title: D155Y Substitution of SARS-CoV-2 ORF3a Weakens Binding with Caveolin-1: An in silico Study

abstract: The clinical manifestation of the recent pandemic COVID-19, caused by the novel SARS-CoV-2 virus, varies from mild to severe respiratory illness. Although environmental, demographic and co-morbidity factors have an impact on the severity of the disease, contribution of the mutations in each of the viral genes towards the degree of severity needs a deeper understanding for designing a better therapeutic approach against COVID-19. Open Reading Frame-3a (ORF3a) protein has been found to be mutated at several positions. In this work, we have studied the effect of one of the most frequently occurring mutants, D155Y of ORF3a protein, found in Indian COVID-19 patients. Using computational simulations we demonstrated that the substitution at 155th changed the amino acids involved in salt bridge formation, hydrogen-bond occupancy, interactome clusters, and the stability of the protein compared with the other substitutions found in Indian patients. Protein-protein docking using HADDOCK analysis revealed that substitution D155Y weakened the binding affinity of ORF3a with caveolin-1 compared with the other substitutions, suggesting its importance in the overall stability of ORF3a-caveolin-1 complex, which may modulate the virulence property of SARS-CoV-2.


title: D155Y Substitution of SARS-CoV-2 ORF3a Weakens Binding with Caveolin-1

category: bioinformatics


title: Improved architectures for flexible DNA production using retrons across kingdoms of life

abstract: Exogenous DNA is a critical molecular tool for biology. This is particularly true for gene editing, where exogenous DNA can be used as a template to introduce precise changes to the sequence of a cells genome. This DNA is typically synthesized or assembled in vitro and then delivered to target cells. However, delivery can be inefficient, and low abundance of template DNA may be one reason that precise editing typically occurs at a low rate. It has recently been shown that producing DNA inside cells can using reverse transcriptases can increase the efficiency of genome editing. One tool to produce that DNA is a retron, a bacterial retroelement that has an endogenous role in phage defense. However, little effort has been directed at optimizing the retron for production of designed sequences when used as a component of biotechnology. Here, we identify modifications to the retron non-coding RNA that result in more abundant reverse transcribed DNA. We also test architectures of the retron operon that enable efficient reverse transcription across kingdoms of life from bacteria, to yeast, to cultured human cells. We find that gains in DNA production using modified retrons are portable from prokaryotic to eukaryotic cells. Finally, we demonstrate that increased production of RT-DNA results in more efficient genome editing in both prokaryotic and eukaryotic cells. These experiments provide a general framework for production of DNA using a retron for biotechnological applications.

category: synthetic biology


title: Occurrence of COVID-19 symptoms during SARS-CoV-2 infection defines waning of humoral immunity

abstract:
Approximately half of the SARS-CoV-2 infections occur without apparent symptoms, raising questions regarding long-term humoral immunity in asymptomatic individuals. Plasma levels of immunoglobulin G (IgG) and M (IgM) against the viral spike or nucleoprotein were determined for 25,091 individuals enrolled in a surveillance program in Wuhan, China. We compared 405 asymptomatic individuals with 459 symptomatic COVID-19 patients. The well-defined duration of the SARS-CoV-2 endemic in Wuhan allowed a side-by-side comparison of antibody responses following symptomatic and asymptomatic infections without subsequent antigen re-exposure. IgM responses rapidly declined in both groups. However, both the prevalence and durability of IgG responses and neutralizing capacities correlated positively with symptoms. Regardless of sex, age, and body weight, asymptomatic individuals lost their SARS-CoV-2-specific IgG antibodies more often and rapidly than symptomatic patients. These findings have important implications for immunity and favour immunization programs including individuals after asymptomatic infections.

One-Sentence SummaryPrevalence and durability of SARS-CoV-2-specific IgG responses and neutralizing capacities correlate with COVID-19 symptoms.

category: immunology


title: Effect of a Monascus sp. red yeast rice extract on germination of bacterial spores

abstract: The pink-red colour of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant colour of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for colouring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2 % v/v. For B. subtilis, total inhibition of spore germination was observed only after addition of 4 % v/v RYR extract to the medium containing 1.3% w/w NaCl.

category: microbiology


title: Rapid response of nitrogen cycling gene transcription to labile carbon amendments in a soil microbial community

abstract:
Episodic inputs of labile carbon (C) to soil can rapidly stimulate nitrogen (N) immobilization by soil microorganisms. However, the transcriptional patterns that underlie this process remain unclear. In order to better understand the regulation of N cycling in soil microbial communities, we conducted a 48 h laboratory incubation with an agricultural soil where we stimulated the uptake of inorganic N by amending the soil with glucose. We analyzed the metagenome and metatranscriptome of the microbial communities at four timepoints that corresponded with changes in N availability. The relative abundances of genes remained largely unchanged throughout the incubation. In contrast, glucose addition rapidly increased transcription of genes encoding for ammonium and nitrate transporters, enzymes responsible for N assimilation into biomass, and genes associated with the N regulatory network. This upregulation coincided with an increase in transcripts associated with glucose breakdown and oxoglutarate production, demonstrating a connection between C and N metabolism. When concentrations of ammonium were low, we observed a transient upregulation of genes associated with the nitrogen fixing enzyme nitrogenase. Transcripts for nitrification and denitrification were downregulated throughout the incubation, suggesting that dissimilatory transformations of N may be suppressed in response to labile C inputs in these soils. These results demonstrate that soil microbial communities can respond rapidly to changes in C availability by drastically altering the transcription of N cycling genes.

IMPORTANCEA large portion of activity in soil microbial communities occurs in short time frames in response to an increase in C availability, affecting the biogeochemical cycling of nitrogen. These changes are of particular importance as nitrogen represents both a limiting nutrient for terrestrial plants as well as a potential pollutant. However, we lack a full understanding of the short-term effects of labile carbon inputs on the metabolism of microbes living in soil. Here, we found that soil microbial communities responded to labile carbon addition by rapidly transcribing genes encoding proteins and enzymes responsible for inorganic nitrogen acquisition, including nitrogen fixation. This work demonstrates that soil microbial communities respond within hours to carbon inputs through altered gene expression. These insights are essential for improved understanding of the microbial processes governing soil organic matter production, decomposition, and nutrient cycling in natural and agricultural ecosystems.


title: Green Fluorescent Carbon Dots as Targeting Probes for LED-Dependent Bacterial Killing

abstract: The emergence of antimicrobial resistance represents a significant health and economic challenge worldwide. The slow pace of antibacterial discovery necessitates strategies for optimal use of existing agents, including effective diagnostics able to drive informed prescribing; and development of alternative therapeutic strategies that go beyond traditional small-molecule approaches. Thus, the development of novel probes able to target bacteria for detection and killing, and that can pave the way to effective theranostic strategies, is of great importance. Here we demonstrate that metal-free green-emitting fluorescent carbon dots (FCDs) synthesized from glucosamine HCl and m-phenylenediamine, and featuring 2,5-deoxyfructosazine on a robust amorphous core, can label both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) bacterial pathogens within 10 minutes of exposure. Moreover, effective killing of Gram-positive and -negative bacteria can be induced by combining FCD treatment with irradiation by LED light in the visible range. Cell-based, electron microscopy and Tandem Mass Tag (TMT) proteomic experiments indicate that FCD administration in combination with LED exposure gives rise to local heating, ROS production, and membrane- and DNA-damage, suggesting multiple routes to FCD-mediated bacterial killing. Our data identify FCDs as materials that combine facile synthesis from low-cost precursors with labelling and light-dependent killing of clinically important bacterial species, and that thus warrant further exploration as the potential bases for novel theranostics.


title: A spatial and cellular distribution of neurotropic virus infection in the mouse brain revealed by fMOST and single cell RNA-seq

abstract: Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both human and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques. In this study, we explore the use of fluorescence micro-optical sectioning tomography (fMOST) and single cell RNA sequencing (scRNA-seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of recombinant RABV expressing enhanced green fluorescent protein (EGFP) under different infection routes, and a three-dimensional view of the distribution of RABV in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA-seq. Our fMOST data provide the first evidence that RABV can infect multiple nuclei related to fear independent of different infection routes. More surprisingly, scRNA-seq data indicate that besides neurons RABV can infect macrophages and NK cells in vivo. Collectively, this study draws a comprehensively spatial and cellular map of RABV infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of neurotropic viruses.


