title: Pharmacological activation of PIEZO1 in human red blood cells prevents Plasmodium falciparum invasion

abstract: An inherited gain-of-function variant (E756 del) in the mechanosensitive cationic channel PIEZO1 was recently shown to confer a significant protection against severe malaria. Here, we demonstrate in vitro that human red blood cell (RBC) infection by Plasmodium falciparum is prevented by the pharmacological activation of PIEZO1. The PIEZO1 activator Yoda1 inhibits RBC invasion, without affecting parasite intraerythrocytic growth, division or egress. RBC dehydration, echinocytosis and intracellular Na+/K+ imbalance are unrelated to the mechanism of protection. Inhibition of invasion is maintained, even after a prolonged wash out of Yoda1. Similarly, the chemically unrelated activators Jedi1 and Jedi2 potently inhibit parasitemia, further indicating a PIEZO1-dependent mechanism. Notably, Yoda1 treatment significantly reduced RBC surface receptors of P. falciparum, and decreased merozoite attachment and subsequent RBC deformation. Altogether these data indicate that the pharmacological activation of Piezo1 in human RBCs inhibits malaria infection by impairing P. falciparum invasion.


title: High-throughput, multiplex microfluidic test strip for the determination of antibiotic susceptibility in uropathogenic E. coli with smartphone detection

abstract: Antibiotic resistance in urinary tract infections is a major global challenge and improved cost-effective and high throughput antibiotic susceptibility tests (AST) are urgently needed to inform correct antibiotic selection. We evaluated a high throughput microfluidic test strip for AST and minimum inhibitory concentration (MIC) determination in 20 urinary pathogenic E. coli (UPEC) isolates using six commonly prescribed or therapeutically beneficial antibiotics. The microfluidic MIC performs broth microdilution in 1 microliter volume capillaries, 100 X smaller than standard broth microdilution. Each test strip contains 10 parallel capillaries which are dipped into a single well of a 96 well plate, significantly increasing throughput over a microtitre plate. When tested with clinical UPEC isolates at standardised inoculum density, these devices gave 100% essential agreement (+/- 1 doubling dilution of antibiotic) to the gold standard microplate broth microdilution method described by CLSI. Although for some antibiotic/isolate combinations an earlier endpoint readout reduced accuracy, MIC test strips read at a 6h endpoint still gave 69 - 100 % essential agreement depending on the antibiotic. Growth could be detected significantly earlier than 6h, but with a trade-off between speed vs accuracy. These high-throughput, multiplexed test strips could be used to increase throughput and give faster results than microplates while retaining the core broth microdilution methodology of gold standard techniques for AST and MIC determination.

category: microbiology


title: Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide insight into Copper Import

abstract:
CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS), and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12 transmembrane helices (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role, and remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His and Cys residues of CcoA we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggested a model linking Cu import for cbb3-Cox biogenesis with a thiol: disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function.

ImportanceCopper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here we show that CcoA undergoes a thiol: disulfide oxidoreduction cycle, which is important for its Cu import activity.


title: A crystal structure of alphavirus nonstructural protein 4 (nsP4) reveals an intrinsically dynamic RNA-dependent RNA polymerase

abstract:
Alphaviruses such as Ross River virus (RRV), chikungunya virus, Sindbis virus (SINV), and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 and 1.9 [A]. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogendeuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Key PointsO_LICrystal structures of alphavirus nsP4 RNA polymerase domain from RRV and SINV.
C_LIO_LInsP4 protein is highly dynamic with an intrinsically disordered N-terminal domain.
C_LIO_LIOptimized RNA elongation activity assay to facilitate antiviral discovery.
C_LI

category: molecular biology


title: Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold.


title: Identification of ubiquitin variants that inhibit the E2 ubiquitin conjugating enzyme, Ube2k

abstract: Transfer of ubiquitin to substrate proteins regulates most processes in eukaryotic cells. E2 enzymes are a central component of the ubiquitin machinery, and generally determine the type of ubiquitin signal generated and thus the ultimate fate of substrate proteins. The E2, Ube2k, specifically builds degradative ubiquitin chains on diverse substrates. Here we have identified protein-based reagents, called ubiquitin variants (UbVs), that bind tightly and specifically to Ube2k. Crystal structures reveal that the UbVs bind to the E2 enzyme at a hydrophobic cleft that is distinct from the active site and previously identified ubiquitin binding sites. We demonstrate that the UbVs are potent inhibitors of Ube2k and block both ubiquitin charging of the E2 enzyme, and E3-catalysed ubiquitin transfer. The binding site of the UbVs suggests they directly clash with the ubiquitin activating enzyme, while potentially disrupting interactions with E3 ligases via allosteric effects. Our data reveal the first protein-based inhibitors of Ube2k and unveil a hydrophobic groove that could be an effective target for inhibiting Ube2k and other E2 enzymes.

category: biochemistry


title: Novel antibodies for the simple and efficient enrichment of native O-GlcNAc modified peptides

abstract: Antibodies against post-translational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides, and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1,300 unique O-GlcNAc-modified peptides and over 1,000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.


title: DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

abstract: Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

category: cancer biology


title: Spectral dependency of the human pupillary light reflex. Influences of pre-adaptation and chronotype.

abstract: Non-visual photoreceptors (ipRGCs) and rods both exert a strong influence on the human pupil, yet pupil models regularly use cone-derived sensitivity as their basis. This inconsistency is further exacerbated by the fact that circadian effects can modulate the wavelength sensitivity. We assessed the pupillary reaction to monochromatic light stimuli in the mesopic range. Pupil size for eighty-three healthy participants with normal color vision was measured in nine experimental protocols with varying series of continuous or discontinuous light stimuli under Ganzfeld conditions, presented after 90 seconds of dark adaptation. One hundred and fifty series of stimulation were conducted across three experiments, and were analyzed for wavelength-dependency on the pupillary constriction amplitude (PCA), conditional on experimental settings and individual traits. Traits were surveyed by questionnaire; color vision was tested by Ishihara plates or the Lanthony D15 test. Data were analyzed with generalized additive mixed models (GAMM). The pupillary constriction amplitude response is consistent with L+M-cone derived sensitivity when the series of light stimuli is continuous, i.e., is not interrupted by periods of darkness, but not otherwise. The results also show that a mesopic illuminance weighing led to an overall best prediction of pupillary constriction compared to other types of illuminance measures. IpRGC influence on PCA is not readily apparent from the results. When we explored the interaction of chronotype and time of day on the wavelength dependency, differences consistent with ipRGC influence became apparent. The models indicate that subjects of differing chronotype show a heightened or lowered sensitivity to short wavelengths, depending on their time of preference. IpRGC influence is also seen in the post-illumination pupil reflex if the prior light-stimulus duration is one second. However, shorter wavelengths than expected become more important if the light-stimulus duration is fifteen or thirty seconds. The influence of sex on PCA was present, but showed no interaction with wavelength. Our results help to define the conditions, under which the different wavelength sensitivities in literature hold up for monochromatic light settings. The chronotype effect might signify a mechanism for strengthening the individuals chronotype, but demands replication in a controlled study.

category: physiology


title: Elucidating Tumor-stromal Metabolic Crosstalk in Colorectal Cancer through Integration of Constraint-Based Models and LC-MS Metabolomics

abstract: Colorectal cancer (CRC) is a major cause of morbidity and mortality in the United States. Tumor-stromal metabolic crosstalk in the tumor microenvironment promotes CRC development and progression, but exactly how stromal cells, in particular cancer-associated fibroblasts (CAFs), affect the metabolism of tumor cells remains unknown. Here we take a data-driven approach to investigate the metabolic interactions between CRC cells and CAFs, integrating constraint-based modeling and metabolomic profiling. Using metabolomics data, we perform unsteady-state parsimonious flux balance analysis to infer flux distributions for central carbon metabolism in CRC cells treated with or without CAF-conditioned media. We find that CAFs reprogram CRC metabolism through stimulation of glycolysis, the oxidative arm of the pentose phosphate pathway (PPP), and glutaminolysis as well as inhibition of the tricarboxylic acid cycle. To identify potential therapeutic targets, we simulate enzyme knockouts and find that inhibiting the hexokinase and glucose-6-phosphate dehydrogenase reactions exploits the CAF-induced dependence of CRC cells on glycolysis and oxidative PPP. Our work gives mechanistic insights into the metabolic interactions between CRC cells and CAFs and provides a framework for testing hypotheses towards CRC-targeted therapies.

