title: Transmission of human influenza A virus in pigs selects for adaptive mutations on the HA gene

abstract:
Influenza A viruses (IAV) cause respiratory diseases in many host species, including humans and pigs. The spillover of IAV between swine and humans has been a concern for both public health and the swine industry. With the emergence of the triple reassortant internal gene (TRIG) constellation, establishment of human-origin IAVs in pigs has become more common, leading to increased viral diversity. However, little is known about the adaptation processes that are needed for a human-origin IAV to transmit and become established in pigs. We generated a reassortant IAV containing surface gene segments from a human IAV strain and internal gene segments from the 2009 pandemic and TRIG IAV lineages and demonstrated that it can replicate and transmit in pigs. Sequencing and variant calling analysis identified a mutant that emerged during replication in pigs, which was mapped to a region near the receptor binding site of the hemagglutinin (HA). The variant was present in all contact pigs and replicated more efficiently in differentiated swine tracheal cells compared to the virus containing the wildtype human-origin HA. These results show that variants are selected quickly after replication of human-origin HA in pigs, leading to improved fitness in the swine host, likely contributing to transmission.

ImportanceInfluenza A viruses (IAV) cause respiratory disease in several species, including humans and pigs. The bidirectional transmission of IAV between humans and pigs plays a significant role in the generation of novel viral strains, greatly impacting viral epidemiology. However, little is known about the evolutionary processes that allow human IAV to become established in pigs. In this study, we generated reassortant viruses containing human seasonal HA and NA on different constellations of internal genes and tested their ability to replicate and transmit in pigs. We demonstrated that a virus containing a common internal gene constellation currently found in U.S. swine was able to transmit efficiently via the respiratory route. We identified a specific amino acid mutation that was fixed in the respiratory contact pigs that was associated with improved replication in primary swine tracheal epithelial cells, suggesting it was crucial for the transmissibility of the human virus in pigs.


title: FLAGELLAR MOTILITY DURING E. COLI BIOFILM FORMATION PROVIDES A COMPETITIVE DISADVANTAGE WHICH RECEDES IN THE PRESENCE OF CO-COLONIZERS.

abstract: In nature, bacteria form biofilms in very diverse environments, involving a range of specific properties and exhibiting competitive advantages for surface colonization. However, the underlying mechanisms are difficult to decipher. In particular, the contribution of cell flagellar motility to biofilm formation remains unclear. Here, we examined the ability of motile and nonmotile E. coli cells to form a biofilm in a well-controlled geometry, both in a simple situation involving a single-species biofilm and in the presence of co-colonizers. Using a millifluidic channel, we determined that motile cells have a clear disadvantage in forming a biofilm, exhibiting a long delay as compared to nonmotile cells. By monitoring biofilm development in real time, we observed that the decisive impact of flagellar motility on biofilm formation consists in the alteration of surface access time. Implementing a simple mathematical model to calculate surface access velocity for both motile (diffusion) and nonmotile (settling) cells, we discovered that the competitive advantage strongly depends on the geometry of the environment to be colonized. Interestingly, this advantage reverts for the smallest geometries, with a crossover at a height of 100 m. We also report that the difference between and nonmotile cells in the ability to form a biofilm diminishes in the presence of cocolonizers, which could be due to motility inhibition through the consumption of key resources by the co-colonizers. We conclude that the impact of flagellar motility on surface colonization closely depends on the environment geometry and the population features, suggesting a unifying vision of the role of cell motility in surface colonization and biofilm formation.

category: microbiology


title: TransCRISPR - sgRNA design tool for CRISPR/Cas9 experiments targeting transcription factor motifs

abstract: Transcription factors (TFs) regulate gene expression via binding to specific sequence motifs in promoters or enhancers. They are involved in various biological processes and their aberrant action may lead to multiple pathological states. To better understand the function of a given TF, it is necessary to identify its crucial binding sites and target genes, which is not a trivial task. CRISPR/Cas9 has been recently successfully applied in the study of TFs. Here, we present an online tool, transCRISPR, created to search for TF binding motifs in the user-provided query and design optimal sgRNAs targeting them. Users can obtain sgRNAs for chosen TFs motifs, for up to tens of thousands of target regions in the human or mouse genome, either for the Cas9 or dCas9 system. TransCRISPR provides user-friendly tables and visualizations, summarizing features of identified motifs and designed sgRNAs such as genomic localization, quality scores, closest transcription start sites, and others. TransCRISPR is available at https://transcrispr.igcz.poznan.pl/transcrispr/.