title: High diversity and heterogeneity define microbial communities across an active municipal landfill

abstract: Global waste production is increasing rapidly, with the majority of waste destined for landfills. Microbial communities in landfills transform waste and generate methane in an environment unique from other built and natural environments. Previous work has largely considered landfill microbial diversity only at the phylum level, identifying complex and variable communities. The extent of shared organismal diversity across landfills or over time and at more precise levels of classification remains unknown. We used 16S rRNA gene amplicon and metagenomic sequencing to examine the taxonomic and functional diversity of the microbial communities inhabiting a Southern Ontario landfill. The diversity of microbial populations in leachate and groundwater samples was correlated with geochemical conditions to determine drivers of microbial heterogeneity. Across the landfill, 25 bacterial and archaeal phyla were present at >1% relative abundance within at least one landfill sample. The Patescibacteria, Bacteroidota, Firmicutes, and Proteobacteria had the highest relative abundances, with most other phyla present at low (<5%) abundance. Below the phylum level, very few populations were identified at multiple sites, with only 121 of 8,030 populations present at five or more sites. This indicates that, although phylum-level signatures are conserved, individual landfill microbial populations vary widely. Significant differences in geochemistry occurred across the leachate and groundwater wells sampled, with calcium, iron, magnesium, boron, meta and para xylenes, ortho xylenes, and ethylbenzene concentrations contributing most strongly to observed site differences. This study illustrates that leachate microbial communities are much more complex and diverse within landfills than previously reported, with implications for waste management best practices.


title: Comparison of SARS-CoV-2 VOC 202012/01 (UK variant) and D614G variant transmission by different routes in Syrian hamsters

abstract: Many SARS-CoV-2 variants of concern has been reported recently which were linked to increased transmission. In our earlier study on virus shedding using VOC 202012/01(UK variant) and D614G variant in hamster model, we observed significantly higher viral RNA shedding through nasal wash in case of UK variant. Hence, we compared the transmission of both the UK and D614G variant by various routes in Syrian hamsters to understand whether the high viral RNA shedding could enhance the transmission efficiency of the variant. The current study demonstrated comparable transmission efficiency of both UK and D614G variants of SARS-CoV-2 in Syrian hamsters.


title: Thriving of hyperthermophilic microbial communities from a deep-sea sulfidic hydrothermal chimney under electrolithoautotrophic conditions with nitrate as electron acceptor.

abstract: Recent studies have shown the presence of an abiotic electrical current across the walls of deep-sea hydrothermal chimneys, allowing the growth of electroautotrophic microbial communities. To understand the role of the different phylogenetic groups and metabolisms involved, this study focused on an electrotrophic enrichment, with nitrate as electron acceptor. The biofilm density, the community composition, the organic products released, and the electrical consumption were monitored by FISH confocal microscopy, qPCR, Metabarcoding, MNR and potentiostat measurements. A statistic analysis by PCA showed the correlation between the different parameters in 3 distinct temporal phases. The Archaeoglobales have been shown to play a key role in the development of the community, as first colonizers and producing pyruvate, therefor used as organic source for heterotrophs. Some Thermococcales showed the ability to perform electrofermentation of this pyruvate into acetate and H2. Finally, through subcultures of the community, we showed the development of a larger biodiversity over time. This observed phenomenon could explain the biodiversity development in hydrothermal context where energy sources are transient and unstable.


title: Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA

abstract:
DiSulfide Bond forming proteins (DSB) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide bond protein A (DsbA) catalyzes the formation of disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, we identified two fragments - 1 (bromophenoxy propanamide) and 2 (4-methoxy-N-phenylbenzenesulfonamide), that bind to the DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. Crystal structures of the oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 suggests that both fragments bind to a hydrophobic pocket that is formed by a change in the side chain orientation of tyrosine 110. This conformational change opens a "cryptic" pocket that is not evident in the apo-protein structure. This binding location was supported by 2D-NMR studies which identified a chemical shift perturbation of the tyrosine 110 backbone amide resonance of more than 0.05 ppm upon addition of 2 mM of fragment 1 and over 0.04 ppm upon addition of 1 mM of fragment 2. Although binding was detected by both X-ray crystallography and NMR, the binding affinity (KD) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the modelled crystal structures which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have high binding affinity due to their size and the relatively small surface area that can be involved in intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. Identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.

SynopsisDescribes the binding properties of two drug-like fragments to a conformationally dynamic site in the disulfide-bond forming protein A from Burkholderia pseudomallei.

category: molecular biology


title: Beyond fish eDNA metabarcoding: Field replicates disproportionately improve the detection of stream associated vertebrate species

abstract: Fast, reliable, and comprehensive biodiversity monitoring data are needed for environmental decision making and management. Recent work on fish environmental DNA (eDNA) metabarcoding shows that aquatic diversity can be captured fast, reliably, and non-invasively at moderate costs. Because water in a catchment flows to the lowest point in the landscape, often a stream, it can often collect traces of terrestrial species via surface or subsurface runoff along its way or when specimens come into direct contact with water (e.g., for drinking purposes). Thus, fish eDNA metabarcoding data can provide information on fish but also on other vertebrate species that live in riparian habitats. This additional data may offer a much more comprehensive approach for assessing vertebrate diversity at no additional costs. Studies on how the sampling strategy affects species detection especially of stream-associated communities, however, are scarce. We therefore performed an analysis on the effects of biological replication on both fish as well as (semi-)terrestrial species detection. Along a 2 km stretch of the river Mulde (Germany), we collected 18 1-L water samples and analyzed the relation of detected species richness and quantity of biological replicates taken. We detected 58 vertebrate species, of which 25 were fish and lamprey, 18 mammals, and 15 birds, which account for 50%, 24%, and 7% of all native species to the German federal state of Saxony-Anhalt. However, while increasing the number of biological replicates resulted in only 25% more detected fish and lamprey species, mammal, and bird species richness increased disproportionately by 69% and 84%, respectively. Contrary, PCR replicates showed little stochasticity. We thus emphasize to increase the number of biological replicates when the aim is to improve general species detections. This holds especially true, when the focus is on rare aquatic taxa or on (semi-)terrestrial species, the so-called  bycatch. As a clear advantage, this information can be obtained without any additional sampling or laboratory effort when the sampling strategy is chosen carefully. With the increased use of eDNA metabarcoding as part of national fish bioassessment and monitoring programs, the complimentary information provided on bycatch can be used for biodiversity monitoring and conservation on a much broader scale.


title: TNFAIP8 is a novel phosphoinositide-binding inhibitory regulator of Rho GTPases that promotes cancer cell migration

abstract: More than half of human tumors exhibit aberrantly dysregulated phosphoinositide signaling, yet how this is controlled remains not fully understood. While somatic mutations of PI3K, PTEN and Ras account for many cases of the hyperactivated lipid signals, other mechanisms for these dysfunctions in cancer are also being discovered. We report here that TNFAIP8 interacts with PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and is likely to be hijacked by cancer cells to facilitate directional migration during malignant transformation. TNFAIP8 maintains the quiescent cellular state by sequestering inactive Rho GTPases in the cytosolic pool, which can be set free upon chemoattractant activation at the leading edge. Consequently, loss of TNFAIP8 results in severe defects of chemotaxis and adhesion. Thus, TNFAIP8, whose expression can be induced by inflammatory cytokines such as TNF from tumor microenvironment, represents a molecular bridge from inflammation to cancer by linking NF-{kappa}B pathway to phosphoinositide signaling. Our study on the conserved hydrophobic cavity structure will also advise in silico drug screening and development of new TNFAIP8-based strategies to combat malignant human diseases.

category: cell biology


title: HIGH VIRULENCE SELECTS FOR FAST HOST RESISTANCE EVOLUTION

abstract: Parasites and pathogens vary strikingly in their virulence and the resulting selection they impose on their hosts. While the evolution of different virulence levels is well studied, the evolution of host resistance in response to different virulence levels is less understood and as of now mainly based on observations and theoretical predictions with few experimental tests. Increased virulence can increase selection for host resistance evolution if resistance costs are outweighed by the benefits of avoiding infection. To test this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of two variants of the filamentous phage, VALG{Phi}8, that differ in their virulence. The bacterial host exhibited two alternative defence strategies against future phage infection: (1) super infection exclusion (SIE) whereby phage-infected cells were immune to subsequent infection at a cost of reduced growth, and (2) surface receptor mutations (SRM) in genes encoding the MSHA type-IV pilus providing resistance to infection by preventing phage attachment. While SIE emerged rapidly against both phages, SRM evolved faster against the high virulence compared to the low virulence phage. Using a mathematical model of our system we show that increasing virulence strengthens selection for SRM due to the higher costs of infection suffered by SIE immune hosts. In both the experiments and the model, higher levels of SRM in the host population drove more rapid phage extinction. Thus, by accelerating the evolution of host resistance, more virulent phages caused shorter epidemics.