category: systems biology


title: Frontal language areas do not emerge in the absence of temporal language areas: A case study of an individual born without a left temporal lobe

abstract: Language relies on a left-lateralized fronto-temporal brain network. How this network emerges ontogenetically remains debated. We asked whether frontal language areas emerge in the absence of temporal language areas through a  deep-data investigation of an individual (EG) born without her left temporal lobe. Using fMRI methods that have been validated to elicit reliable individual-level responses, we find that--as expected for early left hemisphere damage--EG has a fully functional language network in her right hemisphere (comparable to that in n=145 controls) and performs normally on language assessments. However, we detect no response to language in EGs left frontal lobe (replicated across two sessions, 3 years apart). Another network--the multiple demand network--is robustly present in frontal lobes bilaterally, suggesting that EGs left frontal cortex can support non-linguistic cognition. The existence of temporal language areas therefore appears to be a prerequisite for the emergence of the frontal language areas.

category: neuroscience


title: Differences in social brain function in autism spectrum disorder are linked to the serotonin transporter

abstract:
Alterations in the serotonergic control of brain pathways responsible for facial-emotion processing in people with autism spectrum disorder (ASD) may be a target for intervention. However, the molecular underpinnings of autistic-neurotypical serotonergic differences are challenging to access in vivo. Receptor-Enriched Analysis of functional Connectivity by Targets (REACT) has helped define molecular-enriched fMRI brain networks based on a priori information about the spatial distribution of neurochemical systems from available PET templates. Here, we used REACT to estimate the dominant fMRI signal related to the serotonin transporter (5-HTT) distribution during processing of aversive facial expressions of emotion processing in adults with and without ASD. We first predicted a group difference in baseline (placebo) functioning of this system. We next used a single 20 mg oral dose of citalopram, i.e. a serotonin reuptake inhibitor, to test the hypothesis that network activity in people with and without ASD would respond differently to inhibition of 5-HTT. To confirm the specificity of our findings, we also repeated the analysis with 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT4 receptor maps.

We found a baseline group difference in the 5-HTT-enriched response to faces in the ventromedial prefrontal cortex. A single oral dose of citalopram  shifted the response in the ASD group towards the neurotypical baseline but did not alter response in the control group.

Our findings suggest that the 5HTT-enriched functional network is dynamically different in ASD during processing of socially relevant stimuli. Whether this acute neurobiological response to citalopram in ASD translates to a clinical target will be an important next step.


title: The legacy of maternal SARS-CoV-2 infection on the immunology of the neonate

abstract: Despite extensive and ongoing studies of SARS-CoV-2 and evidence that pregnant women are at increased risk of severe COVID-19, the effect of maternal infection on the developing infant remains unclear. To determine the potential impact of exposure to SARS-CoV-2 in utero on the neonate, we have assessed the immunological status of infants born to mothers with confirmed SARS-CoV-2 infection during gestation. No evidence of vertical transmission of SARS-CoV-2 was observed, but transfer of maternal SARS-CoV-2 specific IgG to infants was apparent, although to a lesser extent in cases of active or recent maternal infection. Infants born to mothers with recent/ongoing infection had elevated circulating pro-inflammatory cytokines and enhanced percentages of innate immune cells compared to that seen in infants born to uninfected mothers. In tandem, higher frequencies of FOXP3+ regulatory T cells and circulating IL-10 demonstrated a further nuance to the neonatal effector response. Interestingly, cytokine functionality was enhanced in infants born to mothers exposed to SARS-CoV-2 at any time during pregnancy. This indicates that maternal SARS-CoV-2 infection influences in utero priming of the fetal immune system.

category: immunology


title: Complete genomes of clade G6 Saccharibacteria suggest a divergent ecological niche and lifestyle

abstract:
AO_SCPLOWBSTRACTC_SCPLOWSaccharibacteria (formerly TM7) have reduced genomes, a small size, and appear to have a parasitic lifestyle dependent on a bacterial host. Although there are at least 6 major clades of Saccharibacteria inhabiting the human oral cavity, cultured isolates or complete genomes of oral Saccharibacteria have been previously limited to the G1 clade. In this study, nanopore sequencing was used to obtain three complete genome sequences from clade G6. Phylogenetic analysis suggested the presence of at least 3-5 distinct species within G6, with two discrete taxa represented by the 3 complete genomes. G6 Saccharibacteria were highly divergent from the more well-studied clade G1, and had the smallest genomes and lowest GC-content of all Saccharibacteria. Pangenome analysis showed that although 97% of shared pan-Saccharibacteria core genes and 89% of G1-specific Core Genes had putative functions, only 50% of the 244 G6-specific Core Genes had putative functions, highlighting the novelty of this group. Compared to G1, G6 encoded divergent metabolic pathways. G6 genomes lacked an F1F0 ATPase, the pentose phosphate pathway, and several genes involved in nucleotide metabolism, which were all core genes for G1. G6 genomes were also unique compared to G1 in that they encoded lactate dehydrogenase, adenylate cyclase, limited glycerolipid metabolism, a homolog to a lipoarabinomannan biosynthesis enzyme, and the means to degrade starch. These differences at key metabolic steps suggest a distinct lifestyle and ecological niche for clade G6, possibly with alternative hosts and/or host-dependencies, which would have significant ecological, evolutionary, and likely pathogenic, implications.

IO_SCPLOWMPORTANCEC_SCPLOWSaccharibacteria are ultrasmall, parasitic bacteria that are common members of the oral microbiota and have been increasingly linked to disease and inflammation. However, the lifestyle and impact on human health of Saccharibacteria remains poorly understood, especially for the 5 clades (G2-G6) with no complete genomes or cultured isolates. Obtaining complete genomes is of particular importance for Saccharibacteria, because they lack many of the "essential" core genes used for determining draft genome completeness and few references exist outside of clade G1. In this study, complete genomes of 3 G6 strains, representing two candidate species, were obtained and analyzed. The G6 genomes were highly divergent from G1, and enigmatic, with 50% of the G6 core genes having no putative functions. The significant difference in encoded functional pathways is suggestive of a distinct lifestyle and ecological niche, probably with alternative hosts and/or host-dependencies, which would have major implications in ecology, evolution, and pathogenesis.


title: Two Type VI Secretion Systems in Vibrio coralliilyticus RE22Sm exhibit differential target specificity for bacteria prey and oyster larvae

abstract:
Vibrio coralliilyticus is an extracellular bacterial pathogen and a causative agent of vibriosis in larval oysters. Host mortality rates can quickly reach 100% during vibriosis outbreaks in oyster hatcheries. Type VI Secretion Systems (T6SS) are rapidly polymerizing, contact dependent injection apparatus for prey cell intoxication and play important roles in pathogenesis. DNA sequencing of V. coralliilyticus RE22Sm indicated the likely presence of two functional T6SSs with one on each of two chromosomes. Here, we investigated the antibacterial and anti-eukaryotic roles of the two T6SSs (T6SS1 and T6SS2) against E. coli Sm10 cells and Crassostrea virginica larvae, respectively. Mutations in hcp and vgrG genes were created and characterized for their effects upon bacterial antagonism and eukaryotic host virulence. Mutations in hcp1 and hcp2 resulted in significantly reduced antagonism against E. coli Sm10, with the hcp2 mutation demonstrating the greater impact. In contrast, mutations in vgrG1 or vgrG2 had little effect on E. coli killing. In eastern oyster larval challenge assays, T6SS1 mutations in either hcp1 or vgrG1 dramatically attenuated virulence against C. virginica larvae. Strains with restored wild type hcp or vgrG genes reestablished T6SS-mediated killing to that of wild type V. coralliilyticus RE22Sm. These data suggest that the T6SS1 of V. coralliilyticus RE22Sm principally targets eukaryotes and secondarily bacteria, while the T6SS2 primarily targets bacterial cells and secondarily eukaryotes. Attenuation of pathogenicity was observed in all T6SS mutants, demonstrating the requirement for proper assembly of the T6SS systems to maintain maximal virulence.

ImportanceVibriosis outbreaks lead to large-scale hatchery losses of oyster larvae (product and seed) where Vibrio sp. associated losses of 80 to 100 percent are not uncommon. Practical and proactive biocontrol measures can be taken to help mitigate larval death by Vibrio sp. by better understanding the underlying mechanisms of virulence in V. coralliilyticus. In this study, we demonstrate the presence of two Type VI Secretion Systems (T6SS) in V. coralliilyticus RE22Sm and interrogate the roles of each T6SS in bacterial antagonism and pathogenesis against a eukaryotic host. Specifically, we show that the loss of T6SS1 function results in the loss of virulence against oyster larvae.


title: Characteristics of genetically diverse Antarctic sea ice bacteriophages allow adaptation to changing environmental conditions

abstract:
Despite generally appreciated significant roles of microbes in sea ice and polar waters, detailed studies of virus-host systems from such environments have been so far limited by only a few isolates. Here, we investigated infectivity under various conditions, infection cycles, and genetic diversity of Antarctic sea ice bacteriophages: PANV1, PANV2, OANV1, and OANV2. The phages infect common sea ice bacteria belonging to the genera Paraglaciecola or Octadecabacter. Although the phages are marine and cold-active, replicating at 0-5 {degrees}C, they all survived temporal incubations at [≥]30 {degrees}C and remained infectious without any salts or supplemented only with magnesium, suggesting a robust virion assembly maintaining integrity under a wide range of conditions. Host recognition in the cold proved to be effective, and the release of progeny viruses occurred as a result of cell lysis. The analysis of viral genome sequences showed that nearly half of the gene products of each virus are unique, highlighting that sea ice harbors unexplored virus diversity. Based on predicted genes typical for tailed double-stranded DNA phages, we suggest placing the four studied viruses in the class Caudoviricetes. Searching against viral sequences from metagenomic assemblies revealed that related viruses are not restricted to Antarctica, but also found in distant marine environments.