category: molecular biology


title: Parental histone distribution at nascent strands controls homologous recombination during DNA damage tolerance

abstract: The advance and stability of replication forks rely on a tight co-regulation of the processes of DNA synthesis and nucleosome assembly. We have addressed the relevance of parental histone recycling in the mechanisms of DNA damage tolerance (DDT) - homologous recombination (HR) and translesion synthesis (TLS) - that assist replication forks under conditions that block their advance. We show that mutants affected in the deposition of parental histones are impaired in the recombinational repair of the single-strand DNA gaps generated during DDT, with the defects being more severe in mutants impaired in the lagging strand-specific deposition pathway. These recombinational defects are not due to a deficit of parental histones at the nascent strands but to an excess of parental nucleosomes at the invaded strand that destabilizes the sister chromatid junction formed after strand invasion. In conclusion, parental histone distribution at stressed forks regulates HR and provides a potential mechanism for the choice between HR and TLS that would depend on whether DNA synthesis is blocked at the lagging or the leading strand.


title: Revealing determinants of translation efficiency via whole-gene codon randomisation and machine learning

abstract: Codon usage refers to the occurrence of synonymous codons in protein-coding genes. It is known for decades that codon usage contributes to translation efficiency and hence to protein production levels. However, its role in protein synthesis is still only partly understood. This lack of understanding hampers the design of synthetic genes for efficient protein production. In this study, we developed a method to generate a large, synonymous codon library of the gene encoding the red fluorescent protein (RFP). After expression in Escherichia coli, 1459 clones of this library were selected of which we measured protein production levels and determined the full coding sequences. Using different machine learning approaches, this data was used to reveal correlations between codon usage and protein production. Interestingly, protein production levels can be relatively accurately predicted (Pearson correlation of 0.762) by a Random Forest model, which only relies on the sequence information for the first 8 codons. This study clearly demonstrated the key role of codons at the start of the coding sequence. As such, it provides not only important fundamental insights on the influence of codon usage on protein production but also relevant clues on optimising the design of efficiently translated synthetic genes.


title: CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever

abstract: Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional: the intracellular dimeric NS1 (iNS1) forms part of the viral replication complex, the extracellular multi-oligomeric secreted NS1 (sNS1) is a major factor contributing to DHF. The structure of the iNS1 is well studied but not sNS1. Here we show the tetrameric (stable and loose conformation) and hexameric structures of sNS1. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain - elongated {beta}-sheet or {beta}-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics.


title: Distinct regions of H. pylori's bactofilin regulate protein interactions to control cell shape

abstract: The helical shape of H. pylori cells promotes robust stomach colonization, however, how the helical shape of H. pylori cells is determined is unresolved. Previous work identified helical-cell-shape-promoting protein complexes containing a peptidoglycan-hydrolase (Csd1), a peptidoglycan precursor synthesis enzyme (MurF), a non-enzymatic homologue of Csd1 (Csd2), non-enzymatic transmembrane proteins (Csd5 and Csd7), and a bactofilin (CcmA). Bactofilins are highly conserved, spontaneously polymerizing cytoskeletal bacterial proteins. We sought to understand CcmA's function in generating the helical shape of H. pylori cells. Using CcmA deletion analysis, in vitro polymerization, and in vivo co-immunoprecipitation experiments we identified that the bactofilin domain and N-terminal region of CcmA are required for helical cell shape and the bactofilin domain of CcmA is sufficient for polymerization and interactions with Csd5 and Csd7. We also found that CcmA's N-terminal region inhibits interaction with Csd7. Deleting the N-terminal region of CcmA increases CcmA-Csd7 interactions and destabilizes the peptidoglycan-hydrolase Csd1. Using super-resolution microscopy, we found that Csd5 recruits CcmA to the cell envelope and promotes CcmA enrichment at the major helical axis. Thus, CcmA helps organize cell-shape-determining proteins and peptidoglycan synthesis machinery to coordinate cell wall modification and synthesis, promoting the curvature required to build a helical cell.


title: Single-cell transcriptomics to define Plasmodium falciparum stage-transition in the mosquito midgut

abstract: Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory vector mosquito provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 hours post blood feeding, including the zygote and ookinete stages. This study revealed the transcriptional trajectories of the ApiAP2 family of transcription factors, and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structure-based functional predictions we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody or peptide-based transmission suppression strategies.


title: Description of Halopseudomonas maritima sp. nov., based on phylogenomic analysis

abstract: A novel bacterium, strain RR6T of the genus Halopseudomonas, was isolated from the sea sand from Paradip port area, India. Cells are Gram-stain-negative, rod-shaped, aerobic, non-sporulating and motile. The major cellular fatty acids of RR6T were C10:0 3OH, C12:0, C16:1 w7c/16:1 w6c, 18:1 w7c and/or 18:1 w6c and C16:0. The predominant polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), unidentified phospholipid (PL) and unidentified lipids (L1-L2). The genome size of the strain RR6 was 3.93 Mb. The genomic G+C content was 61.3%. Gene ontology study showed a significant fraction of genes were associated with biological processes (53%), followed by molecular function (36%) and cellular components (11%). Comparisons of 16S rRNA gene sequences revealed 99.73 - 99.87 % sequence similarity with the closely related type strains of the genus Halopseudomonas. The average nucleotide identity (ANI) and average amino acid identity (AAI) of RR6T with reference strains of closely related species of the genus Halopseudomonas were below 95-96, and the corresponding in-silico DNA-DNA hybridization (DDH) values were below 70 %. A phylogenomic tree based on core genome analysis supported these results. Genotypic and phenotypic characteristics of RR6T indicate that the strain represents a novel species of the genus Halopseudomonas and the name Halopseudomonas maritima sp. nov. is proposed. The type strain is RR6T (= NBRC 115418T = TBRC 15628T).