category: evolutionary biology


title: HIGHER VIRAL VIRULENCE SELECTS FOR FASTER EVOLUTION OF HOST RESISTANCE


title: Higher phage virulence accelerates the evolution of host resistance


title: Biochemical and Immuno-Histochemical Localization of Type IIA Procollagen in Annulus Fibrosus of Mature Bovine Intervertebral Disc

abstract: For next generation tissue-engineered constructs and regenerative medicine to succeed clinically, the basic biology and extracellular matrix composition of tissues that these repair techniques seek to restore have to be fully determined. Using the latest reagents coupled with tried and tested methodologies, we continue to uncover previously undetected structural proteins in mature intervertebral disc. In this study we show that the "embryonic" type IIA procollagen isoform (containing a cysteine-rich amino propeptide) was biochemically detectable in the annulus fibrosus of both calf and mature steer intervertebral discs, but not in the nucleus pulposus where the type IIB isoform was predominantly localized. Specifically, the triple-helical type IIA procollagen isoform immunolocalized in the outer margins of the inner annulus fibrosus. Triple helical processed type II collagen exclusively localized within the interlamellae regions and with type IIA procollagen in the intra-lamellae regions. Mass spectrometry of the 1 (II) collagen chains from the region where type IIA procollagen localized showed high 3-hydroxylation of Proline-944, a post-translational modification that is correlated with thin collagen fibrils as in the nucleus pulposus. The findings implicate small diameter fibrils of type IIA procollagen in select regions of the annulus fibrosus where it likely contributes to the organization of collagen bundles and structural properties within the type I-type II collagen transition zone.

category: biochemistry


title: A simple micropreparative gel electrophoresis technique for purification of proteins, nucleic acids, and bioconjugates

abstract: The biochemical and biomedical fields hinge on the ability to effectively separate and purify biological macromolecules. Though this need is largely addressed with a variety of chromatographic and electrophoretic purification techniques, such techniques are usually laborious, time-consuming, and often require complex and costly instalments that are inaccessible to most laboratories. In this work, we introduce a simple micro-preparative (MP) method based on polyacrylamide gel electrophoresis (PAGE) to purify biological samples containing proteins, nucleic acids, and complex bioconjugates. Using a conventional vertical slab system, we demonstrate the extraction of purified DNA, proteins, and DNA-protein bioconjugates from their respective mixtures using MP-PAGE. We apply this system to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using specialized commercial devices. We also demonstrate the purification of folded enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, comparable to purities achieved using a two-step size exclusion and immobilized metal-ion affinity chromatography purification procedure. Finally, we demonstrate the successful isolation of an EYFP-DNA bioconjugate sample that otherwise could not be processed using the two-step chromatography procedure. MP-PAGE thus offers a rapid and versatile means of purifying a variety of biomolecules without the need for specialized equipment.


title: Norstictic acid is a selective allosteric transcriptional regulator

abstract: Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (~900 [A]2) and have little topology, and thus do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprised of a highly dynamic loop flanking one canonical binding surface and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.


title: Systematic Profiling of Temperature- and Retinal-Sensitive Rhodopsin Variants by Deep Mutational Scanning

abstract: Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of "temperature-sensitive" variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and an investigational corrector (9-cis-retinal) on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures is correlated with the response to retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants within this helix appear to disrupt a network of hydrogen bonds that stabilizes a native helical kink. By comparison, mutants that alter a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest this insensitivity primarily arises from an abundance of mutations that enhance its membrane integration, stabilize its native conformation, and/ or perturb the retinal binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings elucidate various factors that mediate the sensitivity of genetic variants to temperature and to small molecule correctors.

category: biophysics


title: The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens

abstract: Vibrio natriegens is known as the worlds fastest growing organism with a doubling time of less than 10 minutes. This incredible growth speed empowers V. natriegens as a chassis for synthetic and molecular biology, potentially replacing E. coli in many applications. While first genetic parts have been built and tested for V. natriegens, a comprehensive toolkit containing well-characterized and standardized parts did not exist. To close this gap, we created the Marburg Collection - a highly flexible Golden Gate Assembly-based cloning toolbox optimized for the emerging chassis organism V. natriegens. The Marburg Collection overcomes the paradigm of plasmid construction - integrating inserts into a backbone - by enabling the de novo assembly of plasmids from basic genetic parts. This allows users to select the plasmid replication origin and resistance part independently, which is highly advantageous when limited knowledge about the behavior of those parts in the target organism is available. Additional design highlights of the Marburg Collection are novel connector parts, which facilitate modular circuit assembly and, optionally, the inversion of individual transcription units to reduce transcriptional crosstalk in multigene constructs. To quantitatively characterize the genetic parts contained in the Marburg Collection in V. natriegens, we developed a reliable microplate reader measurement workflow for reporter experiments and overcame organismspecific challenges. We think the Marburg Collection with its thoroughly characterized parts will provide a valuable resource for the growing V. natriegens community.


title: Sex differences in concentrations of HMGB1 and numbers of pigmented monocytes in infants with malaria

abstract: Females generally mount more robust innate and adaptive immune responses and demonstrate a higher rate of morbidity, and prevalence of autoimmune diseases by comparison with males. In malaria, females demonstrate higher concentrations of antibodies and rates of severe adverse events and mortality following natural infections and malaria vaccination. Although monocytes/macrophages play a crucial role in disease and protection in malaria, no studies have investigated sex differences in their functions in production of proinflammatory cytokines and chemokines in malaria-infected subjects. Here, we show significant sex differences in serum concentrations of HMGB1, a non-histone chromatin-associated protein, and numbers of pigmented monocytes, which are both markers of severe malaria, in infants <5 years old from a malaria endemic region in Northern Uganda. Female infants with clinical malaria had significantly higher HMGB1 concentrations than male infants, and female infants with asymptomatic malaria had significantly lower numbers of pigmented monocytes than male infants with asymptomatic malaria. There was (1) a significant correlation between HMGB1 concentrations and pigmented monocyte numbers in female but not male infants; and (2) a significant correlation between HMGB1 concentrations and parasite densities in female but not male infants. These findings suggest that female infants with clinical malaria might be at a greater risk of morbidity characterized by higher serum HMGB1 levels.


title: Copper intoxication in group B Streptococcus triggers a mis-metallation signature and activation of the cop operon leading to enhanced virulence

abstract:
Bacteria can utilize Copper (Cu) as a trace element to support cellular processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B streptococcus (GBS), and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of GBS mutants deficient in either the copA Cu exporter, or the copY repressor, were severely compromised in Cu-stress conditions. GBS survival of Cu stress reflected a mechanism of CopY de-repression of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA-sequencing revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication, via regulation by the Cu-sensing transcriptional repressor, copY. Cu stress responses in GBS reflect a transcriptional signature that heightens virulence and represents an important part of the bacterias ability to survive in different environments.

ImportanceUnderstanding how bacteria manage cellular levels of metal ions, such as copper, helps to explain how microbial cells can survive in different stressful environments. We show how the opportunistic pathogen group B Streptococcus (GBS) achieves homeostasis of intracellular copper through the activities of the genes that comprise the cop operon, and describe how this helps GBS survive in stressful environments, including in the mammalian host during systemic disseminated infection.


title: Overabundance of Asaia and Serratia bacteria is associated with deltamethrin insecticide susceptibiltiy in Anopheles coluzzii from Agboville, Cote d'Ivoire

abstract:
BackgroundInsecticide resistance among mosquito species is now a pervasive phenomenon, which threatens to jeopardise global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterised and compared the microbiota of Anopheles (An.) coluzzii in relation to their deltamethrin resistance and exposure profiles.

ResultsComparisons between 2-3 day old deltamethrin resistant and susceptible mosquitoes, demonstrated significant differences in microbiota diversity (PERMANOVA, pseudo-F = 19.44, p=0.0015). Ochrobactrum, Lysinibacillus and Stenotrophomonas genera, each of which comprised insecticide degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals (Shannon index: H=13.91, q=0.0003, Faiths phylogenetic diversity: H=6.68, q=0.01), with Asaia and Serratia dominating microbial profiles. There was no significant difference in deltamethrin exposed and unexposed 5-6 day old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. Serratia and Asaia were also dominant in 5-6 day old mosquitoes, regardless of exposure or phenotype, and had reduced microbial diversity compared with 2-3 day old mosquitoes.