ImportanceVery little is known about sea ice microbes despite the significant role of sea ice in the global oceans as well as microbial input into biogeochemical cycling. Studies on the sea ice viruses have been typically limited to -omics-based approaches and microscopic examinations of sea ice samples. Up to date, only four cultivable viruses have been isolated from Antarctic sea ice. Our study of these unique isolates advances the understanding of the genetic diversity of viruses in sea ice environments, their interactions with host microbes, and possible links to other biomes. Such information contributes to more accurate future sea ice biogeochemical models.


title: Virus-host interactions and genetic diversity of Antarctic sea ice bacteriophages


title: Zones of Cellular Damage Around Pulsed-Laser Wounds

abstract: After a tissue is wounded, cells surrounding the wound adopt distinct wound-healing behaviors to repair the tissue. Considerable effort has been spent on understanding the signaling pathways that regulate immune and tissue-resident cells as they respond to wounds, but these signals must ultimately originate from the physical damage inflicted by the wound. Tissue wounds comprise several types of cellular damage, and recent work indicates that different types of cellular damage initiate different types of signaling. Hence to understand wound signaling, it is important to identify and localize the types of wound-induced cellular damage. Laser ablation is widely used by researchers to create reproducible, aseptic wounds in a tissue that can be live-imaged. Because laser wounding involves a combination of photochemical, photothermal and photomechanical mechanisms, each with distinct spatial dependencies, cells around a pulsed-laser wound will experience a gradient of damage. Here we exploit this gradient to create a map of wound-induced cellular damage. Using genetically-encoded fluorescent proteins, we monitor damaged cellular and sub-cellular components of epithelial cells in living Drosophila pupae in the seconds to minutes following wounding. We hypothesized that the regions of damage would be predictably arrayed around wounds of varying sizes, and subsequent analysis found that all damage radii are linearly related over a 3-fold range of wound size. Thus, around laser wounds, the distinct regions of damage can be estimated after measuring any one. This report identifies several different types of cellular damage within a wounded epithelial tissue in a living animal. By quantitatively mapping the size and placement of these different types of damage, we set the foundation for tracing wound-induced signaling back to the damage that initiates it.

category: cell biology


title: A genome-wide CRISPR screen identifies regulators of beta cell function involved in type 2 diabetes risk

abstract: Identification of the genes and processes mediating genetic association signals for complex disease represents a major challenge. Since many of the genetic signals for type 2 diabetes exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of- function screen in human pancreatic beta cells. We focused on the regulation of insulin content as a disease-relevant readout of beta cell function. We identified 580 genes influencing this phenotype: integration with genetic and genomic data provided experimental support for 20 candidate type 2 diabetes effector transcripts including the autophagy receptor CALCOCO2. Our study highlights how cellular screens can augment existing multi-omic efforts to accelerate biological and translational inference at GWAS loci.

category: genomics


title: Structural basis of polyamine transport by human ATP13A2 (PARK9)

abstract:
Polyamines are small, organic polycations that are ubiquitous and essential to all forms of life. Currently, how polyamines are transported across membranes is not understood. Recent studies have suggested that ATP13A2 and its close homologs, collectively known as P5B-ATPases, are polyamine transporters at endo-/lysosomes. Loss-of-function mutations of ATP13A2 in humans cause hereditary early-onset Parkinsons disease. To understand the polyamine transport mechanism of ATP13A2, we determined high-resolution cryo-EM structures of human ATP13A2 in five distinct conformational intermediates, which together represent a near-complete transport cycle of ATP13A2. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, where polyamines may exit within the cytosolic leaflet of the membrane. Our study provides important mechanistic insights into polyamine transport and a framework to understand functions and mechanisms of P5B-ATPases.

HighlightsCryo-EM structures of human ATP13A2 in five distinct conformations at 2.5-3.7 [A] resolutions.

Unique features of ATP13A2 in comparison to other P-type ATPases.

Structure of the substrate-binding pocket of ATP13A2 and the molecular basis of polyamine binding.

Conformational changes along the transport cycle and proposed model for polyamine transport.


title: Integration and comparison of multi-omics profiles of NGLY1 deficiency plasma and cellular models to identify clinically relevant molecular phenotypes

abstract: NGLY1 (N-glycanase 1) deficiency is a rare congenital recessive disorder of protein deglycosylation unaddressed by the current standard of care. Using combined metabolomics and proteomics profiling, we show that NGLY1 deficiency activates the immune response and disturbs lipid metabolism, biogenic amine synthesis, and glutathione metabolism. These alterations were also observed in NGLY1 deficient patient-derived induced pluripotent stem cells (iPSCs) and differentiated neural progenitor cells (NPCs), which serve as personalized cellular models of the disease. These findings provide molecular insight into the pathophysiology of NGLY1 deficiency and suggest potential therapeutic strategies.


title: Distinct cellular immune signatures in acute Zika virus infection are associated withhigh or low persisting neutralizing antibody titers

abstract:
Although the formation of a durable neutralizing antibody response after an acute viral infection is a key component of protective immunity, little is known about why some individuals generate high versus low neutralizing antibody titers to infection or vaccination. Infection with Zika virus (ZIKV) during pregnancy can cause devastating fetal outcomes, and efforts to understand natural immunity to this infection are essential for optimizing vaccine design. In this study, we leveraged the high-dimensional single-cell profiling capacity of mass cytometry (CyTOF) to deeply characterize the cellular immune response to acute and convalescent ZIKV infection in a cohort of blood donors in Puerto Rico incidentally found to be viremic during the 2015-2016 epidemic in the Americas. During acute ZIKV infection, we identified widely coordinated responses across innate and adaptive immune cell lineages. High frequencies of multiple activated innate immune subsets, as well as activated follicular helper CD4+ T cells and proliferating CD27-IgD-B cells, during acute infection were associated with high titers of ZIKV neutralizing antibodies at 6 months post-infection. On the other hand, low titers of ZIKV neutralizing antibodies were associated with immune features that suggested a cytotoxic-skewed immune "set-point." Our study offers insight into the cellular coordination of immune responses and identifies candidate cellular biomarkers that may offer predictive value in vaccine efficacy trials for ZIKV and other acute viral infections aimed at inducing high titers of neutralizing antibodies.

One Sentence SummaryMass cytometry reveals acute ZIKV infection cellular immune signatures that predict high or low neutralizing antibody titers 6 months post-infection.


title: Reconstruction of metagenome-assembled genomes from aquaria

abstract: We report 11 metagenome-assembled genomes (MAGs) reconstructed from freshwater and saltwater aquaria including representatives of Polynucleobacter, Anaerolinae, Roseobacter, Flavobacteriia, Octadecabacter, Mycobacterium and Candidate Phyla Radiation (CPR) members. These MAGs can serve as a resource for aquatic research and elucidating the role of CPR taxa in the built environment.


title: ErpY-like lipoprotein of Leptospira outsmart host complement regulation by acquiring complement regulators, activating alternate pathway, and intervening membrane attack complex

abstract: The survival of pathogenic Leptospira in the host pivots on its proficiency to circumvent the immune response. These pathogens evade the complement system in serum by enticing and amassing the serum complement regulators onto their surface. ErpY-like lipoprotein, a surface-exposed protein of Leptospira spp., is conserved and exclusively present in the pathogenic spirochete. The recombinant form of this protein is comprehended to interact with multiple extracellular matrix (ECM) components and serum proteins like soluble complement regulators factor H (FH) and factor I (FI). Here, we document that the supplementation of recombinant ErpY-like protein (40 {micro}g/mL) in the host (humans) serum augments the viability of E. coli and saprophytic L. biflexa by more than 2-fold. Pure complement regulators FH and FI, when bound to rErpY-like protein, preserve their respective cofactor and protease activity mandated to cleave the complement component C3b. The supplementation of rErpY-like protein (40 {micro}g/mL) in serum ensued in [~]90 % reduction of membrane attack complex (C5b-9/MAC) deposition through alternate complement pathway (AP) activation. However, rErpY-like protein could moderately reduce ([~]16%) MAC deposition in serum through the classical pathway (CP). In addition, the rErpY-like protein solely activated the AP, suggesting its role in the rapid consumption and depletion of the complement components. Blocking the pathogenic L. interrogans surface with anti-rErpY resulted in an increase in MAC formation on the bacterial surface, indicating a specific role of the ErpY-like lipoprotein in complement-mediated immune evasion. This study underscores the role of the ErpY-like lipoprotein of Leptospira in complement evasion.


