bliss cumulative synergy volume range 0.13-234.32). In line with the in vitro findings, synergistic interactions were confirmed by in vivo experiments. The fungal kidney burden decreases were more than 3 log volumes in mice treated with combination of 1 mg/kg caspofungin and 20 mg/kg isavuconazole daily; this difference was statistically significant compared with control mice (p<0.001). Despite the favourable effect of isavuconazole in combination with caspofungin, further studies are needed to confirm the therapeutic advantage of this combination when treating an infection caused by C. auris.


title: Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans

abstract:
Microbial pathogens grow in a wide range of different morphologies that provide distinct advantages for virulence. In the fungal pathogen Candida albicans, adenylyl cyclase (Cyr1) is thought to be a master regulator of the switch to invasive hyphal morphogenesis and biofilm formation. However, faster growing cyr1{Delta}/{Delta} pseudorevertant (PR) mutants were identified that form hyphae in the absence of cAMP. Isolation of additional PR mutants revealed that their improved growth was due to loss of one copy of BCY1, the negative regulatory subunit of protein kinase (A) from the left arm of chromosome 2. Furthermore, hyphal morphogenesis was improved in some of PR mutants by multigenic haploinsufficiency resulting from loss of large regions of the left arm of chromosome 2, including global transcriptional regulators. Interestingly, hyphal-associated genes were also induced in a manner that was independent of cAMP. This indicates that basal protein kinase A activity is an important prerequisite to induce hyphae, but activation of the cAMP pathway is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase 1 family member Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.

AUTHOR SUMMARYThe human fungal pathogen Candida albicans switches between budding and filamentous hyphal morphologies to gain advantages for virulence and survival in the host. Although adenylyl cyclase has been thought to be a master regulator that controls this switch, we identified C. albicans pseudorevertant mutants that grow better and form hyphae in the absence of adenylyl cyclase and cAMP. The mutant cells were also able to induce hyphal-associated genes in the absence of cAMP that are needed for virulence. Integrating information from different omics approaches identified cAMP-independent mechanisms that promote hyphal growth. This includes phosphoproteomic studies that revealed key roles for the Cdc28 cyclin-dependent kinase and casein kinase 1 in promoting hyphal growth. In addition, integrating transcriptomic and proteomic data revealed that post-transcriptional mechanisms regulate the levels of a set of key transcription factors that are important for hyphal induction, suggesting a novel form of translational regulation. These studies better define the pathways that stimulate C. albicans to switch from budding to hyphal growth, which is important for invasion into tissues, escape from the immune system, and biofilm formation.


title: Zooplankton trophic dynamics drive carbon export efficiency

abstract:
The magnitude and efficiency of particulate carbon export from the ocean surface depends not only on net primary production (NPP) but also on how carbon is consumed, respired, and repackaged by organisms. We contend that several of these processes can be captured by the size spectrum of the plankton community. However, most global models have relatively simple food-web structures that are unable to generate plankton sizespectra. Moreover, the life-cycles of multicellular zooplankton are typically not resolved, restricting the ability of models to represent time-lags that are known to impact carbon export and its efficiency (pe-ratio). Here, we use a global mechanistic size-spectrum model of the marine plankton community to investigate how particulate export and pe-ratio relate to the community size spectrum, community composition, and time-lags between predators and prey. The model generates emergent food-webs with associated size distributions for organisms and detrital particles. To resolve time-lags between phytoplankton and zooplankton, we implement the life-cycle of multicellular zooplankton (here represented by copepods). The simulation successfully captures observed patterns in biomass and energy fluxes across regions. We find that carbon export correlates best with copepod biomass and trophic level, whereas the pe-ratio correlates best with the exponent of the size spectrum and sea surface temperature (SST). Community metrics performed better than NPP or SST for both deep export and pe-ratio. Time-lags between phytoplankton and copepods did not strongly affect export or pe-ratio. We conclude by discussing how can we reconcile size-spectrum theory with field sampling.