ConclusionsOur findings revealed significant alterations of An. coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of Serratia and Asaia was consistent with 16S rRNA sequencing, suggesting that population level field screening of the bacterial microbiota may be feasibly integrated into wider resistance monitoring if reliable and reproducible markers associated with phenotype can be identified.


title: Metagenomic insight into the plant growth-promoting potential of a diesel-degrading bacterial consortium for enhanced rhizoremediation application

abstract: The slow rate of natural attenuation of organic pollutants, together with unwanted environmental impacts of traditional remediation strategies, has necessitated the exploration of plant-microbe systems for enhanced bioremediation applications. The identification of microorganisms capable of promoting both plant growth and hydrocarbon degradation is crucial to the success of plant-based remediation techniques. Through successive enrichments of a soil sample from a historic oil-contaminated site in Wietze, Germany, we isolated a plant growth-promoting and hydrocarbon-degrading bacterial consortium. Metagenome analysis of the consortium led to the identification of genes and taxa putatively associated with these processes. The majority of the coding DNA sequences involved in these reactions were affiliated to Acidocella aminolytica and Acidobacterium capsulatum. In microcosm experiments performed in association with Medicago sativa L., the consortium achieved 91% rhizodegradation of diesel fuel hydrocarbons within 60 days, indicating its potential for biotechnological applications in the remediation of sites contaminated by organic pollutants.


title: Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropod

abstract: Arthropod endosymbiont Wolbachia pipientis is part of a global biocontrol strategy to reduce the replication of mosquito-borne RNA viruses such as alphaviruses. We previously demonstrated the importance of a host cytosine methyltransferase, DNMT2, in Drosophila and viral RNA as a cellular target during pathogen-blocking. Here we report on the role of DNMT2 in Wolbachia-induced alphavirus inhibition in Aedes species. Expression of DNMT2 in mosquito tissues, including the salivary glands, is elevated upon virus infection. Notably, this is suppressed in Wolbachia-colonized animals, coincident with reduced virus replication and decreased infectivity of progeny virus. Ectopic expression of DNMT2 in cultured Aedes cells is proviral, increasing progeny virus infectivity, and this effect of DNMT2 on virus replication and infectivity is dependent on its methyltransferase activity. Finally, examining the effects of Wolbachia on modifications of viral RNA by LC-MS show a decrease in the amount of 5-methylcytosine modification consistent with the down-regulation of DNMT2 in Wolbachia colonized mosquito cells and animals. Collectively, our findings support the conclusion that disruption of 5-methylcytosine modification of viral RNA is a vital mechanism operative in pathogen blocking. These data also emphasize the essential role of epitranscriptomic modifications in regulating fundamental alphavirus replication and transmission processes.


title: Bacterial small RNAs and host epigenetic effectors of a transgenerational memory of pathogens in C. elegans.

abstract: The inheritance of memories is adaptive for survival. Microbes interact with all organisms challenging their immunity and triggering behavioral adaptations. Some of these behaviors induced by bacteria can be inherited although the mechanisms of action are largely unexplored. In this work, we use C. elegans and its bacteria to study the transgenerational RNA dynamics of an interspecies crosstalk leading to a heritable behavior. Heritable responses to bacterial pathogens in the nematode include avoidance and pathogen-induced diapause (PIDF), a state of suspended animation to evade the pathogen threat. We identify a small RNA RsmY, involved in quorum sensing from P. aeruginosa as required for initiation of PIDF. Histone methyltransferase SET-18/SMYD3 is also needed for PIDF initiation in C. elegans. In contrast, SET-25/EHMT2 is necessary for the maintenance of the memory of pathogen exposure in the transgenerational lineage. This work can be a starting point to understanding microbiome-induced inheritance of acquired traits.


title: RNA chaperones Hfq and ProQ play a key role in the virulence of the plant pathogenic bacterium Dickeya dadantii

abstract: Dickeya dadantii is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the transcription of its genes encoding the main virulence functions, little information is available on the post-transcriptional regulation of these functions. We investigated the involvement of the RNA chaperones Hfq and ProQ in the production of the main D. dadantii virulence functions. Phenotypic assays on the hfq and proQ mutants showed that inactivation of hfq resulted in a growth defect, a modified capacity for biofilm formation and strongly reduced motility, and in the production of degradative extracellular enzymes (proteases, cellulase and pectate lyases). Accordingly, the hfq mutant failed to cause soft rot on chicory leaves. The proQ mutant had reduced resistance to osmotic stress, reduced extracellular pectate lyase activity compared to the wild-type strain, and reduced virulence on chicory leaves. Most of the phenotypes of the hfq and proQ mutants were related to the low amounts of mRNA of the corresponding virulence factors. Complementation of the double mutant hfq-proQ by each individual protein and cross-complementation of each chaperone suggested that they might exert their effects via partially overlapping but different sets of targets. Overall, it clearly appeared that the two Hfq and ProQ RNA chaperones are important regulators of pathogenicity in D. dadantii. This underscores that virulence genes are regulated post transcriptionally by non-coding RNAs.


title: Genetic Variant in 3' Untranslated Region of the Mouse Pycard Gene Regulates Inflammasome Activity

abstract: Quantitative trait locus mapping for interleukin-1{beta} release after inflammasome priming and activation was performed on bone marrow-derived macrophages (BMDM) from an AKRxDBA/2 strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at single nucleotide polymorphism (SNP) in their 3 untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels of Pycard mRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increased Pycard mRNA stability without an increased transcription rate. CRIPSR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change the Pycard 3UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reduced Pycard expression and inflammasome activity after cells were differentiated into macrophages due to reduced Pycard mRNA stability.

category: genetics


title: Recurrent chromosomal translocations in sarcomas create a mega-complex that mislocalizes NuA4/TIP60 to Polycomb target loci

abstract: Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with Endometrial Stromal Sarcomas (ESS) and Ossifying FibroMyxoid Tumors (OFMT) leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a co-activator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a mega-complex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically- associating domain including part of the HOXD cluster. This is linked to aberrant gene expression, most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1, implicated with a PRC2 component in the most frequent translocation in ESS, JAZF1-SUZ12, is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating similar outcomes as EPC1-PHF1. Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas employ the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes leading to mislocalization of histone marks and aberrant polycomb target gene expression.


title: Development and Validation of Fluorescently Labeled, Functional Type I Collagen Molecules

abstract: While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are quantified for the first time. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ~1.2 labels preserves them. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labelled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development, is endocytosed rapidly by cells and is translocated into synthesized matrix networks within 24 hours.


title: STRESS, an automated geometrical characterization of deformable particles for in vivo measurements of cell and tissue mechanical stresses

abstract:
From cellular mechanotransduction to the formation of embryonic tissues and organs, mechanics has been shown to play an important role in the control of cell behavior and embryonic development. Most of our existing knowledge of how mechanics affects cell behavior comes from in vitro studies, mainly because measuring cell and tissue mechanics in 3D multicellular systems, and especially in vivo, remains challenging. Oil microdroplet sensors, and more recently gel microbeads, use surface deformations to directly quantify mechanical stresses within developing tissues, in vivo and in situ, as well as in 3D in vitro systems like organoids or multicellular spheroids. However, an automated analysis software able to quantify the spatiotemporal evolution of stresses and their characteristics from particle deformations is lacking. Here we develop STRESS (Surface Topography Reconstruction for Evaluation of Spatiotemporal Stresses), an analysis software to quantify the geometry of deformable particles of spherical topology, such as microdroplets or gel microbeads, that enables the automatic quantification of the temporal evolution of stresses in the system and the spatiotemporal features of stress inhomogeneities in the tissue. As a test case, we apply these new code to measure the temporal evolution of mechanical stresses using oil microdroplets in developing zebrafish tissues. Starting from a 3D timelapse of a droplet, the software automatically calculates the statistics of local anisotropic stresses, decouples the deformation modes associated with tissue- and cell-scale stresses, obtains their spatial features on the droplet surface and analyzes their spatiotemporal variations using spatial and temporal stress autocorrelations. The automated nature of the analysis will help users obtain quantitative information about mechanical stresses in a wide range of 3D multicellular systems, from developing embryos or tissue explants to organoids.

Author summaryThe measurement of mechanical stresses in 3D multicellular systems, such as living tissues, has been very challenging because of a lack in technologies for this purpose. Novel microdroplet techniques enable direct, quantitative in situ measurements of mechanical stresses in these systems. However, computational tools to obtain mechanical stresses from 3D images of microdroplets in an automated and accurate manner are lacking. Here we develop STRESS, an automated analysis software to analyze the spatiotemporal characteristics of mechanical stresses from microdroplet deformations in a wide range of systems, from living embryonic tissues and tissue explants to organoids and multicellular spheroids.


title: The anthropometric and physical qualities of womens rugby league Super League and international players; identifying differences in playing position and standard

abstract: Participation in womens rugby league has been growing since the foundation of the English womens rugby league Super League in 2017. However, the evidence base to inform womens rugby league remains sparse. This study provides the largest quantification of anthropometric and physical qualities of womens rugby league players to date, identifying differences between positions (forwards & backs) and playing standard (Womens Super League [WSL] vs. International). The height, weight, body composition, lower body strength, jump height, speed and aerobic capacity of 207 players were quantified during the pre-season period. Linear mixed models and effects sizes were used to determine differences between positions and standards. Forwards were significantly (p < 0.05) heavier (forwards: 82.5 {+/-} 14.8kg; backs: 67.7 {+/-} 9.2kg) and have a greater body fat % (forwards: 37.7 {+/-} 6.9%; backs: 30.4 {+/-} 6.3%) than backs. Backs had significantly greater lower body power measured via jump height (forwards: 23.5 {+/-} 4.4cm; backs: 27.6 {+/-} 4.9cm), speed over 10m (forwards: 2.12 {+/-} 0.14s; backs: 1.98 {+/-} 0.11s), 20m (forwards: 3.71 {+/-} 0.27s; backs: 3.46 {+/-} 0.20s), 30m (forwards: 5.29 {+/-} 0.41s; backs: 4.90 {+/-} 0.33s), 40m (forwards: 6.91 {+/-} 0.61s; backs: 6.33 {+/-} 0.46s) and aerobic capacity (forwards: 453.4 {+/-} 258.8m; backs: 665.0 {+/-} 298.2m) than forwards. Additionally, international players were found to have greater anthropometric and physical qualities in comparison to their WSL counterparts. This study adds to the limited evidence base surrounding the anthropometric and physical qualities of elite womens rugby league players. Comparative values for anthropometric and physical qualities are provided which practitioners may use to evaluate the strengths and weaknesses of players, informing training programs to prepare players for the demands of womens rugby league.