title: Global analysis of the zinc homeostasis network in Pseudomonas aeruginosa and its gene expression dynamics

abstract: Zinc is one of the most important trace elements for life and its deficiency, like its excess, can be fatal. In the bacterial opportunistic pathogen Pseudomonas aeruginosa, Zn homeostasis is not only required for survival, but also for virulence and antibiotic resistance. Thus, the bacterium possesses multiple Zn import/export/storage systems. In this work, we determine the expression dynamics of the entire P. aeruginosa Zn homeostasis network at both transcript and protein levels. Precisely, we followed the switch from a Zn-deficient environment, mimicking the initial immune strategy to counteract bacterial infections, to a Zn-rich environment, representing the phagocyte metal boost used to eliminate an engulfed pathogen. Thanks to the use of the NanoString technology, we timed the global silencing of Zn import systems and the orchestrated induction of Zn export systems. We show that the induction of Zn export systems is hierarchically organized as a function of their impact on Zn homeostasis. Moreover, we identify PA2807 as a novel Zn resistance component in P. aeruginosa and highlight new regulatory links among Zn-homeostasis systems. Altogether, this work unveils a sophisticated and adaptive homeostasis network, which complexity is key in determining a pathogen spread in the environment and during host-colonization.


title: Short- and long-term effects of amoxicillin/clavulanic acid or doxycycline on the gastrointestinal microbiome of growing cats

abstract: Antibiotic treatment in early life influences gastrointestinal (GI) microbial composition and function. In humans, the resultant intestinal dysbiosis is associated with an increased risk for certain diseases later in life. The objective of this study was to determine the temporal effects of antibiotic treatment on the GI microbiome of young cats. Fecal samples were collected from cats randomly allocated to receive either amoxicillin/clavulanic acid (20 mg/kg q12h) for 20 days (AMC group; 15 cats) or doxycycline (10 mg/kg q24h) for 28 days (DOX group;15 cats) as part of the standard treatment of upper respiratory tract infection. In addition, feces were collected from healthy control cats (CON group;15 cats). All cats were approximately two months of age at enrolment. Samples were collected on days 0 (baseline), 20 or 28 (AMC and DOX, respectively; last day of treatment), 60, 120, and 300. DNA was extracted and sequencing of the 16S rRNA gene and qPCR assays were performed. Fecal microbial composition was different on the last day of treatment for AMC cats, and 1 month after the end of antibiotic treatment for DOX cats, compared to CON cats. Species richness was significantly greater in DOX cats compared to CON cats on the last day of treatment. Abundance of Enterobacteriales was increased, and that of Erysipelotrichi was decreased in cats of the AMC group on the last day of treatment compared to CON cats. The abundance of the phylum Proteobacteria was increased in cats of the DOX group on days 60 and 120 compared to cats of the CON group. Only minor differences in abundances between the treatment groups and the control group were present on day 300. Both antibiotics appear to delay the developmental progression of the microbiome, and this effect is more profound during treatment with amoxicillin/clavulanic acid and one month after treatment with doxycycline. Future studies are required to determine if these changes influence microbiome function and whether they have possible effects on disease susceptibility in cats.


title: An early signaling transcription factor regulates differentiation in Giardia

abstract:
Transcriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. In response to encystation stimuli, Giardia lamblia cells shift from G1+S phase of the cell cycle to G2+M. By 2-4 hours, cyst wall proteins are upregulated, indicating that key regulatory steps occur within the first four hours of encystation. However, all characterized transcription factors (TFs) in Giardia have only been investigated at later time points of encystation. How TFs initiate encystation and link it to the cell cycle remains enigmatic. Here, we systematically screened six putative early upregulated TFs for nuclear localization, established their dynamic expression profiles, and determined their functional role in regulating encystation. We found a critical repressor, GLP4 that increases rapidly after 30 min of encystation stimuli and downregulates encystation specific markers, including Cyst Wall Proteins (CWPs) and enzymes in the cyst N-acetyl galactosamine (GalNAc) pathway. Depletion of GLP4 increases cyst production. Importantly, we observe that G2+M cells exhibit higher levels of CWP1 resulting from the activation of MYB2, a TF previously linked to encystation in Giardia. GLP4 upregulation occurs in G1+S cells, suggesting a role in repressing MYB2 and encystation specific genes in the G1+S phase of the cell cycle. Furthermore, we demonstrate that depletion of GLP4 upregulates MYB2 and promotes encystation while overexpression of GLP4 downregulates MYB2 and represses encystation. Together, these results suggest that Giardia employs a dose-dependent transcriptional response that involves the cell cycle regulated repressor GLP4 to orchestrate MYB2 and entry into the encystation pathway.

ImportanceTransition between life cycle stages is a common feature among parasitic pathogens and its regulation must be optimized to balance persistence of infection with transmission. The early transcription factors (TFs) regulating commitment to differentiate are totally unknown in Giardia. In this work, we identified GLP4, a previously uncharacterized GARP-like TF, as an early-acting transcriptional repressor that inhibits G1+S cells from entering the encystation pathway. GLP4 is therefore a key regulator controlling the balance between proliferative growth and terminal differentiation into infective cysts.

category: developmental biology


title: An early signaling GARP transcription factor regulates the entry point for differentiation in Giardia


title: A cell cycle-dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia


title: Metabolic design in a model of extreme mammalian metabolism, the North American least shrew (Cryptotis parva)

abstract: Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the Least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused-ion-beam EM imaging and tandem-mass-tag MS proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogenous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism.

category: zoology


title: In vivo selection of nanobodies to target the tumor immune microenvironment for immunotherapy development

abstract: Cancer immunotherapy has changed the landscape of treatment for some cancers, but variable responses reflect limitations imposed by the tumor microenvironment (TME)1-3. To facilitate the development of novel immunotherapies, we used an unbiased in vivo selection approach using a VHH nanobody library to identify immunologic targets in the TME. Using next generation sequencing (NGS), we demonstrate the parallel enrichment of immune cell-binding nanobodies that target immune cell subsets within the TME. We selected from enriched nanobodies - Cl.1 and Cl.2 - that bind to the CD11b+ and CD11c+ populations and we identified the desmosome complex as the target for Cl.1, suggesting the nanobodies can target key immune populations and may involve unvalidated targets. To demonstrate nanobodies could functionally alter target immune cells in vivo, we showed by single cell RNA sequencing (scRNAseq) that enriched nanobodies can increase activation transcripts in CD8+ T cells and dendritic cells. This approach is being used to develop novel cancer immunotherapies, but the methods can be broadly applicable to biologic discovery experiments in various physiologic or pathologic contexts.

category: bioengineering


title: New fossils of Australopithecus sediba reveal a nearly complete lower back

abstract: Adaptations of the lower back to bipedalism are frequently discussed but infrequently demonstrated in early fossil hominins. Newly discovered lumbar vertebrae contribute to a near-complete lower back of Malapa Hominin 2 (MH2), offering additional insights into posture and locomotion in Australopithecus sediba. We show that MH2 demonstrates a lower back consistent with human-like lumbar lordosis and other adaptations to bipedalism, including an increase in the width of intervertebral articular facets from the upper to lower lumbar column ("pyramidal configuration"). This contrasts with recent work on lordosis in fossil hominins, where MH2 was argued to demonstrate no appreciable lordosis ("hypolordosis") similar to Neandertals. Our three-dimensional geometric morphometric (3D GM) analyses show that MH2s nearly complete middle lumbar vertebra is human-like in shape but bears large, cranially-directed transverse processes, implying powerful trunk musculature. We interpret this combination of features to indicate that A. sediba used its lower back in both human-like bipedalism and ape-like arboreal positional behaviors, as previously suggested based on multiple lines of evidence from other parts of the skeleton and reconstructed paleobiology of A. sediba.

category: paleontology


title: The Evolutionary History of Small RNAs in the Solanaceae

abstract:
The Solanaceae or "nightshade" family is an economically important group that harbors a remarkable amount of diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the familys small RNA genomic landscape, we downloaded over 255 publicly available small RNA datasets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major small RNA classes: (1) microRNAs (miRNAs), typically 20-22 nt RNAs generated from a hairpin precursor and functioning in gene silencing, and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs (hc-siRNAs) typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs (phasiRNAs) and trans-acting siRNAs (tasiRNAs) generated via miRNA-directed cleavage of a Pol II-derived RNA precursor. Our analyses described thousands of small RNA loci, including poorly-understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these small RNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar small RNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities.