Plain Language SummaryPlankton are tiny but extremely abundant aquatic organisms. Plankton lock CO2 away from the atmosphere as they sink to the deep ocean, where carbon can be stored for hundreds of years. However, how much carbon is locked away and for how long depends on how organisms eat, defecate, and respire. We argue that these processes are reflected in the size composition of the plankton community. The size composition shows a clear relationship between the number of organisms and their body-size. The steepness of this "size-abundance relationship" describes the balance between small vs. large organisms, and has been argued to reflect how energy is transferred from small to large organisms. Since large organisms create fast-sinking particles, the size-abundance relationship could be used to estimate how much carbon is being stored in the deep ocean. Here we use a computer simulation of the global plankton community to investigate how the removal of carbon relates to the plankton community and the steepness of the sizeabundance relationship. The model successfully captures patterns observed in nature. We found that the size-abundance relationship, together with the quantity of large zooplankton better explained carbon export than other measures typically used, such as photosynthesis and temperature.

Key PointsO_LIWe use a global mechanistic size-spectrum model to investigate the relation between particulate export and plankton community metrics.
C_LIO_LIWe find a good correlation between export efficiency and the exponent of the size spectrum.
C_LIO_LITotal carbon export correlated well with copepod biomass and trophic level of active copepods in the model.
C_LI

category: ecology


title: Linking plankton size spectra and community composition to carbon export and its efficiency


title: Segmentation of Tissues and Proliferating Cells in Light-Sheet Microscopy Images using Convolutional Neural Networks

abstract:
Background and ObjectiveA variety of genetic mutations are known to affect cell proliferation and apoptosis during organism development, leading to structural birth defects such as facial clefting. Yet, the mechanisms how these alterations influence the development of the face remain unclear. Cell proliferation and its relation to shape variation can be studied in high detail using Light-Sheet Microscopy (LSM) imaging across a range of developmental time points. However, the large number of LSM images captured at cellular resolution precludes manual analysis. Thus, the aim of this work was to develop and evaluate automatic methods to segment tissues and proliferating cells in these images in an accurate and efficient way.

MethodsWe developed, trained, and evaluated convolutional neural networks (CNNs) for segmenting tissues, cells, and specifically proliferating cells in LSM datasets. We compared the automatically extracted tissue and cell annotations to corresponding manual segmentations for three specific applications: (i) tissue segmentation (neural ectoderm and mesenchyme) in nuclear-stained LSM images, (ii) cell segmentation in nuclear-stained LSM images, and (iii) segmentation of proliferating cells in Phospho-Histone H3 (PHH3)-stained LSM images.

ResultsThe automatic CNN-based tissue segmentation method achieved a macro-average F-score of 0.84 compared to a macro-average F-score of 0.89 comparing corresponding manual segmentations from two observers. The automatic cell segmentation method in nuclear-stained LSM images achieved an F-score of 0.57, while comparing the manual segmentations resulted in an F-score of 0.39. Finally, the automatic segmentation method of proliferating cells in the PHH3-stained LSM datasets achieved an F-score of 0.56 for the automated method, while comparing the manual segmentations resulted in an F-score of 0.45.

ConclusionsThe proposed automatic CNN-based framework for tissue and cell segmentation leads to results comparable to the inter-observer agreement, accelerating the LSM image analysis. The trained CNN models can also be applied for shape or morphological analysis of embryos, and more generally in other areas of cell biology.


title: Transplanting increases the leaf production rate in rice, consequently modifying plant development and growth