category: physiology


title: Drift as a driver of language change: An artificial language experiment

abstract: Over half a century ago, George Zipf observed that more frequent words tend to be older. Corpus studies since then have confirmed this pattern, with more frequent words being replaced and regularized less often than less frequent words. Two main hypotheses have been proposed to explain this: that frequent words change less because selection against innovation is stronger at higher frequencies, or that they change less because stochastic drift is stronger at lower frequencies. Here, we report the first experimental test of these hypotheses. Participants were tasked with learning a miniature language consisting of two nouns and two plural markers. Nouns occurred at different frequencies and were subjected to treatments that varied drift and selection. Using a model that accounts for participant heterogeneity, we measured the rate of noun regularization, the strength of selection, and the strength of drift in participant responses. Results suggest that drift alone is sufficient to generate the elevated rate of regularization we observed in low-frequency nouns, adding to a growing body of evidence that drift may be a major driver of language change.


title: Massive loss of transcription factors and the initial diversification of placental mammals

abstract: As one of the most successful categories of organisms, mammals occupy a variety of niches on earth as a result of macroevolution. Transcription factors (TFs), the basic regulators of gene expression, may also evolve during mammalian phenotypic diversification and macroevolution. To examine the relationship between TFs and mammalian macroevolution, we analyzed 140,821 de novo-identified TFs and their birth and death histories from 96 mammalian species. Gene tree vs. species tree reconciliation revealed that mammals experienced an upsurge in TF losses around 100 million years ago and also near the K-Pg boundary, thus implying a relationship with the divergence of placental animals. From approximately 100 million years ago to the present, losses dominated TF events without a significant change in TF gains. To quantify the effects of this TF pruning on mammalian macroevolution, we analyzed rates of molecular evolution and expression profiles of regulated target genes. Surprisingly, TF loss decelerated, rather than accelerated, molecular evolutionary rates of their target genes, suggesting increased functional constraints. Furthermore, an association study revealed that massive TF losses are significantly positively correlated with solitary behavior, nocturnality, reproductive-seasonality and insectivory life history traits, possibly through rewiring of regulatory networks.


title: SongExplorer: A deep learning workflow for discovery and segmentation of animal acoustic communication signals

abstract: Many animals produce distinct sounds or substrate-borne vibrations, but these signals have proved challenging to segment with automated algorithms. We have developed SongExplorer, a web-browser based interface wrapped around a deep-learning algorithm that supports an interactive workflow for (1) discovery of animal sounds, (2) manual annotation, (3) supervised training of a deep convolutional neural network, and (4) automated segmentation of recordings. Raw data can be explored by simultaneously examining song events, both individually and in the context of the entire recording, watching synced video, and listening to song. We provide a simple way to visualize many song events from large datasets within an interactive low-dimensional visualization, which facilitates detection and correction of incorrectly labelled song events. The machine learning model we implemented displays higher accuracy than existing heuristic algorithms and similar accuracy as two expert human annotators. We show that SongExplorer allows rapid detection of all song types from new species and of novel song types in previously well-studied species.

category: animal behavior and cognition


title: A Statistical Model for Describing and Simulating Microbial Community Profiles

abstract: Many methods have been developed for statistical analysis of microbial community profiles, but due to the complex nature of typical microbiome measurements (e.g. sparsity, zero-inflation, nonindependence, and compositionality) and of the associated underlying biology, it is difficult to compare or evaluate such methods within a single systematic framework. To address this challenge, we developed SparseDOSSA (Sparse Data Observations for the Simulation of Synthetic Abundances): a statistical model of microbial ecological population structure, which can be used to parameterize real-world microbial community profiles and to simulate new, realistic profiles of known structure for methods evaluation. Specifically, SparseDOSSAs model captures marginal microbial feature abundances as a zero-inflated log-normal distribution, with additional model components for absolute cell counts and the sequence read generation process, microbemicrobe, and microbe-environment interactions. Together, these allow fully known covariance structure between synthetic features (i.e. "taxa") or between features and "phenotypes" to be simulated for method benchmarking. Here, we demonstrate SparseDOSSAs performance for 1) accurately modeling human-associated microbial population profiles; 2) generating synthetic communities with controlled population and ecological structures; 3) spiking-in true positive synthetic associations to benchmark analysis methods; and 4) recapitulating an end-to-end mouse microbiome feeding experiment. Together, these represent the most common analysis types in assessment of real microbial community environmental and epidemiological statistics, thus demonstrating SparseDOSSAs utility as a general-purpose aid for modeling communities and evaluating quantitative methods. An open-source implementation is available at http://huttenhower.sph.harvard.edu/sparsedossa2.


title: Pseudohydrosme bogneri sp. nov. (Araceae), a spectacular Critically Endangered (Possibly Extinct) species from Gabon, long confused with Anchomanes nigritianus.

abstract: A Gabonese taxon cultivated for decades in the botanic gardens of Europe as Anchomanes nigritianus is shown to be a new species to science, and on current evidence, is best placed as the fourth species of the Gabonese-centred, poorly known genus Pseudohydrosme. Data on the morphological separation between Anchomanes and Pseudohydrosme are reviewed. Although phylogenomic studies may show in future that the two genera need to be merged, for the moment their separation is reinforced on morphological grounds. Anchomanes lacks the spathe tube, ovoid-globose, 2 - 4 locular pistil and thick, lobed stigma on a symmetric, stout style that we show to characterise the redelimited Pseudohydrosme. (Anchomanes has oblong, polygonal, 1-locular pistils, stigmas asymmetric, sessile, thin and disc-like or on asymmetrical conical styles and are pointed or brush-like). In addition, Pseudohydrosme (where known) has stipitate (versus sessile) fruits and on current evidence lacks the lacticifers recorded from Anchomanes. We test the hypothesis that the taxon is a new species to science, naming it as Pseudohydrosme bogneri, and conclude that it is Critically Endangered (Possibly Extinct) using the IUCN 2012 standard. Pseudohydrosme bogneri appears to be the tenth documented probable global extinction of a plant species that has occurred among the narrowly endemic plant species of the Libreville area, Gabon.

category: plant biology


title: Functional selectivity for naturalistic social interaction perception in the human superior temporal sulcus

abstract:
Recognizing others social interactions is a crucial human ability. Using simple stimuli, previous studies have shown that social interactions are selectively processed in the superior temporal sulcus (STS), but prior work with movies has suggested that social interactions are processed in the medial prefrontal cortex (mPFC), part of the theory of mind network. It remains unknown to what extent social interaction selectivity is observed in real world stimuli when controlling for other covarying perceptual and social information, such as faces, voices, and theory of mind. The current study utilizes a functional magnetic resonance imaging (fMRI) movie paradigm and advanced machine learning methods to uncover the brain mechanisms uniquely underlying naturalistic social interaction perception. We analyzed two publicly available fMRI datasets, collected while both male and female human participants (n = 17 and 18) watched two different commercial movies in the MRI scanner. By performing voxel-wise encoding and variance partitioning analyses, we found that broad social-affective features predict neural responses in social brain regions, including the STS and mPFC. However, only the STS showed robust and unique selectivity specifically to social interactions, independent from other covarying features. This selectivity was observed across two separate fMRI datasets. These findings suggest that naturalistic social interaction perception recruits dedicated neural circuity in the STS, separate from the theory of mind network, and is a critical dimension of human social understanding.