ONE-SENTENCE SUMMARYWe use over 255 publicly-available small RNA datasets to characterize the small RNA landscape for the Solanaceae family.

category: plant biology


title: GIGANTEA gene expression influence leaf senescence in Populus in two different ways

abstract:
GIGANTEA (GI) genes have a central role in plant development and influence several processes such as light signaling, circadian rhythm and abiotic stress tolerance. Hybrid aspen T89 (Populus tremula x tremuloides) trees with low GI expression through RNAi have a severely compromised growth. In order to study the effect of reduced GI expression on leaf traits with special emphasis on leaf senescence, we grafted GI-RNAi scions onto wild type (WT) rootstocks and managed to restore scions growth. The RNAi line had distorted leaf shape and reduced photosynthesis, probably caused by modulating phloem or stomatal function, increased starch accumulation, higher carbon-to-nitrogen (C/N) ratio and a reduced capacity to withstand moderate light stress. GI-RNAi also induced senescence under long day (LD) and moderate light conditions. Furthermore, the GI-RNAi lines were affected in their capacity to respond to "autumn environmental cues" inducing senescence, a type of leaf senescence with characteristics different from senescence induced directly by stress under LD conditions. Whereas Overexpression of GI delayed senescence. The two different effects on leaf senescence were not affected by the expression of FT (Flowering locus T), were "local" - they followed the genotype of the branch independent on the position in the tree - and trees with modified gene expression grown in the field were affected in a similar way as under controlled conditions. Taken together, GI plays a central role to sense the environmental changes during autumn and determine the appropriate timing for leaf senescence in Populus.

One sentence summaryLeaf senescence is a complex process that is not well understood, but this paper shows that changing the expression of one gene could influence leaf senescence in Populus trees in two separate ways.


title: A novel bacteriophage with broad host-range against Clostridioides difficile ribotype 078 elucidates the phage receptor.

abstract:
Bacteriophage represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent the dysbiosis of the colonic microbiota associated with the treatment of CDI with antibiotics. Whilst numerous phages have been isolated, none have been characterised with broad host-range activity towards PCR ribotype (RT) 078 C. difficile strains despite their considerable relevance to medicine and agriculture. In this study, we isolated four novel C. difficile Myoviruses: {Phi}CD08011, {Phi}CD418, {Phi}CD1801 and {Phi}CD2301. Their characterisation revealed that each was comparable with other C. difficile phages described in the literature, with the exception of {Phi}CD1801 which exhibited a broad host-range activity towards RT 078, infecting 15/16 (93.8%) of the clinical isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. In an attempt to advance these efforts, we conducted a series of fundamental experiments that identified the C. difficile SlpA, the major constituent of the C. difficile surface-layer (S-layer), as the phage receptor. Thus, we demonstrated that {Phi}CD1801 could only bind to RT 012 or RT 027 strains in the presence of a plasmid-borne S-layer cassette corresponding to RT 078. Armed with this information, efforts should now be directed towards the isolation of phages with broad host-range activity against each of the fourteen described S-layer cassette types which could form the basis of an effective cocktail active against a wide range of C. difficile isolates.

ImportanceResearch into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridium difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far, has aimed to find phages with the broadest possible host-range. Although, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad-host range activity are yet to be discovered. In this study, we isolate 4 novel Myoviruses including {Phi}CD1801, which exerts the broadest host-range activity towards RT 078 reported in the literature. Through the application of {Phi}CD1801 to robust binding assays, we elucidate SlpA as the phage receptor on the bacterial cell surface. Our finding suggests that an optimal "one size fits all" combinatorial phage cocktail, could theoretically comprise 14 phages, each targeting one of the 14 described S-layer cassettes of C. difficile.


title: L-arginine ameliorates defective autophagy in GM2 gangliosidoses by mTOR modulation

abstract: Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause fatal and progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by {beta}-hexosaminidase A (HexA) leads to the accumulation of its specific substrate, GM2 ganglioside, in neuronal lysosomes. Despite the development of a florid storage phenotype, the role of autophagy and its regulation by the mammalian target of rapamycin (mTOR) has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. Pathological autophagosomes, with enhanced expression of the p62/SQSTM1 protein, suggested impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed inappropriately diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities - and immediately suggests an avenue for therapeutic exploration in this cruel disease. We also contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of systemic treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy


title: Subepithelial Myofibroblasts Are Critical Regulators of Intestinal Epithelial Restoration

abstract: Intestinal epithelial migration is supposed to occur in a passive manner driven by the mitotic pressure exerted either by the cryptal stem cells, under physiological conditions, or the newly formed epithelium, upon damage. However, whether interactions between different neighboring cell types and with the matrix contribute to epithelial movement remain elusive. Here, we developed a novel three-dimensional in vitro intestinal mucosa model of gap closure, that includes both the epithelium and the basement membrane cellular compartments in a spatially relevant manner, to show that intestinal subepithelial myofibroblasts (ISEMFs) play a crucial role in epithelial restoration. ISEMF-derived biochemical cues boost epithelial proliferation and maintain epithelial barrier integrity. While, at the wounded area, ISEMF actively migrate to the epithelial front where they activate and generate [a]-SMA contractile stress fibers along the direction of the epithelial cells promoting epithelial migration. Furthermore, ISEMF also deposit collagen paths that act as "guide rails" for directing IEC migration. Thus, the mere physical presence of ISEMFs can greatly accelerate restoration in wound healing, which suggests that ISEMFs should be recognized as new potential therapeutic targets.


title: TALPID3/KIAA0586 regulates multiple aspects of neuromuscular patterning during gastrointestinal development in animal models and human

abstract: TALPID3/KIAA0586 is an evolutionary conserved protein, which plays an essential role in protein trafficking. Its role during gastrointestinal (GI) and enteric nervous system (ENS) development has not been studied previously. Here, we analysed chicken, mouse and human embryonic GI tissues with TALPID3 mutations. The GI tract of TALPID3 chicken embryos was shortened and malformed. Histologically, the gut smooth muscle was mispatterned and enteric neural crest cells were scattered throughout the gut wall. Analysis of the Hedgehog pathway and gut extracellular matrix provided causative reasons for these defects. Interestingly, chicken intra-species grafting experiments and a conditional knockout mouse model showed that ENS formation did not require TALPID3, but was dependent on correct environmental cues. Surprisingly, the lack of TALPID3 in enteric neural crest cells (ENCC) affected smooth muscle and epithelial development in a non cell-autonomous manner. Analysis of human gut fetal tissues with a KIAA0586 mutation showed strikingly similar findings compared to the animal models demonstrating conservation of TALPID3 and its necessary role in human GI tract development and patterning


title: BRD9-containing non-canonical BAF complexes safeguard cell identity and prevent reprogramming

abstract: Epigenetic reprogramming requires extensive remodeling of chromatin landscapes to silence cell-type specific gene expression programs. ATP-dependent chromatin-remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of Bromodomain-containing protein 9 (BRD9) and the associated ncBAF (non-canonical BRG1-associated factors) complex in reprogramming remains unknown. Here, we show that genetic suppression of BRD9 as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase the efficiency by which induced pluripotent stem cells (iPSCs) can be generated from human somatic cells. Chemical inhibition and acute degradation of BRD9 phenocopied this effect. Interestingly, we find that BRD9 is dispensable for establishment and maintenance of human pluripotency but required for mesendodermal lineage commitment during differentiation. Mechanistically, BRD9 inhibition downregulates somatic cell type-specific genes and decreases chromatin accessibility at somatic enhancers. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.


title: RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells.

abstract: Embryonic stem cell (ESC) fate decisions are regulated by a complex molecular circuitry that requires tight and coordinated gene expression regulations at multiple levels from chromatin organization to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key regulatory pathways that efficiently control stem cell homeostasis. However, the molecular mechanisms underlying the regulation of these pathways remain largely unknown to date. Here, we analyzed the expression, in mouse ESCs, of over 300 genes involved in ribosome biogenesis and we identified RSL24D1 as the most differentially expressed between self-renewing and differentiated ESCs. RSL24D1 is highly expressed in multiple mouse pluripotent stem cell models and its expression profile is conserved in human ESCs. RSL24D1 is associated with nuclear pre-ribosomes and is required for the maturation and the synthesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors, including POU5F1 and NANOG, as well as components of the polycomb repressive complex 2 (PRC2). Consistently, RSL24D1 is required for mouse ESC self-renewal and proliferation. Taken together, we show that RSL24D1-dependant ribosome biogenesis is required to both sustain the expression of pluripotent transcriptional programs and silence developmental programs, which concertedly dictate ESC homeostasis.