abstract:
In Asia, direct sowing and water savings are major trends in previously transplanted and flooded irrigated rice systems because of the higher cost of wages and increasing water shortage. Previous experiments showed that the leaf appearance rate varies between aerobic and flooded cropping systems. This study aimed to further understand how the planting method affects the development rate, flowering time, and yield of lowland irrigated rice crops. A two-year experiment was undertaken at the International Rice Research Institute, Philippines, using three contrasting rice varieties and three planting methods (transplanted, wet direct-seeded, and dry direct-seeded; at a density of 25 plants m-2) in a field submerged in 3-5 cm water from two weeks after the transplanting date. The effect of the planting method was similar in the three varieties, mostly without interaction between the two factors. In 2013, the leaf number of seedlings grown in seedling trays was two leaves behind that of direct-seeded plants at the time of transplanting. However, the young transplanted plants recovered quickly; produced new leaves at a faster rate (with a shorter phyllochron); reached panicle initiation, flag-leaf emergence and flowering time 1 week later; and developed more leaves compared to the direct-seeded plants. In 2014, growing in the nursery induced no delay in leaf appearance due to temperatures lower than those in 2013; therefore, the planting method did not affect the leaf appearance rate. Thus, plant development was primarily delayed by the density stress in the seedling trays under warm temperatures; however, the transplanted rice plants had lower plastochron duration than the direct-seeded plants, which made up for the initial delay in leaf appearance. In both years and at similar plant density, the transplanted plants produced more tillers bearing larger upper leaves that led to a higher leaf area index; however, grain yields were similar for the transplanted and direct-seeded crops.

Highlights- In seedling trays, leaf appearance stopped at the appearance of the fourth leaf.
- After transplanting, leaf appearance resumed at a faster rate than in direct-seeded plants.
- Transplanted plants had delayed panicle initiation and flowering time, more tillers, and more and larger leaves per tiller, but similar grain yield compared to direct-seeded plants at similar planting density.


title: A H3K9me2-Binding Protein AGDP3 Limits DNA Methylation and Transcriptional Gene Silencing in Arabidopsis

abstract: DNA methylation is critical for tuning gene expression to prevent potentially deleterious gene-silencing. The Arabidopsis DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic loci. However, the mechanism recruiting ROS1 to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor required for ROS1-mediated DNA demethylation, and targets ROS1 to specific loci. We found that AGDP3 could bind to the H3K9me2 mark by its AGD12 cassette. The crystal structure of the AGDP3 AGD12 in complex with an H3K9me2 peptide reveals the molecular basis for the specific recognition, that the dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. Interestingly, a histidine residue located in the methylysine binding aromatic cage enables AGDP3 pH-dependent H3K9me2 binding capacity. Considering the intracellular pH correlates with the histone acetylation status, our results provide the molecular mechanism for the regulation of ROS1 DNA demethylase by the gene silencing H3K9me2 mark and the potential crosstalk with active histone acetylation mark.


title: Nurturing Diversity and Inclusion in AI in Biomedicine through a Virtual Summer Program for High School Students

abstract: Artificial Intelligence (AI) has the power to improve our lives through a wide variety of applications, many of which fall into the healthcare space; however, a lack of diversity is contributing to flawed systems that perpetuate gender and racial biases, and limit how broadly AI can help people. The UCSF AI4ALL program was established in 2019 to address this issue by promoting diversity and inclusion in AI. The program targets high school students from underrepresented backgrounds in AI and gives them a chance to learn about AI with a focus on biomedicine. In 2020, the UCSF AI4ALL three-week program was held entirely online due to the COVID-19 pandemic. Thus students participated virtually to gain experience with AI, interact with diverse role models in AI, and learn about advancing health through AI. Specifically, they attended lectures in coding and AI, received an in-depth research experience through hands-on projects exploring COVID-19, and engaged in mentoring and personal development sessions with faculty, researchers, industry professionals, and undergraduate and graduate students, many of whom were women and from underrepresented racial and ethnic backgrounds. At the conclusion of the program, the students presented the results of their research projects at our final symposium. Comparison of pre- and post-program survey responses from students demonstrated that after the program, significantly more students were familiar with how to work with data and to evaluate and apply machine learning algorithms. There was also a nominally significant increase in the students knowing people in AI from historically underrepresented groups, feeling confident in discussing AI, and being aware of careers in AI. We found that we were able to engage young students in AI via our online training program and nurture greater inclusion in AI.