Significance StatementSocial interaction perception guides our daily behavior, yet it is unknown how our brain processes real-world social interaction scenes. Here, we demonstrate that social brain areas, including the superior temporal sulcus (STS) and medial prefrontal cortex (mPFC), are sensitive to broad social-affective information in naturalistic movies, replicating prior results with controlled paradigms. We show for the first time however, that the STS, but not mPFC, selectively processes social interactions in natural movies, independent of other co-occurring perceptual and social features, including motion, presence of faces, speech, and mentalizing about others. Our results suggest that social interaction perception is a crucial dimension of social understanding represented in the human brain.

category: neuroscience


title: Your pleasure is mine; when people share amusical emotional experience during a livemusic performance in a concert hall.

abstract: Over the years, several publications have proposed that musical sound could be an ancestral emotional way of communication, thus positing an ancestral biological function for music. Understanding how musical emotions, and the pleasure derived from music regardless of the musical valence, can be shared between individuals is a fascinating question, and investigating it can shed light on the function of musical reward. Is the pleasure felt at the individual level transmitted on a collective level? And if so, how? We investigated these questions in a natural setting during an international competition for orchestra conductors. Participants (n=15) used a dedicated smartphone app to report their subjective emotional experiences in real time during a concert. We recorded participants electrodermal activity (EDA) and cerebral activity with electroencephalography (EEG). The overall behavioral real time ratings suggest a possible social influence on the reported and felt pleasure. The physically closer the participants, the more similar their reported pleasure. We estimated the inter-individual cerebral coherence, which indicates the degree of mutual cerebral information between pairs of participants in the frequency domain. The results show that when people simultaneously reported either high or low pleasure, their cerebral activities were closer than for simultaneous neutral pleasure reports. Participants skin conductance levels were also more coupled when reporting higher emotional degrees simultaneously. More importantly, the participants who were physically closer had higher cerebral coherence, but only when they simultaneously reported intense pleasure. We propose that mechanisms of emotional contagion and/or emotional resonance could explain why a form of  emotional connecting force could arise between people.


title: Purkinje Cell Activity During Suppression of Voluntary Eye Movements in Rhesus Macaques

abstract:
Volitional suppression of responses to distracting external stimuli enables us to achieve our goals. This volitional inhibition of a specific behavior is supposed to be mainly mediated by the cerebral cortex. However, recent evidence supports the involvement of the cerebellum in this process. It is currently not known whether different parts of the cerebellar cortex play differential or synergistic roles in planning and execution of this behavior. Here, we measured Purkinje cell (PC) responses in the medial and lateral cerebellum in two rhesus macaques during a pro- and antisaccade task. During an antisaccade trial, non-human primates were instructed to make a saccadic eye movement away from a target, rather than towards it, as in prosaccade trials. Our data shows that the cerebellum plays an important role not only during execution of the saccades, but also during the volitional inhibition of eye movements towards the target. Simple Spike (SS) modulation during the instruction and execution period of pro- and antisaccades was prominent in PCs of both medial and lateral cerebellum. However, only the SS activity in the lateral cerebellar cortex contained information about trial identity and showed a stronger reciprocal interaction with complex spikes. Moreover, SS activity of different PC groups modulated bidirectionally in both regions, but the PCs that showed facilitating and suppressive activity were predominantly associated with instruction and execution, respectively. These findings show that different cerebellar regions and PC groups contribute to goal-directed behavior and volitional inhibition, but with different propensities, highlighting the rich repertoire of cerebellar control in executive functions.

Significance StatementThe antisaccade task is commonly used in research and clinical evaluation as a test of volitional and flexible control of behavior. It requires volitional suppression of prosaccades, a function that has been attributed to the neocortex. However, recent findings indicate that cerebellum also contributes to this behavior. We recorded from neurons in the medial and lateral cerebellum to evaluate their responses in this task. We found that both regions significantly modulated their activity during this task, but only cells in the lateral cerebellum encoded the stimulus identity in each trial. These results indicate that the cerebellum actively contributes to the control of flexible behavior and that lateral and medial cerebellum play different roles during volitional eye movements.


title: Purkinje Cell Activity in Medial and Lateral Cerebellum During Suppression of Voluntary Eye Movements in Rhesus Macaques


title: Epithelial Regeneration Ability of Crohn's Disease Assessed Using Patient-Derived Intestinal Organoids

abstract:
BackgroundThe intrinsic limitation of cell lines and animal models limits our understanding of epithelial regeneration capability in Crohns disease (CD). Therefore, we aimed to study epithelial regeneration ability in CD using an intestinal organoid model. Further, since tumor necrosis factor alpha (TNF) is a major proinflammatory effector during CD pathogenesis, we also investigated TNF-induced alteration of regeneration ability in CD patient-derived intestinal organoids.

MethodsHuman intestinal organoids were constructed in a three-dimensional intestinal crypt culture of enteroscopic biopsy samples from control subjects and patients with CD. The epithelial regeneration ability of intestinal organoids was assessed using organoid reconstitution, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), and wound healing assays.

ResultsEx vivo cultures of ileal crypt cells revealed that organoid formation rate of CD patients were reduced compared with that of control subjects (p <.001). CD patient-derived organoids sub-cultured for more than 6 passages showed stable organoid reconstitution and identical morphological features. The organoid constitution and MTT assay revealed that the viability of TNF-treated CD patient-derived organoids were significantly lower than that of TNF-treated control organoids (p <.05 for each). The number of EdU+ proliferative cells was significantly lower in TNF-treated CD patient-derived organoids than in TNF-treated control organoids (p <.05). The wound-healing ability of TNF-treated CD patient-derived organoids was significantly lower than that of TNF-treated control organoids (p <.001).

ConclusionsThe clinical trials are disabled to settle this issue, our results indicated that the epithelial regenerative ability is impaired in patients with CD, especially in TNF-enriched condition.


title: In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M

abstract:
Photoswitchable reagents to modulate microtubule stability and dynamics are an exciting tool approach towards micron- and millisecond-scale control over endogenous cytoskeleton-dependent processes. When these reagents are globally administered yet locally photoactivated in 2D cell culture, they can exert precise biological control that would have great potential for in vivo translation across a variety of research fields and for all eukaryotes. However, photopharmacologys reliance on the azobenzene photoswitch scaffold has been accompanied by a failure to translate this temporally- and cellularly-resolved control to 3D models or to in vivo applications in multi-organ animals, which we attribute substantially to the metabolic liabilities of azobenzenes.

Here, we optimised the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based instead on the styrylbenzothiazole (SBT) photoswitch scaffold, that are non-responsive to the major fluorescent protein imaging channels and so enable multiplexed imaging studies. We applied these reagents to 3D systems (organoids, tissue explants) and classic model organisms (zebrafish, clawed frog) with one- and two-protein imaging experiments. We successfully used systemic treatment plus spatiotemporally-localised illuminations in vivo to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in these systems with cellular precision and second-level resolution. These nanomolar, in vivo-capable photoswitchable reagents can prove a game-changer for high-precision cytoskeleton research in cargo transport, cell motility, cell division and development. More broadly, their straightforward design can also inspire the development of similarly capable optical reagents for a range of protein targets, so bringing general in vivo photopharmacology one step closer to productive realisation.


title: Plasma membrane damage limits replicative lifespan in yeast and human fibroblasts

abstract: Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. Here, using yeast and human fibroblasts, we show that cellular senescence, irreversible cell cycle arrest contributing to organismal aging, is the third outcome of PMD. To identify the genes essential for PMD response, we performed a systematic yeast genome-wide screen. The screen identified 48 genes. The top hits in the screen are the endosomal sorting complexes required for transport (ESCRT) genes. Strikingly, the replicative lifespan regulator genes are enriched in our 48 hits, and indeed, PMD limits the replicative lifespan in budding yeast; the ESCRT activator AAA-ATPase VPS4-overexpression extends it. In normal human fibroblasts, PMD induces premature senescence via p53. Our study demonstrates that PMD limits replicative lifespan in two different eukaryotic cell types and highlights an underappreciated but ubiquitous senescent cell subtype, namely PMD-dependent senescent cells.


title: Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts


title: Curvature sensing steers actin-driven cell migration

abstract: Cell migration is a hallmark out-of-equilibrium process in biology. In addition to persistent self-propelled motion, many cells display remarkable adaptive behaviors when they navigate complex environments within the body. Combining theory and experiments, we identify a curvature-sensing mechanism underlying obstacle avoidance in immune-like cells. The genetic perturbation of this machinery leads to a reduced capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. We propose that the active polymerization of the actin cytoskeleton at the advancing edge of migrating cells is locally inhibited by the curvature-sensitive BAR protein Snx33 in regions with inward plasma membrane curvature. This coupling between actin and membrane dynamics leads to a mechanochemical instability that generates complex protrusive patterns at the cellular front. Adaptive motility thus arises from two simultaneous curvature-dependent effects, i) the specific reduction of propulsion in regions where external objects deform the plasma membrane and ii) the intrinsic patterning capacity due to the membrane-actin coupling that promotes spontaneous changes in the cells protrusions. Our results show how cells utilize actin- and plasma membrane biophysics to sense their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment.


title: Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles


title: Higher-order interactions among coinfecting parasites and a microbial mutualist impact disease progression

abstract: Interactions among parasites and other microbes within hosts can impact disease progression, yet study of such interactions has been mostly limited to pairwise combinations of microbes. Given the diversity of microbes within hosts, higher-order interactions among more than two microbial species may also impact disease. To test this hypothesis, we performed inoculation experiments that investigated interactions among two fungal parasites, Rhizoctonia solani and Colletotrichum cereale, and a systemic fungal endophyte, Epichloe coenophiala, within a grass host. Both pairwise and higher-order interactions impacted disease progression. While the endophyte did not directly influence R. solani growth or C. cereale symptom development, the endophyte modified the interaction between the two parasites. The magnitude of the facilitative effect of C. cereale on the growth of R. solani tended to be greater when the endophyte was present. Moreover, this interaction modification strongly affected leaf mortality. For plants lacking the endophyte, parasite co-inoculation did not increase leaf mortality compared to single-parasite inoculations. In contrast, for endophyte-infected plants, parasite co-inoculation increased leaf mortality compared to inoculation with R. solani or C. cereale alone by 1.9 or 4.9 times, respectively. Together, these results show that disease progression can be strongly impacted by higher-order interactions among microbial symbionts.

category: ecology


title: TOWARDS QUANTITATIVE FORECASTING OF SPECIES YIELDS WITH INCOMPLETE INFORMATION ON MODEL PARAMETERS

abstract:
Predicting both the absolute and the relative abundance of species in a spatial patch is of paramount interest in areas like, agriculture, ecology and environmental science.