title: Enhancing Prime Editing Efficiency and Flexibility with Tethered and Split pegRNAs

abstract: Prime editing (PE) has advantages for small insertion, deletion or point mutations without double-stranded DNA breaks. The 3-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity. Here we generated stem-loop PEs (sPEs) by adding stem-loop aptamers at the 3-terminal of pegRNA, which can be tethered to Cas9 nickase resulting in tethered PEs (tPEs). sPEs and tPEs increased the small insertion, deletion or point mutations efficiency by 2-4-fold on average in HEK293, U2OS and HeLa cells. We split the modified pegRNAs into sgRNA and prime RNA. The resulting split pegRNA prime editors (SnPEs) maintain the PE activity and increase flexibility.


title: Development of ultra-low-input nanoRibo-seq enables quantification of translational control, revealing broad uORF translation by subtype-specific neurons

abstract: While increasingly powerful approaches enable investigation of transcription using small samples of RNA, approaches to investigate translational regulation in small populations of specific cell types, and/or (sub)-cellular contexts are lacking. Comprehensive investigation of mRNAs actively translated into proteins from ultra-low input material would provide important insight into molecular machinery and mechanisms underlying many cellular, developmental, and disease processes in vivo. Such investigations are limited by the large input required for current state-of-the-art Ribo-seq. Here, we present an optimized, ultra-low input "nanoRibo-seq" approach using 102 - 103-fold less input material than standard approaches, demonstrated here in subtype-specific neurons. nanoRibo-seq requires as few as 2.5K neurons, and exhibits rigorous quality control features: 1) strong enrichment for CDS versus UTRs and non-CDS; 2) narrow, distinct length distributions over CDS; 3) ribosome P-sites predominantly in-frame to annotated CDS; and 4) sufficient ribosome-protected fragment (RPF) coverage across thousands of mRNAs. As proof-of-concept, we calculate translation efficiencies from paired Ribo-seq and alkaline fragmented control libraries from "callosal projection neurons" (CPN), revealing divergence between mRNA abundance and RPF abundance for hundreds of genes. Intriguingly, we identify substantial translation of upstream ORFs in the 5 UTRs of genes involved in axon guidance and synapse assembly. nanoRibo-seq enables previously inaccessible investigation of translational regulation by small, specific cell populations in normal or perturbed contexts.


title: Cryo-EM Structure of R-loop monoclonal antibody S9.6 in recognizing RNA:DNA hybrids

abstract: The three-stranded chromatin structure R-loop is commonly found in the genomes of different species, and functions as the double-edged sword in gene expression and genome stability. The monoclonal antibody S9.6 specifically recognizes RNA:DNA hybrids, and has been widely used as a powerful tool to detect R-loops genome-widely. However, the structure basis and the molecular recognition mechanism of S9.6 to the nucleic acid substrates is still limited. Here, applying cryo-electron microscopy, we have determined a 4.9 [A] structure of S9.6 antigen-binding fragment (Fab) complexed with RNA:DNA hybrids. We found that the native Fab cleaved from S9.6 antibody has much higher affinity to RNA:DNA hybrids than to the double-strand RNAs, and the minimum length of the hybrids should be more than 8 base-pair. The structure of Fab binding to the hybrids suggested one loop of S9.6 heavy chain inserts into the minor groove in the RNA:DNA hybrids, and the other three loops flank to the hybrids. The top of four loop all enrich with aromatic or positive charge residues which are potentially responsible for nucleic acids binding specificity. Our results revealed the recognition mechanism of S9.6 on R-loops, which directs the future engineering of S9.6, and thus could further promote R-loop biology studies.


title: m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site

abstract: Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5 splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIO1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5 splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3 splice site usage and reinforces splicing fidelity at elevated temperature. We generalise these findings to reveal two major classes of 5 splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5 splice sites crucial to alternative splicing.