category: scientific communication and education


title: Genomic environments scale the activities of diverse core promoters

abstract: A classical model of gene regulation is that enhancers provide specificity while core promoters provide a modular site for the assembly of the basal transcriptional machinery. However, examples of core promoter specificity have led to an alternate hypothesis in which specificity is achieved by core promoters with different sequence motifs that respond differently to genomic environments containing different enhancers and chromatin landscapes. To distinguish between these models, we measured the activities of hundreds of diverse core promoters in four different genomic locations and, in a complementary experiment, six different core promoters at thousands of locations across the genome. While genomic locations had large effects on expression, the intrinsic activities of different classes of promoters were preserved across genomic locations, suggesting that core promoters are modular regulatory elements whose activities are independently scaled up or down by different genomic locations. This scaling of promoter activities is non-linear and depends on the genomic location and the strength of the core promoter. Our results support the classical model of regulation in which diverse core promoter motifs set the intrinsic strengths of core promoters, which are then amplified or dampened by the activities of their genomic environments.

category: systems biology


title: IsoSolve: an integrative framework to improve isotopic coverage and consolidate isotopic measurements by MS and/or NMR

abstract: Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS)and nuclear magnetic resonance (NMR)spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased, and the coverage of isotopic space improved, by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic dataset by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to i) clarify and improve the coverage of isotopic space, ii) evaluate the complementarity and redundancy of different techniques, iii) consolidate isotopic datasets, iv) design experiments, and v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSysLISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open source software that can be readily integrated into data analysis pipelines.


title: A VLP-based vaccine targeting ANGPTL3 lowers plasma triglycerides in mice

abstract:
Elevated triglycerides (TGs) are an important risk factor for the development of coronary heart disease (CHD) and in acute pancreatitis. Angiopoietin-like proteins 3 (ANGPTL3) and 4 (ANGPTL4) are critical regulators of TG metabolism that function by inhibiting the activity of lipoprotein lipase (LPL), which is responsible for hydrolyzing triglycerides in lipoproteins into free fatty acids. Interestingly, individuals with loss-of-function mutations in ANGPTL3 and ANGPTL4 have low plasma TG levels, have a reduced risk of CHD, and are otherwise healthy. Consequently, interventions targeting ANGPTL3 and ANGPTL4 have emerged as promising new approaches for reducing elevated TGs. Here, we developed virus-like particle (VLP) based vaccines that target the LPL binding domains of ANGPTL3 and ANGPTL4. ANGPTL3 VLPs and ANGPTL4 VLPs are highly immunogenic in mice and vaccination with ANGPTL3 VLPs, but not ANGPTL4 VLPs, was associated with reduced steady state levels of TGs. Immunization with ANGPTL3 VLPs rapidly cleared circulating TG levels following an oil gavage challenge and enhanced plasma LPL activity. These data indicate that targeting ANGPTL3 by active vaccination is potential alternative to other ANGPTL3-inhibiting therapies.

HighlightsO_LIANGPTL3 and ANGPTL4 are mediators of lipoprotein metabolism that inhibit lipoprotein lipase (LPL) activity.
C_LIO_LIVaccination using virus-like particles (VLPs) targeting ANGPTL3 and ANGPTL4 elicits high-titer IgG antibody responses.
C_LIO_LIImmunization with ANGPTL3 VLPs lowers steady-state plasma triglycerides and enhances LPL activity.
C_LI

category: immunology


title: The force loading rate drives cell mechanosensing through both reinforcement and fluidization

abstract: Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton undergoes fluidization and softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon occurs at the organ level. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved.


title: Quantitative Theory for the Diffusive Dynamics of Liquid Condensates

abstract: To unravel the biological functions of membraneless liquid condensates it is crucial to develop a quantitative understanding of the physics underlying their dynamics. Key processes within such condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date we lack a physics-based quantitative framework for the dynamics of labeled condensate components. Here, we derive the corresponding theory, building on the physics of phase separation, and quantitatively validate this framework via experiments. We show that using our theory we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Strikingly, our theory can also be used to determine the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This bypasses recently described quenching artefacts in the dense phase, which can underestimate partition coefficients by orders of magnitude. Our experimentally verified theory opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities and quantifying rates of biochemical reactions in liquid condensates.