title: Diverse mating phenotypes impact the spread of wtf meiotic drivers in S. pombe

abstract: Meiotic drivers are genetic loci that break Mendels law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe, a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity to inbreed varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed level of inbreeding slows, but does not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

category: evolutionary biology


title: Ancestral diversity improves discovery and fine-mapping of genetic loci for anthropometric traits - the Hispanic/Latino Anthropometry Consortium

abstract: Hispanic/Latinos have been underrepresented in genome-wide association studies (GWAS) for anthropometric traits despite notable anthropometric variability with ancestry proportions, and a high burden of growth stunting and overweight/obesity in Hispanic/Latino populations. This address this knowledge gap, we analyzed densely-imputed genetic data in a sample of Hispanic/Latino adults, to identify and fine-map common genetic variants associated with body mass index (BMI), height, and BMI-adjusted waist-to-hip ratio (WHRadjBMI). We conducted a GWAS of 18 studies/consortia as part of the Hispanic/Latino Anthropometry (HISLA) Consortium (Stage 1, n=59,769) and validated our findings in 9 additional studies (HISLA Stage 2, n=9,336). We conducted a trans-ethnic GWAS with summary statistics from HISLA Stage 1 and existing consortia of European and African ancestries. In our HISLA Stage 1+2 analyses, we discovered one novel BMI locus, as well two novel BMI signals and another novel height signal, each within established anthropometric loci. In our trans-ethnic meta- analysis, we identified three additional novel BMI loci, one novel height locus, and one novel WHRadjBMI locus. We also identified three secondary signals for BMI, 28 for height, and two for WHRadjBMI. We replicated >60 established anthropometric loci in Hispanic/Latino populations at genome-wide significance--representing up to 30% of previously-reported index SNP anthropometric associations. Trans-ethnic meta-analysis of the three ancestries showed a small-to-moderate impact of uncorrected population stratification on the resulting effect size estimates. Our novel findings demonstrate that future studies may also benefit from leveraging differences in linkage disequilibrium patterns to discover novel loci and additional signals with less residual population stratification.

category: genetics


title: The Mitochondrial Genome and Epigenome of the Golden Lion Tamarin from Fecal DNA using Nanopore Adaptive Sequencing

abstract: The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3 side of several coding regions. Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.


title: Long-term repair of porcine articular cartilage using cryopreservable, clinically compatible human embryonic stem cell-derived chondrocytes

abstract: Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.


title: RNA folding using quantum computers

abstract: The 3-dimensional fold of an RNA molecule is largely determined by patterns of intramolecular hydrogen bonds between bases. Predicting the hydrogen bonding network from the sequence, also referred to as RNA secondary structure prediction or RNA folding, is a nondeterministic polynomial-time (NP)-complete computational problem. The structure of the molecule is strongly predictive of its functions and biochemical properties, and therefore the ability to accurately predict the structure is a crucial tool for biochemists. Many methods have been proposed to efficiently sample possible secondary structure patterns. Classic approaches employ dynamic programming, and recent studies have explored approaches inspired by evolutionary algorithms. This work demonstrates leveraging quantum computing hardware to predict the secondary structure of RNA. A Hamiltonian written in the form of a Binary Quadratic Model (BQM) is derived to drive the system toward maximizing the number of base pairs while simultaneously maximizing the average length of the stems. An Adiabatic Quantum Computer (AQC) is compared to a Replica Exchange Monte Carlo (REMC) algorithm programmed with the same objective function, with the AQC being shown to be highly competitive at rapidly identifying low energy solutions. The method proposed in this study was compared to three algorithms from literature and was found to have the highest success rate.

category: bioinformatics


title: Widespread occurrence of hybrid internal-terminal exons in human transcriptomes

abstract: Alternative RNA processing is a major mechanism for diversifying the human transcriptome. Messenger RNA isoform differences are predominantly driven by alternative first exons, cassette internal exons and alternative last exons. Despite the importance of classifying exons to understand isoform structure, there is a lack of tools to look at isoform-specific exon usage using RNA-sequencing data. We recently observed that alternative transcription start sites often arise near annotated internal exons, creating "hybrid" exons that can be used as both first or internal exons. To investigate the creation of hybrid exons, we built the HIT (Hybrid-Internal-Terminal) exon pipeline that systematically classifies exons depending on their isoform-specific usage. Using a combination of junction reads coverage and probabilistic modeling, the HIT index identified thousands of hybrid first-internal and internal-last exons that were previously misclassified. Hybrid exons are enriched in long genes with at least ten internal exons, have longer flanking introns and strong splice sites. The usage of hybrid exons varies considerably across human tissues, but they are predominantly used in brain, testis and colon cells. Notably, genes involved in RNA splicing have the highest fraction of intra-tissue hybrid exons. Further, we found more than 100,000 inter-tissue hybrid exons that changed from internal to terminal exons across tissues. By developing the first method that can classify exons according to their isoform contexts, our findings demonstrate the existence of hybrid exons, expand the repertoire of tissue-specific terminal exons and uncover unexpected complexities of the human transcriptome.


title: Systematic evaluation of NIPT aneuploidy detection software tools with clinically validated NIPT samples

abstract:
MotivationNon-invasive prenatal testing (NIPT) is a powerful screening method for fetal aneuploidy detection, relying on laboratory and computational analysis of cell-free DNA. Although several published computational NIPT analysis tools are available, no comprehensive and direct accuracy comparison of these tools is published. Here, we evaluate and determine the precision of five commonly used computational NIPT aneuploidy analysis tools, considering diverse sequencing depth (coverage) and fetal DNA fraction (FF) on clinically validated NIPT samples.

MethodsWe evaluated computational NIPT aneuploidy analysis tools WisecondorX, NIPTeR, NIPTmer, RAPIDR, and GIPseq, on the same set of clinically validated samples, subsampled to different sequencing coverages between 1.25-20M reads per sample (RPS). These clinically validated samples consisted of 423 samples, including 19 samples with fetal chromosome 21 trisomy (T21, Down syndrome), eight trisomy 18 (T18, Edwards syndrome) and three trisomy 13 (T13, Patau syndrome) samples. For each software and sequencing coverage, we determined the number of false-negative and false-positive trisomy/euploidy calls. For a uniform trisomy detection interpretation, we defined a framework based on the percent-point function for determining the cut-off threshold for calling aneuploidy based on the sample Z-score and the reference group Z-score distribution. We also determined the effect of the naturally occurring arbitrary read placement driven uncertainty on T21 detection at very low sequencing coverage and the effect of cell-free fetal DNA fraction (FF) on the accuracy of these computational tools in the case of various sequencing coverages.

ResultsThis is the first head-to-head comparison of NIPT aneuploidy detection tools for the low-coverage whole-genome sequencing approach. We determined that, with the currently available software tools, the minimum sequencing coverage with no false-negative trisomic cases was 5M RPS. Secondly, for these compared tools, the number of false-negative trisomic cases could be reduced if the trisomy call cut-off threshold considers the Z-score distribution of euploid reference samples. Thirdly, we observed that in the case of low FF, both aneuploidy Z-score and FF inference was considerably less accurate, especially in NIPT assays with 5M RPS or lower coverage.

ConclusionsWe determined that all compared computational NIPT tools were affected by lower sequencing depth, resulting in systematically increasing the proportions of false-negative trisomy results as the sequencing depth decreased. Trisomy detection for lower coverage NIPT samples (e.g. 2.5M RPS) is technically possible but can increase the proportion of false-positive and false-negative trisomic cases, especially in the case of low FF.


title: Molecular basis for kinin selectivity and activation of the human bradykinin receptors

abstract: Bradykinin and kallidin are endogenous kinin peptide hormones that belong to the kallikrein-kinin system and are essential to the regulation of blood pressure, inflammation, coagulation, and pain control. Des-Arg10-kallidin, the carboxy-terminal des-Arg metabolite of kallidin, and bradykinin selectively activate two G protein-coupled receptors, type 1 and type 2 bradykinin receptors (B1R and B2R), respectively. The hyperactivation of bradykinin receptors, termed "bradykinin storm", is associated with pulmonary edema in COVID-19 patients, suggesting that bradykinin receptors are important targets for COVID-19 intervention. Here we report two G protein complex structures of B1R and B2R bound to des-Arg10-kallidin and bradykinin. Combined with functional analysis, our structures reveal the mechanism of ligand selectivity and specific activation of the bradykinin receptor. These findings also provide a framework for guiding drug design targeting bradykinin receptors for the treatment of inflammation, cardiovascular disorders, and COVID-19.

category: biophysics


title: A Universal Approach to Analyzing Transmission Electron Microscopy with ImageJ

abstract:
2Transmission electron microscopy (TEM) is a scientific research standard for producing nanometer-resolution ultrastructural images of subcellular components within cells and tissues. Mitochondria, endoplasmic reticulum (ER), lysosomes, and autophagosomes are organelles of particular interest to those investigating metabolic disorders. However, there is no clear consensus amongst regarding the best methods for quantifying the features of organelles in TEM images. In this protocol, we propose a standardized approach to accurately measure the morphology of these important subcellular structures using the free program ImageJ, developed by the National Institutes of Health (NIH). Specifically, we detail procedures for obtaining mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology. We further provide methods for measuring interactions between the mitochondria and ER and measuring the length and width of lysosomes and autophagosomes. This standardized method can be used to quantify key features of organelle morphology, allowing investigators to produce accurate and reproducible measurements of organelle structures in their experimental samples.