title: Microclimate is a strong predictor of the native and invasive plant-associated soil microbiota on San Cristobal Island, Galapagos archipelago

abstract:
Understanding the major drivers that influence soil bacterial and fungal communities is essential to mitigate the impacts of human activity on vulnerable ecosystems, like those found on the Galapagos Islands. Located ~1000 km off the coast of Ecuador, the volcanically formed islands are situated within distinct oceanic currents, which provide seasonal weather patterns and unique microclimates within small spatial scales across the islands. Although much is known about the impacts of human activity, such as climate change and invasive plant species, on above ground biodiversity of the Galapagos Islands, little is known about the resident soil microbial communities and the drivers that shape these communities. Here, our goal was to investigate the bacterial and fungal communities found in soil located in three distinct microclimates: Mirador (arid), Cerro Alto (semi-arid), and El Junco (humid), and associated with native and invasive plant types. At each site, we collected soil at three depths (rhizosphere, 5 cm, and 15 cm) associated with the invasive plant, Psidium guajava (guava), and native plant types. We determined that the sampling location (microclimate) was the strongest driver of both bacterial and fungal communities (74 and 38%, respectively), with additional minor but significant impacts from plant type and soil depth. This study highlights the continued need to explore microbial communities across diverse environments and demonstrates the weight of different abiotic and biotic factors impacting soil microbial communities across San Cristobal Island in the Galapagos archipelago.

IMPORTANCE/SIGNIFICANCEHuman activity such as climate change, pollution, introduction of invasive species, and deforestation, poses a huge threat to biodiverse environments. Soil microbiota are an essential component to maintaining healthy ecosystems. However, a greater understanding of factors that alter these microbial communities is needed in order to find ways to mitigate and reverse the impacts imposed by human activity. The Galapagos Islands are a unique real-world laboratory, in that the islands biogeography and physical locations in the Pacific Ocean provide distinct microclimates within small geographic distances. Harnessing these distinct environments allowed us to investigate the influence of microclimates, soil depth, and vegetation cover on bacterial and fungal community composition.


title: Comparative analysis about the structure of complete chloroplast genomes in genus Mangifera and accuracy verification about phylogenetic analysis based on gene ycf2 in genus level

abstract: Mango is an evergreen plant belonging to the genus Mangifera of the Anacardiaceae family. Genus Mangifera has 69 species of Mangifera around the world that are mainly distributed in tropical and subtropical countries, including India, Indonesia, the Malay Peninsula, Thailand, and South China. It is a popular tropical fruit known as the "King of Tropical Fruits". However, the study of the structure information of the complete chloroplast genome of Mangifera was microscopic, there was no report about the comparison of SSR, Ka/Ks, codons analysis and RNA editing, so in this study, we sequenced the 6 Mangifera samples and used three different ways to analyze the relationship of 6 species of Mangifera. Then we got some results, through the RNA editing and Ka/Ks calculating, we found the species could be divided into two groups, and the difference between the two groups was protein-coding gene ccsA. Moreover, all RNA editing occurred conversion of C to T and the gene ndhB had the most RNA editing sites in all species. In Ka/Ks analysis, the gene atpB, cemA, clpP, ndhD, petD, petB and ycf15 would be suffered from the positive selection after divergence. We also find the IR regions in these seven samples were very conservation through IR contraction and expansion and Sequence Divergence Analysis. Finally, we tried to confirm the relationship between 7 samples of Mangifera in Angiosperms in 3 different ways. Then we got that ML210 and MP090 had a closer relationship than others, MS796 had a closer relationship with ML210 and MP090 than others. At the same time, the method of phylogenetic analysis based on the gene ycf2 was not more accurate at the genus level than the method based on complete cp genome and proteincoding genes.


title: Nucleoporins facilitate ORC loading onto chromatin

abstract: The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we performed immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis revealed that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, results in decreased ORC2 loading onto chromatin. Depleting Elys also sensitized cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a new connection between ORC, replication initiation and nucleoporins, highlighting a previously unrecognized function of nucleoporins in metazoan replication initiation.


title: How to select the best model from AlphaFold2 structures?

abstract: Among the methods for protein structure prediction, which is important in biological research, AlphaFold2 has demonstrated astonishing accuracy in the 14th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP14). The accuracy is close to the level of experimental structure determination. Furthermore, AlphaFold2 predicts three-dimensional structures and estimates the accuracy of the predicted structures. AlphaFold2 outputs two model accuracy estimation scores, pLDDT, and pTM, enabling the user to judge the reliability of the predicted structures. Original research of AlphaFold2 showed that those scores had good correlations to actual prediction accuracy. However, it was unclear whether we could select a structure close to the native structure when multiple structures are predicted for a single protein. In this study, we generated several hundred structures with different combinations of parameters for 500 proteins and verified the performance of the accuracy estimation scores of AlphaFold2. In addition, we compared those scores with existing accuracy estimation methods. As a result, pLDDT and pTM showed better performance than the existing accuracy estimation methods for AlphaFold2 structures. However, the estimation performance of relative accuracy of the scores was still insufficient, and the improvement would be needed for further utilization of AlphaFold2.

category: bioinformatics


title: Identification of Antimalarial Compounds that Inhibit Apicomplexan AP2 Proteins

abstract:
Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified compounds that interact with this domain. Four compounds were found to compete for DNA binding with AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P. falciparum trophozoites and results in a decrease in abundance of log2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti-Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that are targeted against the ApiAP2 family of proteins. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics.