title: Aerodynamics and motor control of ultrasonic vocalizations for social communication in mice and rats

abstract: Rodent ultrasonic vocalizations (USVs) are crucial to their social communication and a widely used translational tool for linking gene mutations to behavior. To maximize the causal interpretation of experimental treatments, we need to understand how neural control affects USV production. However, both the aerodynamics of USV production and its neural control remain poorly understood. Here we test three intralaryngeal whistle mechanisms - the wall and alar edge impingement, and shallow cavity tone - by combining in vitro larynx physiology and individual-based 3D airway reconstructions with fluid dynamics simulations. Our results show that in the mouse and rat larynx USVs are produced by a glottal jet impinging on the thyroid inner wall. Furthermore, we implemented an empirically based motor control model that predicts motor gesture trajectories of USV call types. Our work provides a quantitative neuromechanical framework to evaluate the contributions of brain and body in shaping USVs, and a first step in linking descending motor control to USV production.


title: Bioacoustics for in situ validation of species distribution modelling: An example with bats in Brazil

abstract: Species distribution modelling (SDM) gained importance on biodiversity distribution and conservation studies worldwide, including prioritizing areas for public policies and international treaties. Useful for large-scale approaches and estimates, is a plus considering that a minor fraction of the planet is adequately sampled. However, SDM needs to be as reliable as possible. Minimizing errors is challenging, but essential, considering the uses and consequences of such models. In situ validation of the SDM outputs should be a key-step - in some cases, urgent. Bioacoustics can be used to validate and refine those outputs, especially if the focal species vocalizations are conspicuous and species-specific. This is the case of echolocating bats. Here, we used extensive acoustic monitoring (>120 validation points, covering >758,000 km2, and >300,000 sound files) to validate MaxEnt outputs for six neotropical bat species in a poorly-sampled region of Brazil. Based on in situ validation, we evaluated four threshold-dependent theoretical evaluation metrics ability in predicting models performance. We also assessed the performance of three widely used thresholds to convert continuous SDMs into presence/absence maps. We demonstrated that MaxEnt produces very different outputs, requiring a careful choice on thresholds and modeling parameters. Although all theoretical evaluation metrics studied were positively correlated with accuracy, we empirically demonstrated that metrics based on specificity-sensitivity and sensitivity-precision are better for testing models, considering that most SDMs are based on unbalanced data. Without independent field validation, we found that using an arbitrary threshold for modelling can be a precarious approach with many possible outcomes, even after getting good evaluation scores. Bioacoustics proved to be important for validating SDMs for the six bat species analyzed, allowing a better refinement of SDMs in large and under-sampled regions, with relatively low sampling effort. Regardless of species assessing method used, our research highlighted the vital necessity of in situ validation for SDMs.


title: Short duration overnight cattle kraaling in natural rangelands: does time after kraal use affect their utilization by wildlife and above ground grass parameters?

abstract: Short duration overnight cattle kraaling in natural rangelands creates nutrients hotspots attractive to a diverse suite of large mammalian herbivores. However, few studies have determined the use of these sites by large mammalian herbivores. We determined the number of animal sightings per day from camera traps and used them as proxies for use of these newly created nutrient hotspots of varying ages (1, 2, 3 and 4 years) and surrounding vegetation. Six mammalian herbivores of different sizes belonging to three feeding guilds viz. grazers (Burchells zebra Equus quagga burchelli and warthog Phacochoerus africanus), mixed feeders (impala Aepyceros melampus and African savanna elephant Loxodonta africana africana) and browsers (northern giraffe Giraffa camelopardalis giraffa and greater kudu Tragelaphus strepsiceros) frequently used these nutrient hotspots. The number of sightings per day of mammalian herbivores was determined during three periods of the year (January - wet season; June - early dry season; October - late dry season) to ascertain their use of these nutrient hotspots. In addition, above ground grass biomass and height was measured and related to grazer sightings. Furthermore, we tested if repeated grazing in the newly created nutrient hotspots stimulated grass compensatory growth. All the mammalian herbivores used the newly created nutrient hotspots similarly throughout the year, with impala the most active users. Grazer and browser use of nutrient hotspots was not influenced by their age, while mixed feeders mostly used the one year old sites. Grazer use of nutrient hotspots was not influenced by aboveground grass biomass and height. Repeated clipping (proxy for grazing) resulted in compensatory aboveground grass biomass growth in nutrient hotspots. Impala benefited the most and zebra the least from the creation of nutrient hotspots in natural rangelands. We conclude that creation of nutrients hotspots through short duration overnight kraaling results in rangeland heterogeneity that improves availability of herbivore foraging sites.