The linear Lotka-Volterra generalized equations (LLVGE) serve for describing the dynamics of communities of species connected by negative as well as positive interspecific interactions.

Here we particularize these LLVGE to the case of single trophic ecological communities, like mixtures of plants, with S >2 species. Thus, by estimating the LLVGE parameters from the yields in monoculture and biculture experiments, the LLVGE are able to produce decently accurate predictions for species yields.

However, a common situation we face is that we dont know all the parameters appearing in the LLVGE. Indeed, for large values of S, only a fraction of the experiments necessary for estimating the model parameters is commonly carried out. We then analyze which quantitative predictions are possible with an incomplete knowledge of the parameters.


title: Effects of stochasticity on the length and behaviour of ecological transients

abstract: There is a growing recognition that ecological systems can spend extended periods of time far away from an asymptotic state, and that ecological understanding will therefore require a deeper appreciation for how long ecological transients arise. Recent work has defined classes of deterministic mechanisms that can lead to long transients. Given the ubiquity of stochasticity in ecological systems, a similar systematic treatment of transients that includes the influence of stochasticity is important. Stochasticity can of course promote the appearance of transient dynamics by preventing systems from settling permanently near their asymptotic state, but stochasticity also interacts with deterministic features to create qualitatively new dynamics. As such, stochasticity may shorten, extend, or fundamentally change a systems transient dynamics. Here, we describe a general framework that is developing for understanding the range of possible outcomes when random processes impact the dynamics of ecological systems over realistic time scales. We emphasize that we can understand the ways in which stochasticity can either extend or reduce the lifetime of transients by studying the interactions between the stochastic and deterministic processes present, and we summarize both the current state of knowledge and avenues for future advances.


title: Differential effects of loss of park7 activity on Iron Responsive Element (IRE) gene sets: Implications for the role of iron dyshomeostasis in the pathophysiology of Parkinson's disease

abstract: Mutation of the gene PARK7 (DJ1) causes monogenic autosomal recessive Parkinsons disease (PD) in humans. Subsequent alterations of PARK7 protein function lead to mitochondrial dysfunction, a major element in PD pathology. Homozygous mutants for the PARK7-orthologous genes in zebrafish, park7, show changes to gene expression in the oxidative phosphorylation pathway, supporting that disruption of energy production is a key feature of neurodegeneration in PD. Iron is critical for normal mitochondrial function, and we have previously used bioinformatic analysis of IRE-bearing transcripts in brain transcriptomes to find evidence supporting the existence of iron dyshomeostasis in Alzheimers disease. Here, we analysed IRE-bearing transcripts in the transcriptome data from homozygous park7-/- mutant zebrafish brains. We found that the set of genes with "high quality" IREs in their 5 untranslated regions (UTRs, the HQ5IRE gene set) was significantly altered in these 4-month-old park7-/- brains. However, sets of genes with IREs in their 3 UTRs appeared unaffected. The effects on HQ5IRE genes are possibly driven by iron dyshomeostasis and/or oxidative stress, but illuminate the existence of currently unknown mechanisms with differential overall effects on 5 and 3 IREs.


title: Apparent sunk cost effect in rational agents

abstract: Rational decision makers aim to maximize their gains, but humans and other animals often fail to do so, exhibiting biases and distortions in their choice behavior. In a recent study of economic decisions, humans, mice, and rats have been reported to succumb to the sunk cost fallacy, making decisions based on irrecoverable past investments in detriment of expected future returns. We challenge this interpretation because it is subject to a statistical fallacy, a form of attrition bias, and the observed behavior can be explained without invoking a sunk cost-dependent mechanism. Using a computational model, we illustrate how a rational decision maker with a reward-maximizing decision strategy reproduces the reported behavioral pattern and propose an improved task design to dissociate sunk costs from fluctuations in decision valuation. Similar statistical confounds may be common in analyses of cognitive behaviors, highlighting the need to use causal statistical inference and generative models for interpretation.


title: Pellaea zygophylla, a new combination for a distinctive & well-known but neglected fern.

abstract: Pellaea ovata is a widespread species, sexual diploid in Texas &northeastern Mexico but an apogamous triploid in northwestern Mexico, south to northern Argentina, &on Hispaniola. The type belongs to the southern, apogamous triploid form. Although these two forms have been discussed repeatedly in the literature, morphological distinctions between them have been overlooked and they have not been recognized taxonomically. However, they are distinct. Pellaea ovata s.s. has puberulent rachides & costae; pinnae usually 2-pinnate with a well-defined main axis &pinnules borne singly; fertile pinnules ovate, cordate basally &rounded apically. The sexual diploid form has rachides &costae glabrous; pinnae pseudo-dichotomously branched &pinnules usually paired; fertile pinnules narrowly rounded-trapeziform, obliquely truncate to cordate basally &truncate apically. Riddell named the sexual diploid form Pteris zygophylla, from which I give it the new combination Pellaea zygophylla.


title: Inferring cell cycle phases from a partially temporal network of protein interactions

abstract: The temporal organisation of biological systems into phases and subphases is often crucial to their functioning. Identifying this multiscale organisation can yield insight into the underlying biological mechanisms at play. To date, however, this identification requires a priori biological knowledge of the system under study. Here, we recover the temporal organisation of the cell cycle of budding yeast into phases and subphases, in an automated way. To do so, we model the cell cycle as a partially temporal network of protein-protein interactions (PPIs) by combining a traditional static PPI network with protein concentration or RNA expression time series data. Then, we cluster the snapshots of this temporal network to infer phases, which show good agreement with our biological knowledge of the cell cycle. We systematically test the robustness of the approach and investigate the effect of having only partial temporal information. Our results show for the first time that a temporal network with only partial temporal information, i.e. for some of the PPIs, is sufficient to infer the temporal organization of a system. The generality of the method makes it suitable for application to other, less well-known biological systems for which the temporal organisation of processes plays an important role.

category: systems biology


title: Sex disparities in influenza: a multiscale network analysis

abstract: Sex differences in the pathogenesis of infectious diseases due to differential immune responses between females and males have been well documented for multiple pathogens. However, the molecular mechanism underlying the observed sex differences in influenza virus infection remains poorly understood. In this study, we used a network-based approach to characterize the blood transcriptome collected over the course of infection with influenza A virus from female and male ferrets to dissect sex-biased gene expression. We identified significant differences in the temporal dynamics and regulation of immune responses between females and males. Our results elucidate sex-differentiated pathways involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory responses, including a female-biased IRE1/XBP1 activation and male-biased crosstalk between metabolic reprogramming and IL-1 and AP-1 pathways. Overall, our study provides molecular insights into sex differences in transcriptional regulation of immune responses and contributes to a better understanding of sex bias in influenza pathogenesis.


title: Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon

abstract: The translocon SecYEG forms the primary protein-conducting channel in the cytoplasmic membrane of bacteria, and the associated ATPase SecA provides the energy for the transport of secretory and cell envelope protein precursors. The translocation requires negative charge at the lipid membrane surface, but its dependence on the properties of the membrane hydrophobic core is not known. Here, we demonstrate that SecA:SecYEG-mediated protein transport is immensely stimulated by unsaturated fatty acids (UFAs). Furthermore, UFA-rich tetraoleoyl-cardiolipin, but not bis(palmitoyloleoyl)-cardiolipin, facilitate the translocation via the monomeric translocon. Biophysical analysis and molecular dynamics simulations show that UFAs determine the loosely packed membrane interface, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced manifold at elevated ionic strength. Tight SecA:lipid interactions convert into the augmented translocation. As bacterial cells actively change their membrane composition in response to their habitat, the modulation of SecA:SecYEG activity via the fatty acids may be crucial for protein secretion over a broad range of environmental conditions.


title: Low-cost drug discovery with engineered E. coli reveals an anti-mycobacterial activity of benazepril

abstract:
Whole-cell screening for Mycobacterium tuberculosis (Mtb) inhibitors is complicated by the pathogens slow growth and biocontainment requirements. Here we present a synthetic biology framework for assaying Mtb drug targets in engineered E. coli. We construct Target Essential Surrogate E. coli (TESEC) in which an essential metabolic enzyme is deleted and replaced with an Mtb-derived functional analog, linking bacterial growth to the activity of the target enzyme. High throughput screening of a TESEC model for Mtb alanine racemase (ALR) revealed benazepril as a targeted inhibitor. In vitro biochemical assays indicated a noncompetitive mechanism unlike that of clinical ALR inhibitors. This is the first report of an antimicrobial activity in an approved Angiotensin Converting Enzyme (ACE) inhibitor and may explain clinical data associating use of ACE inhibitors with reduced Mtb infection risk. We establish the scalability of TESEC for drug discovery by characterizing TESEC strains for four additional targets.