Author SummaryPlasmodium parasites are the causative agent of malaria, which resulted in over 600,000 deaths in 2021. Due to resistance arising for every antimalarial therapeutic deployed to date, new drug targets and druggable pathways must be explored. To address this concern, we used a molecular docking screen to predict competitors of DNA binding by the parasite specific family of Apicomplexan AP2 (ApiAP2) transcription factor proteins for testing in vitro and in vivo. We find that ApiAP2 competing compounds have antimalarial activity consistent with the disruption of gene regulation. This work will further our understanding of both the biological role and targetability of parasite transcriptional regulation.


title: Identification of Antimalarial Compounds that Inhibit Apicomplexan AP2 Proteins in the Human Malaria Parasite Plasmodium falciparum


title: Protist diversity and metabolic strategy in freshwater lakes are shaped by trophic state and watershed land use at a continental scale

abstract:
Protists play key roles in aquatic food webs as primary producers, predators, nutrient recyclers, and symbionts. Yet, a comprehensive view of protist diversity in freshwaters has been challenged by the immense environmental heterogeneity among lakes worldwide. We assessed protist diversity in the surface waters of 366 freshwater lakes across a north temperate to subarctic extent covering nearly 8.4 million km2 of Canada. Sampled lakes represented broad gradients in size, trophic state, and watershed land use. Hypereutrophic lakes contained the least diverse and most distinct protist communities relative to nutrient-poor lakes. Greater taxonomic variation among eutrophic lakes was mainly a product of heterotroph and mixotroph diversity, whereas phototroph assemblages were more similar under high- nutrient conditions. Overall, local physicochemical factors, particularly ion and nutrient concentrations, elicited the strongest responses in community structure, far outweighing the effects of geographic gradients. Despite their contrasting distribution patterns, obligate phototroph and heterotroph turnover was predicted by an overlapping set of environmental factors, while the metabolic plasticity of mixotrophs may have made them less predictable. Notably, protist diversity was associated with variation in watershed soil pH and agricultural crop coverage, pointing to human impact on the land-water interface that has not been previously identified in studies at smaller scales. Our study exposes the importance of both within-lake and external watershed characteristics in explaining protist diversity and biogeography, critical information in further developing an understanding of how freshwater lakes and their watersheds are impacted by anthropogenic stressors.

ImportanceFreshwater lakes are experiencing rapid changes under accelerated anthropogenic stress and a warming climate. Microorganisms underpin aquatic food webs, yet little is known about how freshwater microbial communities are responding to human impact. Here, we assessed the diversity of protists and their myriad ecological roles in lakes varying in size across watersheds experiencing a range of land use pressures by leveraging data from a continental-scale survey of Canadian lakes. We found evidence of human impact on protist assemblages through an association with lake trophic state and extending to agricultural activity and soil characteristics in the surrounding watershed. Furthermore, trophic state appeared to explain the distributions of phototrophic and heterotrophic protists in contrasting ways. Our findings highlight the vulnerability of lake ecosystems to increased land use and the importance of assessing terrestrial interfaces to elucidate freshwater ecosystem dynamics.

category: ecology


title: Non-cancer stem cell-derived Fibromodulin activates Integrin-dependent Notch signaling in endothelial cells to promote tumor angiogenesis and growth

abstract: Cancer stem cells alone can initiate and maintain tumors, but the function of non-cancer stem cells that form the tumor bulk remains poorly understood. Proteomic analysis showed a higher abundance of the extracellular matrix small leucine-rich proteoglycan Fibromodulin (FMOD) in the conditioned medium of non-cancer stem cells (DGCs; differentiated glioma cells) of glioma compared to that of glioma stem-like cells (GSCs). DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth. FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. DGC-secreted FMOD promotes angiogenesis by activating Integrin-dependent Notch signaling in endothelial cells. Furthermore, conditional silencing of FMOD in newly generated DGCs in vivo inhibits the growth of GSC-initiated tumors due to poorly developed vasculature and increases mouse survival. Collectively, these findings demonstrate that DGC-secreted FMOD promotes glioma tumor angiogenesis and growth through paracrine signaling in endothelial cells and identifies a DGC-produced protein as a potential therapeutic target in glioma.

category: cancer biology


title: Toward an optimal contraception dosing strategy

abstract:
Anovulation refers to a menstrual cycle characterized by the absence of ovulation. Exogenous hormones such as synthetic progesterone and estrogen have been used to attain this state to achieve contraception. However, large doses are associated with adverse effects such as increased risk for thrombosis and myocardial infarction. This study utilizes optimal control theory on a modified menstrual cycle model to determine the minimum total exogenous estrogen/progesterone dose, and timing of administration to induce anovulation. The mathematical model correctly predicts the mean daily levels of pituitary hormones LH and FSH, and ovarian hormones E2, P4, and Inh throughout a normal menstrual cycle and reflects the reduction in these hormone levels caused by exogenous estrogen and/or progesterone. Results show that it is possible to reduce the total dose by 92% in estrogen monotherapy, 43% in progesterone monotherapy, and that it is most effective to deliver the estrogen contraceptive in the mid follicular phase. Finally, we show that by combining estrogen and progesterone the dose can be lowered even more. These results may give clinicians insights into optimal formulations and schedule of therapy that can suppress ovulation.