title: Synergism between potassium and nitrate enhances the alleviation of ammonium toxicity in rice seedling root

abstract: Ammonium toxicity in plants is considered a global phenomenon, but the primary mechanisms remain poorly characterized. Here, we showed that although the addition of potassium (K+) or nitrate (NO3-) partially alleviated the inhibition of rice root growth caused by ammonium toxicity, the coexistence of K+ and NO3- clearly improved the alleviation via a synergistic mechanism. The synergism led to significantly improved alleviation effects on root biomass, length, surface area, number and meristem cell number. The aberrant auxin distribution in root tips, rhizosphere acidification level and abnormal cell morphology in the root cap and elongation zone caused by ammonium toxicity could be recovered by this synergism. RNA sequencing and the weighted gene correlation network analysis (WGCNA) revealed that the mechanism of this synergism involves cellulose synthesis, auxin and gibberellin metabolism regulation at the transcription level.


title: RBPMS2 is a conserved regulator of alternative splicing that promotes myofibrillar organization and optimal calcium handling in cardiomyocytes

abstract:
RationaleThe identification of novel cardiomyocyte-intrinsic factors that support heart function will expand the number of candidate genes and therapeutic options for heart failure, a leading cause of death worldwide.

ObjectiveTo identify and characterize conserved regulators of cardiomyocyte function.

Methods and ResultsWe report that the RNA-binding protein RBPMS2 is required for myofibril organization and the regulation of intracellular calcium dynamics in both zebrafish embryos and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A differential expression screen in zebrafish uncovered enrichment of rbpms2 paralogs, rbpms2a and rbpms2b, in the myocardium. Double knock-out (rbpms2-null) embryos suffer from compromised ventricular filling during the relaxation phase of the cardiac cycle, which significantly reduces cardiac output. Whole transcriptome sequencing and validation studies revealed differential alternative splicing of several genes linked to cardiomyopathies in humans, including myosin binding protein C3 (mybpc3) and phospholamban (pln), consistent with a role in causing the observed ventricular deficiencies. Further, RBPMS2-null hiPSC-CMs exhibit myofibril and calcium handling defects that are highly analogous to those observed in the rbpms2-null zebrafish ventricle.

ConclusionsTaken together, our data identify RBPMS2 as a conserved and essential regulator of alternative splicing that is required for myofibrillar organization and optimal calcium handling from zebrafish to humans.

category: developmental biology


title: Genome-wide annotation of gene regulatory elements linked to cell fitness

abstract: Noncoding regulatory elements control gene expression and govern all biological processes. Epigenomic profiling assays have identified millions of putative regulatory elements, but systematically determining the function of each of those regulatory elements remains a substantial challenge. Here we adapt CRISPR-dCas9-based epigenomic regulatory element screening (CERES) technology to screen all >100,000 putative non-coding regulatory elements defined by open chromatin sites in human K562 leukemia cells for their role in regulating essential cellular processes. In an initial screen containing more than 1 million gRNAs, we discovered approximately 12,000 regulatory elements with evidence of impact on cell fitness. We validated many of the screen hits in K562 cells, evaluated cell-type specificity in a second cancer cell line, and identified target genes of regulatory elements using CERES perturbations combined with single cell RNA-seq. This comprehensive and quantitative genome-wide map of essential regulatory elements represents a framework for extensive characterization of noncoding regulatory elements that drive complex cell phenotypes and for prioritizing non-coding genetic variants that likely contribute to common traits and disease risk.