1 SUMMARYWe discuss a standardized method for measuring and quantifying organelle features using transmission electron microscopy and accessing for interactions between subcellular structures; organelles of focus include mitochondria, endoplasmic reticulum, lysosomes, and autophagosomes.


title: Subcutaneous infusion of neurosecretory protein GL promotes fat accumulation in mice

abstract: We recently identified a novel small secretory protein, neurosecretory protein GL (NPGL), in the vertebrate hypothalamus. We revealed that NPGL is involved in energy homeostasis using intracerebroventricular infusion in rodents. However, the effect of NPGL through peripheral administration remains to be elucidated and may be important for therapeutic use. In this study, we performed subcutaneous infusion of NPGL in mice for 12 days and found that it accelerated fat accumulation in white adipose tissue (WAT) without increasing in body mass gain and food intake. The mass of the testis, liver, kidney, heart, and gastrocnemius muscle remained unchanged. Analysis of mRNA expression by quantitative reverse transcription-polymerase chain reaction showed that proopiomelanocortin was suppressed in the hypothalamus by the infusion of NPGL. We observed a decreasing tendency in serum triglyceride levels due to NPGL, while serum glucose, insulin, leptin, and free fatty acids levels were unchanged. These results suggest that the peripheral administration of NPGL induces fat accumulation in WAT via the hypothalamus.


title: A global dataset for the projected impacts of climate change on four major crops

abstract: Reliable estimates of the impacts of climate change on crop production are critical for assessing the sustainability of food systems. Global, regional, and site-specific crop simulation studies have been conducted for nearly four decades, representing valuable sources of information for climate change impact assessments. However, the wealth of data produced by these studies has not been made publicly available. Here, we develop a global dataset by consolidating previously published meta-analyses and data collected through a new literature search covering recent crop simulations. The new global dataset builds on 8314 simulations from 203 studies published between 1984 and 2020. It contains projected yields of four major crops (maize, rice, soybean, and wheat) in 91 countries under major emission scenarios for the 21st century, with and without adaptation measures, along with geographical coordinates, current temperatures, local and global warming levels. This dataset provides a basis for a comprehensive understanding of the impacts of climate change on crop production and will facilitate the rapidly developing data-driven machine learning applications.


title: Sex-specific transcriptome of spinal microglia in neuropathic pain due to peripheral nerve injury

abstract: Recent studies have suggested a sexually dimorphic role of microglia in the maintenance of neuropathic pain in rodents. Here, we utilized RNA sequencing analysis and in vitro primary cultures of microglia to characterize the sex differences in microglia in pain-related regions in nerve injury and chemotherapy-induced peripheral neuropathy mouse models. Whilst mechanical allodynia and behavioral changes were observed in all models, transcriptomic analysis revealed a substantial change in microglial gene expression only within the ipsilateral lumbar spinal cord 7-days after nerve injury. Both sexes upregulated genes associated with inflammation, phagosome, and lysosome activation, though males revealed a prominent global transcriptional shift not observed in female mice, reflecting acute activation. Further, in vitro studies revealed that only male microglia from nerve-injured mice developed a reactive phenotype with increased phagocytotic activity. This study indicates distinct sex differences in spinal microglia and suggests they contribute to the sex-specific pain processing following nerve injury.


title: Revealing conserved mechanisms of neurodegeneration in a colonial chordate

abstract: Loss of the brains functional ability is a common symptom of aging and neurodegenerative diseases1,2. While the genetic and molecular mechanisms underlying human neurodegeneration are studied in-depth3-6, very little is known about the evolutionary origin of these traits and their involvement in loss of nervous system function in aged invertebrate species. Here we study evolutionarily conserved elements of brain degeneration using the colonial chordate model species Botryllus schlosseri. B. schlosseri reproduces both sexually and asexually7, with adult brains regenerating and degenerating multiple times throughout its adult life. Combining microscopy, transcriptomics and behavioral assays, we characterized adult brains from diverse stages and ages. We found that the number of neurons fluctuates each week, reaching a maximum of [~]1000 cells, and thereafter decreasing while the number of immunocytes increases. Comparing the number of neurons in the adult brains of young and old colonies, we found that older brains are smaller and contain fewer cells. Both during weekly degeneration cycles and overall with age, the decrease in neuron number correlates with reduced response to stimuli and with significant changes in the expression of genes with mammalian homologs associated with neural stem cells and neurodegenerative pathways. These results suggest persistent neural stem cell activity across ages and that cellular and molecular mechanisms of neurodegeneration are evolutionary conserved between tunicates and humans.


title: Machine learning prediction of cognition from functional connectivity: Are feature weights reliable?

abstract: Cognitive performance can be predicted from an individuals functional brain connectivity with modest accuracy using machine learning approaches. As yet, however, predictive models have arguably yielded limited insight into the neurobiological processes supporting cognition. To do so, feature selection and feature weight estimation need to be reliable to ensure that important connections and circuits with high predictive utility can be reliably identified. We comprehensively investigate feature weight test-retest reliability for various predictive models of cognitive performance built from resting-state functional connectivity networks in healthy young adults (n=400). Despite achieving modest prediction accuracies (r=0.2-0.4), we find that feature weight reliability is generally poor for all predictive models (ICC<0.3), and significantly poorer than predictive models for overt biological attributes such as sex (ICC {approx} 0.5). Larger sample sizes (n=800), the Haufe transformation, non-sparse feature selection/regularization and smaller feature spaces marginally improve reliability (ICC<0.4). We elucidate a tradeoff between feature weight reliability and prediction accuracy and find that univariate statistics are marginally more reliable than feature weights from predictive models. Finally, we show that measuring agreement in feature weights between cross-validation folds provides inflated estimates of feature weight reliability. We thus recommend for reliability to be estimated out-of-sample, if possible. We argue that rebalancing focus from prediction accuracy to model reliability may facilitate mechanistic understanding of cognition with machine learning approaches.


title: Perceptual clustering in auditory streaming

abstract: Perception relies on being able to segregate stimuli from different objects and causes to perform inference and further processing. For simple binary stimuli (e.g. auditory and visual) we have models of how the human brain can perform such causal inference, but the complexity of the models increases dramatically with more than 2 stimuli. To characterize human perception with more complex stimuli we have developed a Bayesian inference model that allows an unlimited number of stimulus sources to be considered: it is general enough to allow any discrete sequential cues, from any modality. The model uses a non-parametric prior, hence increased complexity of the signal does not necessitate more parameters. The model not only determines the most likely number of sources, but also specifies the source that each signal is associated with. As a test application we show that such a model can explain several phenomena in the auditory stream perception literature, that it gives an excellent fit to experimental data, and that it makes novel predictions that we confirm experimentally. These results have implications not just for human auditory temporal perception but for a large range of other perceptual phenomena.


title: Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor

abstract: The glucagon-like peptide-1 (GLP-1) receptor is a validated drug target for metabolic disorders. Ago-allosteric modulators are capable of acting both as agonists on their own and as efficacy enhancers of orthosteric ligands. However, the molecular details of ago-allosterism remain elusive. Here, we report three cryo-electron microscopy structures of GLP-1R bound to (i) compound 2 (an ago-allosteric modulator); (ii) compound 2 and GLP-1; and (iii) compound 2 and LY3502970 (a small molecule agonist), all in complex with heterotrimeric Gs. The structures reveal that compound 2 is covalently bonded to C347 at the cytoplasmic end of TM6 and triggers its outward movement in cooperation with the ECD whose N terminus penetrates into the GLP-1 binding site. This allows compound 2 to execute positive allosteric modulation through enhancement of both agonist binding and G protein coupling. Our findings offer the structural basis of ago-allosterism at GLP-1R and new knowledge to design better therapeutics.

category: molecular biology


title: DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations

abstract: Chromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequently cause cancer. Yet, the steps linking DSB formation to DSB ligation remain undeciphered. We report that DNA replication timing (RT), mediated by replication origin activity, directly regulates the genesis of lymphomagenic Myc translocations during antibody maturation in B cells. Reduced levels of the replicative helicase, the minichromosome-maintenance (MCM) complex, decreases translocations and globally abrogates the RT program. Ablating a single replication origin at Myc causes an early-to-late RT switch with major loss of translocations, a phenotype that is reversed by restoring early RT. Finally, this novel RT-regulated mechanism occurs after DSB formation and independently of DSB frequency. Thus, RT constitutes a distinct regulatory event in translocation biogenesis linking DSB formation to DSB ligation.