AUTHOR SUMMARYHormonal contraceptives composed of exogenous estrogen and/or progesterone are commonly administered artificial means of birth control. Despite many benefits, adverse side effects associated with high doses such as thrombosis and myocardial infarction, cause hesitation to usage. Our study presents an improved mathematical model for hormonal control of the menstrual cycle and applies optimal control theory to minimize total exogenous estrogen and/or progesterone dose, and determine timing of administration that lead to contraception. We observe a reduction in dosage of about 92% in estrogen monotherapy and 43% in progesterone monotherapy. Our simulations show that it is most effective to deliver the estrogen contraceptive in the mid follicular phase. In addition, we illustrate that combination therapy significantly lower doses further. Our findings may give clinicians insights into optimal dosing scheme for contraception.

category: systems biology


title: Vascular inflammation exposes perivascular cells to SARS-CoV-2 infection

abstract: Pericytes stabilize blood vessels and promote vascular barrier function. However, vessels subjected to pro-inflammatory conditions have impaired barrier function, which has been suggested to potentially expose perivascular cells to SARS-CoV-2. To test this hypothesis, we engineered pericyte-supported vascular capillaries on-a-chip, and determined that the extravasation and binding of spike protein (S1) on perivascular cells of inflamed vessels to be significantly higher that in healthy controls, indicating a potential target to understand COVID-19 vascular complications.

category: bioengineering


title: ViReMaShiny: An Interactive Application for Analysis of Viral Recombination Data

abstract: Recombination is an essential driver of virus evolution and adaption, giving rise to new chimeric viruses, structural variants, sub-genomic RNAs, and Defective-RNAs. Next-Generation Sequencing of virus samples, either from experimental or clinical settings, has revealed a complex distribution of recombination events that contributes to the intrahost diversity. We and others have previously developed alignment tools to discover and map these diverse recombination events in NGS data. However, there is no standard for data visualization to contextualize events of interest and downstream analysis often requires bespoke coding. To address this, we present ViReMaShiny, a web-based application built using the R Shiny framework to allow interactive exploration and point-and-click visualization of viral recombination data provided in BED format generated by computational pipelines such as ViReMa (Viral-Recombination-Mapper).


title: Delineating antibody escape from Omicron variants

abstract: SARS-CoV-2-neutralizing antibodies play a critical role for protection and treatment of COVID-19. Viral antibody evasion therefore threatens essential prophylactic and therapeutic measures. The high number of mutations in the Omicron BA.1 sublineage results in markedly reduced neutralization susceptibility. Consistently, Omicron is associated with lower vaccine effectiveness and a high re-infection rate. Notably, newly emerging Omicron sublineages (BA.1.1, BA.2) have rapidly become dominant. Here, we determine polyclonal serum activity against BA.1, BA.1.1 and BA.2 in 50 convalescent or vaccinated individuals as well as delineate antibody sensitivities on a monoclonal level using 163 antibodies. Our study reveals a significant but comparable reduction of serum activity against Omicron sublineages which markedly increases after booster immunization. However, notable differences in sensitivity to individual antibodies demonstrate distinct escape patterns of BA.1 and BA.2 that also affect antibodies in clinical use. The results have strong implications for vaccination strategies and antibody use in prophylaxis and therapy.

category: immunology


title: SARS-CoV-2 Omicron (BA.1 and BA.2) specific novel CD8+ and CD4+ T cell epitopes targeting spike protein.

abstract: The Omicron (BA.1/B.1.1.529) variant of SARS-CoV-2 harbors an alarming 37 mutations on its spike protein, reducing the efficacy of current COVID-19 vaccines. This study identified CD8+ and CD4+ T cell epitopes from SARS-CoV-2 S protein mutants. To identify the highest quality CD8 and CD4 epitopes from the Omicron variant, we selected epitopes with a high binding affinity towards both MHC I and MHC II molecules and applied other clinical checkpoint predictors including immunogenicity, antigenicity, allergenicity, instability, and toxicity. Subsequently, we found eight Omicron (BA.1/B.1.1.529) specific CD8+ and eleven CD4+ T cell epitopes with a world population coverage of 76.16% and 97.46%, respectively. Additionally, we identified common epitopes across Omicron BA.1 and BA.2 lineages that target mutations critical to SARS-CoV-2 virulence. Further, we identified common epitopes across B.1.1.529 and other circulating SARS-CoV-2 variants, such as B.1.617.2 (Delta). We predicted CD8 epitopes binding affinity to murine MHC alleles to test the vaccine candidates in preclinical models. The CD8 epitopes were further validated using our previously developed software tool PCOptim. We then modeled the three-dimensional structures of our top CD8 epitopes to investigate the binding interaction between peptide-MHC and peptide-MHC-TCR complexes. Importantly, our identified epitopes are targeting the mutations on the RNA-binding domain and the fusion sites of S protein. This could potentially eliminate viral infections and form long-term immune responses compared to rather short-lived mRNA vaccines and maximize the efficacy of vaccine candidates against the current pandemic and potential future variants.