title: Mitochondrial variant enrichment from high-throughput single-cell RNA-seq resolves clonal populations

abstract: Reconstructing lineage relationships in complex tissues can reveal mechanisms underlying development and disease. Recent methods combine single-cell transcriptomics with mitochondrial DNA variant detection to establish lineage relationships in primary human cells, but are not scalable to interrogate complex tissues. To overcome this limitation, here we develop a technology for high-confidence detection of mitochondrial mutations from high-throughput single-cell RNA-sequencing. We use the new method to identify skewed immune cell expansions in primary human clonal hematopoiesis.


title: Maternal cradling bias drives early handedness in infant monkeys: A longitudinal study of grasping lateralization in baboons

abstract:
The most emblematic behavioral manifestation of human brain asymmetries is handedness. While the precise mechanisms behind the development of handedness are still widely debated, empirical evidences highlight that besides genetic factors, environmental factors may play a crucial role. As one of these factors, maternal cradling behavior may play a key role in the emergence of early handedness in the offspring. In the present study we followed 41 olive baboon (Papio anubis) infants living in different social groups with their mother for which direction (e.g., left- or right-arm) and degree of maternal cradling-side bias were available from our previous published study. We assessed hand preferences for an unimanual grasping task at 3 developmental stages: (1) 0-4, (2) 4-6 and (3) 9-10 months of age. We found that individual hand preferences for grasping exist as soon as the first months of age, with a population-level left-handedness predominance, being stable until 6 months; to wit the period during which juveniles are mainly carried by their mothers. More importantly, this early postnatal handedness is positively correlated with maternal cradling lateralization. Interestingly, hand preferences assessed later in the development, once juveniles are no longer carried (i.e., from 9 to 10 months of age), are less consistent with the earlier developmental stages and no longer dependent from the maternal cradling bias. Our findings suggest that the ontogenetic dynamics of the infants hand preference and its changes might ultimately rely on the degree of infant dependence from the mother across development.

Research HighlightsO_LIEarly postnatal individual hand preferences are detected for unimanual food grasping within the first four months of age.
C_LIO_LIEarliest measures of infant hand preference are positively correlated with measures of maternal cradling lateralization.
C_LIO_LIHand preferences assessed later in the development, from 9 to 10 months of age are less consistent with the earlier developmental stages and independent from maternal cradling bias.
C_LI

category: animal behavior and cognition


title: Neuroanatomical organization and functional roles of PVN MC4R pathways in physiological and behavioral regulations

abstract:
ObjectiveThe paraventricular nucleus of hypothalamus (PVN) is an integrative center in the brain orchestrating a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is undoubtedly involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ pathway and its role in diverse physiological and behavioral regulations have not been fully understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and further test their potential functional roles beyond feeding.

MethodsUsing a combination of viral tools, we performed a comprehensive mapping of PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermogenesis, cardiovascular control and other behavioral regulations beyond feeding.

ResultsWe found that PVNMC4R+ neurons broadly innervate many different brain regions known to be important not only for feeding but also for neuroendocrine and autonomic control of thermogenesis and cardiovascular function, including but not limited to preoptic area, median eminence, parabrachial nucleus, locus coeruleus, nucleus of solitary tract, ventrolateral medulla and thoracic spinal cord. Contrary to broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs from limited brain regions, including medial preoptic nucleus, arcuate and dorsomedial hypothalamic nuclei, and supraoptic nucleus. Consistent with broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure and brown adipose tissue thermogenesis. Strikingly, these physiological changes accompanied an unexpected repetitive bedding-removing behavior followed by hypoactivity and resting-like behavior.