SIGNIFICANCE STATEMENTThe challenge of discovering new antibiotics is both scientific and economic. No simple test can determine if a given molecule will be safe and effective in real human patients. Many drug candidates must therefore be advanced for each new antibiotic that reaches the market - a risky and expensive process.

In this work we use synthetic biology to engineer the common laboratory model bacterium E. coli as a tool for early stage antibiotic discovery. As a proof of concept we expressed a known tuberculosis drug target and found a novel inhibitor: benazepril.

Many other drug targets could be screened similarly using the system that we describe. Because E. coli can be grown safely and cheaply, this approach may help to reduce costs and make drug discovery more accessible.


title: Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants

abstract:
Vaccines that induce potent neutralizing antibody (NAb) responses against emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavirus disease 2019 (COVID-19) pandemic. We demonstrated that mouse plasma induced by self-assembling protein nanoparticles (SApNPs) that present 20 rationally designed S2G{Delta}HR2 spikes of the ancestral Wuhan-Hu-1 strain can neutralize the B.1.1.7, B.1.351, P.1, and B.1.617 variants with the same potency. The adjuvant effect on vaccine-induced immunity was investigated by testing 16 formulations for the multilayered I3-01v9 SApNP. Using single-cell sorting, monoclonal antibodies (mAbs) with diverse neutralization breadth and potency were isolated from mice immunized with the receptor binding domain (RBD), S2G{Delta}HR2 spike, and SApNP vaccines. The mechanism of vaccine-induced immunity was examined in mice. Compared with the soluble spike, the I3-01v9 SApNP showed 6-fold longer retention, 4-fold greater presentation on follicular dendritic cell dendrites, and 5-fold stronger germinal center reactions in lymph node follicles.

ONE-SENTENCE SUMMARYWith a well-defined mechanism, spike nanoparticle vaccines can effectively counter SARS-CoV-2 variants.


title: The RNA sensor MDA5 detects SARS-CoV-2 infection

abstract: Human cells respond to infection by SARS-CoV-2, the virus that causes COVID-19, by producing cytokines including type I and III interferons (IFNs) and proinflammatory factors such as IL6 and TNF. IFNs can limit SARS-CoV-2 replication but cytokine imbalance contributes to severe COVID-19. We studied how cells detect SARS-CoV-2 infection. We report that the cytosolic RNA sensor MDA5 was required for type I and III IFN induction in the lung cancer cell line Calu-3 upon SARS-CoV-2 infection. Type I and III IFN induction further required MAVS and IRF3. In contrast, induction of IL6 and TNF was independent of the MDA5-MAVS-IRF3 axis in this setting. We further found that SARS-CoV-2 infection inhibited the ability of cells to respond to IFNs. In sum, we identified MDA5 as a cellular sensor for SARS-CoV-2 infection that induced type I and III IFNs.


title: Antidepressants are complex regulators of lipoprotein(a) macropinocytosis

abstract:
Multiple receptors have been implicated in lipoprotein(a) (Lp(a)) uptake, yet each receptor only partially accounts for the uptake, and no endocytic pathway has been described for Lp(a) internalisation to date. In this study, we define macropinocytosis as the endocytic pathway responsible for internalising Lp(a). In liver and macrophage cells, Lp(a) uptake was dependent on extracellular calcium and inhibited by amiloride or its derivative EIPA (5-(N-ethyl-N-isopropyl)-Amiloride). Uptake of the unique protein component of Lp(a), apolipoprotein(a) (apo(a)) alone was also dependent on macropinocytosis, indicating that it is the likely mediator of Lp(a) uptake. In macrophages, the tricyclic antidepressant, imipramine, and the selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine and sertraline all inhibited Lp(a) uptake, likely due to macrophage de-differentiation effects and dynamin inhibition (in the case of sertraline). In liver cells, imipramine and citalopram strongly stimulated Lp(a) uptake, while sertraline inhibited uptake. Imipramine and citalopram enhanced cell surface binding of Lp(a) rather than upregulating macropinocytosis per se, indicating that immobilisation on the plasma membrane is an important step in Lp(a) macropinocytosis. Serotonin treatment also increased Lp(a) cell surface binding, promoting subsequent macropinocytotic uptake, consistent with imipramine and citalopram boosting extracellular serotonin levels through serotonin transporter inhibition. Imipramine and citalopram increased Lp(a) delivery to Rab11-positive recycling endosomes which promoted recycling of the apo(a) component of Lp(a) into the culture media. These findings support the utility of imipramine and citalopram as promising Lp(a)-lowering therapeutics in people suffering from depression who often have elevated Lp(a) levels and an increased risk of cardiovascular disease.

SummaryElevated lipoprotein(a) (Lp(a)) levels have been found in individuals suffering from depression posing an increased risk of cardiovascular disease (CVD). Efforts to effectively reduce circulating Lp(a) levels have been limited due to an inability to ascribe a clear cellular uptake pathway, despite multiple receptors being implicated in Lp(a) uptake. We report here that Lp(a) uptake is mediated by the fluid-phase endocytic process, macropinocytosis. Furthermore, serotonin and commonly prescribed antidepressants promote Lp(a) uptake by macropinocytosis in liver cells by increasing cell surface association. These findings represent a major paradigm shift in Lp(a) biology since previous studies have attributed Lp(a) uptake to receptor-mediated endocytosis. They also imply that treatment with antidepressants may improve cardiovascular as well as mental health.


title: Serotonin and common antidepressants regulate lipoprotein(a) macropinocytosis via enhanced cell surface binding


title: aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete β-arrestin-2 mediated SphK1-S1PR1-Akt signaling axis

abstract: Endothelial dysfunction is associated with multiple vascular diseases and lacks effective treatments. Activated Protein C (aPC) is a promising biotherapeutic that signals via protease-activated receptor-1 (PAR1) to promote diverse cytoprotective responses, including endothelial barrier stabilization, anti-inflammatory and anti-apoptotic activities, which is facilitated by co-receptors. We showed that aPC-activated PAR1 signals preferentially via {beta}-arrestin-2 ({beta}-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to enhance barrier protection. However, the mechanisms by which aPC/PAR1 promotes other cytoprotective responses are poorly understood. Here we define a novel {beta}-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. We show that PAR1 and S1PR1 co-exist in caveolin-1-rich microdomains basally and aPC markedly increases S1PR1-caveolin-1 co-association. Moreover, aPC stimulates {beta}-arr2-dependent SphK1 activation independent of Dvl2, which is critical for S1PR1 transactivation. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete {beta}-arr2-driven signaling pathways in caveolae.


title: Internalisation and biological activity of nucleic acids delivering cell-penetrating peptide nanoparticles is controlled by the biomolecular corona

abstract: Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g. blood stream. In this study, we compared CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles, and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g. by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be taken into account while developing delivery systems for nucleic acids.


title: Mass Cytometric and Transcriptomic Profiling of Epithelial-Mesenchymal Transitions in Human Mammary Cell Lines

abstract:
Epithelial-mesenchymal transition (EMT) equips breast cancer cells for metastasis and treatment resistance. Inhibition and elimination of EMT-undergoing cells are therefore promising therapy approaches. However, detecting EMT-undergoing cells is challenging due to the intrinsic heterogeneity of cancer cells and the phenotypic diversity of EMT programs. Here, we profiled EMT transition phenotypes in four non-cancerous human mammary epithelial cell lines using a FACS surface marker screen, RNA sequencing, and mass cytometry. EMT was induced in the HMLE and MCF10A cell lines and in the HMLE-Twist-ER and HMLE-Snail-ER cell lines by chronic exposure to TGF{beta}1 or 4-hydroxytamoxifen, respectively. We observed a spectrum of EMT transition phenotypes in each cell line and the spectrum varied across the time course. Our data provide multiparametric insights at single-cell level into the phenotypic diversity of EMT at different time points and in four human cellular models. These insights are valuable to better understand the complexity of EMT, to compare EMT transitions between the cellular models used herein, and for the design of EMT time course experiments.

O_TBL View this table:
org.highwire.dtl.DTLVardef@10d2b87org.highwire.dtl.DTLVardef@1a4cc67org.highwire.dtl.DTLVardef@67bc95org.highwire.dtl.DTLVardef@8df40dorg.highwire.dtl.DTLVardef@1919e7c_HPS_FORMAT_FIGEXP  M_TBL C_TBL Mendeley Data: DOI: 10.17632/pt3gmyk5r2.1

ArrayExpress Data: Accession number E-MTAB-9365