title: Delta-Omicron recombinant SARS-CoV-2 in a transplant patient treated with Sotrovimab

abstract: We identified a Delta-Omicron SARS-CoV-2 recombinant in an unvaccinated, immunosuppressed kidney transplant recipient who had positive COVID-19 tests in December 2021 and February 2022 and was initially treated with Sotrovimab. Viral sequencing in February 2022 revealed a 5 Delta AY.45 portion and a 3 Omicron BA.1 portion with a recombination breakpoint in the spike N-terminal domain, adjacent to the Sotrovimab quaternary binding site. The recombinant virus induced cytopathic effects with characteristics of both Delta (large cells) and Omicron (cell rounding/detachment). Monitoring of immunosuppressed COVID-19 patients treated with antiviral monoclonal antibodies is crucial to detect potential selection of recombinant variants.


title: CARP2 regulates the Golgi dynamics upon EGF stimulation

abstract:
Golgi apparatus regulate diverse cellular functions like protein sorting, vesicular trafficking, secretion, protein modifications like glycosylation etc. In mammalian cells though, Golgi apparatus appear as ribbon architecture, individual stacks laterally linked to each other by tubular structure, its architecture changes dynamically to cater to the needs of the cell under physiological and stress conditions. Loss of Golgi integrity is reported to be associated with pathological conditions like cancer and neurodegeneration. Very little is known of molecular regulators of Golgi dynamics. Here, we demonstrate that CARP2 (Caspase -8 and -10 associated RING containing protein 2), an endosomal ubiquitin ligase and a known regulator of cell migration, modulates the Golgi structure. Stimulation with EGF (Epidermal growth factor) modestly increased CARP2 protein levels. CARP2 exogenous expression or EGF treatment resulted in dispersal of the Golgi apparatus. Conversely, CARP2 deletion suppressed EGF induced Golgi dispersal. CARP2 variants that are defective in their endosomal-association or E3 ligase activity were unable to exhibit Golgi dispersal, indicating importance of both the endosomal localization and the E3 activity for this function. Importantly, we provide evidence that in EGF stimulated cells CARP2 mechanistically functions by targeting one of the Golgi structural proteins, Golgin45 for ubiquitination and degradation. Taken together, our findings unravel the existence of crosstalk between endosomal ubiquitin signaling and the Golgi dynamics.

SignificanceThe Golgi is an organelle that exists in mammals in ribbon form - individual stacks laterally linked with each other - is central to protein and lipid modifications, trafficking and secretion. The Golgi architecture is changed dynamically to cater to the physiological needs of the cells (eg: cell division, migration). Dysfunctional or altered Golgi is reported under pathological conditions like cancer, neurodegenerative diseases etc. This study unravels a complex signaling between endosomal ubiquitin ligase, CARP2 and one of the Golgi structural proteins, Golgin45. Here, we delineate CARP2-Golgin45 signaling as a fundamental mechanism that regulates Golgi dynamics underlying in EGF-stimulated cell migration.

category: cell biology


title: Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase

abstract: Centrioles play critical roles in our cells, being part of centrosomes and cilia, which are important microtubule organising centers (MTOC) with a variety of roles. While centrioles are very stable structures, they disappear in certain cell types upon differentiation, such as in oocytes. Little is known about the regulation of centriole structural integrity. We previously uncovered that the pericentriolar material (PCM), and its recruiter Polo kinase, are required for both the maintenance of centriole structural integrity and centrosome MTOC activity. Using an hypothesis driven RNAi screen, we show that both the cartwheel and the centriole wall play an important role in centrosome integrity. In particular, we uncovered that the centriole wall protein ANA1 is critical for the integrity of both new and mature centrioles, in Drosophila oogenesis as well as in cultured cells. Moreover, our results show that the activity of both Polo and the PCM in centriole integrity depends on ANA1. Our work suggests that the structural integrity of centrioles, once thought to be very stable organelles, depends on the turnover of key components, suggesting new perspectives for understanding the dysfunction of those structures in disease.