ConclusionsOur results clarify the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermogenesis, cardiovascular function and other behavioral regulations.


title: Oncogenic ZMYND11-MBTD1 fusion protein anchors the NuA4/TIP60 histone acetyltransferase complex to the coding region of active genes

abstract:
A chromosomal translocation found in cannibalistic acute myeloid leukemia (AML) leads to an in-frame fusion of the transcription elongation repressor ZMYND11 to MBTD1, a subunit of the NuA4/TIP60 histone acetyltransferase (HAT) complex. In contrast to the NuA4/TIP60 complex, ZMYND11 is linked to repression of actively transcribed genes through recognition of H3.3K36me3. To understand the abnormal molecular events that expression of this ZMYND11-MBTD1 fusion protein can create, we performed its biochemical and functional characterization in comparison to each individual fusion partner. ZMYND11-MBTD1 is stably incorporated into the endogenous NuA4/TIP60 complex but does not bring any additional interactors as the fusion lacks the MYND domain of ZMYND11. Nevertheless, this truncated ZMYND11 moiety in the fusion leads to mislocalization of the NuA4/TIP60 complex on the body of genes normally bound by ZMYND11 in the genome, in a PWWP-H3.3K36me3 interaction-dependent manner. This can be correlated to increased chromatin acetylation and altered gene transcription, most notably on the MYC oncogene, and alternative splicing. Importantly, expression of ZMYND11-MBTD1, but not the individual fusion partners, during embryonic stem cell differentiation, leads to decreased expression of specific differentiation markers, while favoring Myc-driven pluripotency. It also favors self-renewal of hematopoietic stem/progenitor cells. Altogether, these results indicate that the ZMYND11-MBTD1 fusion protein functions primarily by mistargeting the NuA4/TIP60 complex to the body of genes, altering normal transcription of specific genes, likely driving oncogenesis in part through the Myc regulatory network.

Highlights-A recurrent chromosomal translocation detected in cannibalistic acute myeloid leukemia leads to the production of a ZMYND11-MBTD1 fusion protein.
-The ZMYND11-MBTD1 fusion protein is stably incorporated into the endogenous NuA4/TIP60 complex.
-ZMYND11-MBTD1 leads to mistargeting of NuA4/TIP60 activity to the coding region of ZMYND11-target genes, altering gene expression and splicing.
-ZMYND11-MBTD1 binds the MYC gene leading to its upregulation, favoring growth and pluripotency while inhibiting differentiation markers.

category: molecular biology


title: Heparin promotes fibrillation of most phenol soluble modulin peptides from S. aureus: a possible strengthening of the bacterial biofilm

abstract:
Phenol soluble modulins (PSMs) are virulence peptides secreted by different Staphylococcus aureus strains. In addition, PSMs are able to form amyloid fibrils which may strengthen the biofilm matrix. The highly sulfated glycosaminoglycan heparin promotes S.aureus infection but the basis for this is unclear. We hypothesized that heparin promotes PSM fibrillation and in this way aids bacterial colonization. Here we address this hypothesis using a combination of different biophysical techniques along with peptide microarrays. We find that heparin accelerates fibrillation of all -PSMs (except PSM2) and {delta}-toxin, but inhibits {beta}-PSMs fibrillation by blocking nucleation. Given that S. aureus secretes higher levels of -PSMs than {beta}-PSMs peptidess, heparin is likely to overall promote fibrillation. Heparin binding is driven by multiple positively charged lysine residues in -PSMs and {delta}-toxins, whose removal strongly reduces affinity. Binding of heparin does not alter the final fibril conformation. Rather, heparin provides a scaffold to catalyze or inhibit fibrillation. Our findings suggest that heparin may strengthen bacterial biofilm through increased PSM fibrillation.

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title: Localization of muscarinic acetylcholine receptor dependent rhythm generating modules in the Drosophila larval locomotor network

abstract: Mechanisms of rhythm generation have been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. Here we explored how muscarinic acetylcholine receptor (mAChR) dependent rhythm generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. We measured fictive motor patterns in isolated CNS preparations using a combination of Ca2+ imaging and electrophysiology while manipulating mAChR signalling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated rhythm generation in distal regions of the isolated CNS, whereas application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions. Oxotremorine raised baseline Ca2+ levels and potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca2+ levels and abolished rhythmic activity. These results suggest the presence of a bimodal gradient of rhythmogenicity in the larval CNS, with mAChR dependent rhythm generating networks in distal regions separated by medial segments with severely reduced rhythmogenic abilities. This work furthers our understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm generating networks in an emerging genetically tractable locomotor system.