title: Src activation in lipid rafts confers epithelial cells with invasive potential to escape from apical extrusion during cell competition

abstract: Abnormal/cancerous cells within healthy epithelial tissues undergo apical extrusion to protect against carcinogenesis, while they acquire invasive capacity once carcinogenesis progresses. However, the molecular mechanisms by which cancer cells escape from apical extrusion and invade surrounding tissues remain elusive. We found that during competition within epithelial cell layers, Src-transformed cells underwent basal delamination by Src activation within lipid rafts, whereas they were apically extruded when Src was outside of lipid rafts. Comparative analysis of contrasting phenotypes revealed that activation of the Src-STAT3-MMP axis through lipid rafts was required for basal delamination. CUB domain-containing protein 1 (CDCP1) was identified as an Src activating scaffold in lipid rafts, and its overexpression induced basal delamination. In renal cancer spheroids, CDCP1 promoted HGF-dependent invasion by activating the Src-STAT3-MMP axis. Overall, these results suggest that Src activation in lipid raft confers resistance to apical extrusion and invasive potential on epithelial cells to promote carcinogenesis.


title: Seasonal variations of 137Cs concentration in freshwater charr through uptake and metabolism in 1-2 years after the Fukushima accident

abstract: Understanding the factors influencing 137Cs concentrations in freshwater salmonids is crucial for reviving inland fisheries in polluted regions. We studied seasonal variations of 137Cs concentration in charr (Salvelinus leucomaenis) through uptake and metabolism in forested headwaters at Fukushima and Gunma sites. Charr consumed both terrestrial and aquatic animals, and terrestrial prey was predated more in summer at both sites. The 137Cs concentrations in litter, which is a dominant basal food resource of both forest and stream ecosystems, differed between forest and stream due to 137Cs leaching effect on litter submerged in streams. The concentration difference in both litter and prey was greater at Fukushima site than at Gunma site. The estimated prey 137Cs concentration at Fukushima site peaked in summer when terrestrial preys are most available, whereas it remained relatively constant at Gunma site because of the small difference of 137Cs concentrations in between terrestrial and aquatic preys. The specific metabolic rate of charr was commonly changed with stream water temperature, greatest in summer and lowest in winter at both sites. Because both prey 137Cs concentrations and specific metabolic rates peaked in summer, the combination of uptake and metabolism at Fukushima site largely negated seasonal 137Cs fluctuations in charr, whereas specific metabolic rate fluctuations could be the major determinant of charr 137Cs concentrations at Gunma site. Our results suggested that 137Cs concentrations in prey items, whose seasonality are varied due to initial 137Cs fallout volume, were expected to be an important determinant for 137Cs concentrations in charr.

category: ecology


title: Using a two-stage convolutional neural network to rapidly identify tiny herbivorous beetles in the field

abstract: Recently, deep convolutional neural networks (CNN) have been adopted to help beginners identify insect species from field images. However, the application of these methods on the identification of tiny congeneric species moving across heterogeneous background remains difficult. To enable rapid and automatic identification in the field, we customized a method involving real-time object detection of two Phyllotreta beetles. We first performed data augmentation using transformations, syntheses, and random erasing of the original images. We then proposed a two-stage method for the detection and identification of small insects based on CNN, where YOLOv4 and EfficientNet were used as a region proposal network and a re-identification method, respectively. Evaluation of the model revealed that one-step object detection by YOLOv4 alone was not precise (Precision = 0.55) when classifying two species of flea beetles and background objects. In contrast, the two-step CNNs improved the precision (Precision = 0.89) with moderate accuracy (F-measure = 0.55) and acceptable speed (ca. 5 frames per second for full HD images) of detection and identification of insect species in the field. Although real-time identification of tiny insects remains a challenge in the field, our method aids in improving small object detection on a heterogeneous background.


title: Are plants in sown flower strips suitable for communities of wild bees? Pollination network approach in conservation biology

abstract:
O_LIDrastic reductions of insect diversity and abundance are observed in the highly fragmented agricultural landscapes of central Europe. Declines of pollinators may have detrimental effects on the reproduction of wild insect-pollinated plants as well as the yield of crops. In order to mitigate such impacts, sown flower strips on arable land within Agri-Environment Climate Schemes (AECS) are supported across EU countries. However, it is not clear whether sown flower strips provide equivalent benefits to wild flower-visiting insects as semi-natural habitats.
C_LIO_LIHere, we apply plant-pollinator network approach to evaluate the function of sown flower strips for the communities of wild bees. We compared the structural characteristics and the robustness of plant-pollinator networks in sown flower strips and nearby semi-natural habitats. We also quantified the importance of individual plant species for bees based on simulations of plant-pollinator extinction cascades.
C_LIO_LIWe found that assemblages of plants and pollinators were less diverse in sown flower strips than in semi-natural habitats, more generalized, and more nested. However, we did not find any significant differences in network robustness to plant-pollinator coextinctions. Further, simulations revealed a large variation in the functional importance among plant species from both habitats.
C_LIO_LIWe conclude that although the analysis of network robustness suggested that plants in the sown flower strips and semi-natural habitats were functionally equivalent, this masked important differences among the two habitats. From the conservation point of view, semi-natural habitats were superior in supporting a more diverse community of solitary bees and bumblebees.
C_LI


title: Automated segmentation of insect anatomy from micro-CT images using deep learning

abstract: Three-dimensional (3D) imaging, such as micro-computed tomography (micro-CT), is increasingly being used by organismal biologists for precise and comprehensive anatomical characterization. However, the segmentation of anatomical structures remains a bottleneck in research, often requiring tedious manual work. Here, we propose a pipeline for the fully-automated segmentation of anatomical structures in micro-CT images utilizing state-of-the-art deep learning methods, selecting the ant brain as a test case. We implemented the U-Net architecture for 2D image segmentation for our convolutional neural network (CNN), combined with pixel-island detection. For training and validation of the network, we assembled a dataset of semi-manually segmented brain images of 94 ant species. The trained network predicted the brain area in ant images fast and accurately; its performance tested on validation sets showed good agreement between the prediction and the target, scoring 80% Intersection over Union (IoU) and 90% Dice Coefficient (F1) accuracy. While manual segmentation usually takes many hours for each brain, the trained network takes only a few minutes. Furthermore, our network is generalizable for segmenting the whole neural system in full-body scans, and works in tests on distantly related and morphologically divergent insects (e.g., fruit flies). The latter suggest that methods like the one presented here generally apply across diverse taxa. Our method makes the construction of segmented maps and the morphological quantification of different species more efficient and scalable to large datasets, a step toward a big data approach to organismal anatomy.


title: Tightly constrained genome reduction and relaxation of purifying selection during secondary plastid endosymbiosis

abstract: Endosymbiosis, the establishment of a former free-living prokaryotic or eukaryotic cell as an organelle inside a host cell, can dramatically alter the genomic architecture of the endosymbiont. Plastids, the light harvesting organelles of photosynthetic eukaryotes, are excellent models to study this phenomenon because plastid origin has occurred multiple times in evolution. Here, we investigate the genomic signature of molecular processes acting through secondary plastid endosymbiosis - the origination of a new plastid from a free-living eukaryotic alga. We used phylogenetic comparative methods to study gene loss and changes in selective regimes on plastid genomes, focusing on the green lineage that has given rise to three independent lineages with secondary plastids (euglenophytes, chlorarachniophytes, Lepidodinium). Our results show an overall increase in gene loss associated with secondary endosymbiosis, but this loss is tightly constrained by retention of genes essential for plastid function. The data show that secondary plastids have experienced temporary relaxation of purifying selection during secondary endosymbiosis. However, this process is tightly constrained as well, with selection relaxed only relative to the background in primary plastids, but purifying selection remaining strong in absolute terms even during the endosymbiosis events. Selection intensity rebounds to pre-endosymbiosis levels following endosymbiosis events, demonstrating the changes in selection efficiency during different phases of secondary plastid origin. Independent endosymbiosis events in the euglenophytes, chlorarachniophytes, and Lepidodinium differ in their degree of relaxation of selection, highlighting the different evolutionary contexts of these events. This study reveals the selection-drift interplay during secondary endosymbiosis, and evolutionary parallels during the process of organelle origination.


title: Transposable elements drive the evolution of genome streamlining

abstract: Eukaryotes and prokaryotes have distinct genome architectures, with marked differences in genome size, the ratio of coding/non-coding DNA, and the abundance of transposable elements (TEs). As TEs replicate independently of their hosts, the proliferation of TEs is thought to have driven genome expansion in eukaryotes. However, prokaryotes also have TEs in intergenic spaces, so why do prokaryotes have small, streamlined genomes? Using an in silico model describing the genomes of single-celled asexual organisms that co-evolve with TEs, we show that TEs acquired from the environment by horizontal gene transfer can drive the evolution of genome streamlining. The process depends on local interactions and is underpinned by rock-paper-scissor dynamics in which populations of cells with streamlined genomes beat TEs, which beat non-streamlined genomes, in continuous and repeating cycles. Streamlining is maladaptive to individual cells, but delivers lineage-level benefits. Streamlining does not evolve in sexually reproducing populations because recombination partially frees TEs from the deleterious effects they cause.