title: Introducing CELLBLOKS: a novel organ-on-a-chip platform allowing a plug-and-play approach towards building organotypic models

abstract: Human organs are structurally and functionally complex systems. Their function is driven by interactions between many specialised cell types, which is difficult to unravel on a standard petri-dish format. Conventional "petri-dish" approaches to culturing cells are static and self-limiting. However, current organ-on-a-chip technologies are difficult to use, have a limited throughput and lack compatibility with standard workflow conditions. We developed CELLBLOKS(R) as a novel "plug & play" organ-on-a-chip platform that enables straightforward creation of multiple-cell type organ specific microenvironments and demonstrate its advantages by building a liver model representative of live tissue function. CELLBLOKS(R) allows one to systematically test and identify various cell combinations that replicate optimal hepatic relevance. The combined interactions of fibroblasts, endothelial cells and hepatocytes were analysed using hepatic biochemistry (CYP3A4 and urea), cellular proliferation and transporter activities (albumin). The results demonstrate that optimal liver functional can be achieved in cross talk co-culture combinations compared to conventional mono-culture. The optimised CELLBLOKS(R) liver model was tested to analyse drug-induced liver toxicity using tamoxifen. The data suggests that our CELLBLOKS(R) liver model is highly sensitive to toxic insult compared to mono-culture liver model. In summary, CELLBLOKS(R) provides a novel cell culture technology for creating human relevant organotypic models that are easy and straightforward to establish in laboratory settings.


title: Redox sensing by SENP1 augments insulin secretion early after high-fat feeding in mice

abstract:
Pancreatic {beta}-cells respond to metabolic stress by upregulating insulin secretion, however underlying mechanisms remain unclear. In {beta}-cells from overweight humans without diabetes, and mice fed high-fat diet (HFD) for as little as 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. {beta}-cell RNA-seq suggests altered metabolic pathways linked to cytosolic redox following HFD. The increased {beta}-cell exocytosis upon HFD is dependent on a shift towards a reduced intracellular redox state, and increased expression of sentrin-specific protease-1 (Senp1). Mechanistically, allosteric binding of Zn2+ at a site that includes C535 suppresses basal SENP1 activity and unrestrained {beta}-cell exocytosis and increases SENP1 sensitivity to activation by redox signals. Mice with pancreas- or {beta}-cell SENP1 deletion fail to up-regulate exocytosis after 2-day HFD and become rapidly glucose intolerant. This highlights a key role for Zn2+-dependent redox signaling via SENP1 in {beta}-cell functional responses to metabolic stress. (Words: 145)

TeaserEarly after high fat feeding in mice, a cytosolic reducing signal acting via SENP1 increases the capacity of {beta}-cells to secrete insulin.


title: Salicin modifies osteoarthritis progression by binding on IRE1α and inhibiting IRE1α mediated endoplasmic reticulum stress

abstract:
ObjectivesTo investigate the effect and mechanisms of salicin (SA) on osteoarthritis (OA) progression.

MethodsPrimary rat chondrocytes were stimulated with TNF- and treated with or without SA. CCK-8 was utilized to determine the cytotoxicity of SA. RT-qPCR, Western Blotting and immunofluorescence staining were used to detect inflammatory factors, cartilage matrix degeneration markers, cell proliferation and apoptosis markers expression at mRNA and protein levels respectively. EdU assay and flow cytometer analysis were utilized for evaluating cell proliferation and apoptosis. RNA-sequencing, molecular docking, drug affinity responsive target stability and WB were applied to clarify mechanisms. Rat OA model was used to evaluate the effect of intra-articular injection of SA on OA progression.

ResultsNo obvious cytotoxicity was found with the treatment of 10 M SA. SA rescued TNF- induced degeneration of cartilage matrix, inhibition of chondrocytes proliferation, and promotion of chondrocytes apoptosis. In mechanism, we clarified SA could directly bind on IRE1 and occupy IRE1 phosphorylation site, followed with inhibiting IRE1 phosphorylation and regulating IRE1 mediated endoplasmic reticulum (ER) stress by IRE1-I{kappa}B-p65 signaling. Finally, intra-articular injection of SA loaded PLGA could ameliorate OA progression by inhibiting IRE1 mediated ER stress in OA model.

ConclusionsSA alleviates OA by directly binding on ER stress regulator IRE1 and inhibits IRE1 mediated ER stress by IRE1-I{kappa}B-p65 signaling. Topical use of small molecular drug SA holds the potential of modifying OA progression.


title: Conserved degronome features governing quality control-associated proteolysis

abstract: The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identified a large cohort of proteome-derived degrons., some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm established constraints governing degron potency, including the amino acids composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons were identified in viral and human proteins, suggesting for conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of proteins half-life.


title: Cryo-EM reveals an entangled kinetic trap in the folding pathway of a catalytic RNA

abstract: Functional RNAs fold through complex pathways that can contain misfolded "kinetic traps." A complete model of RNA folding requires understanding the formation of such misfolded states, but they are difficult to characterize due to their transient and potentially conformationally dynamic nature. We used cryo-electron microscopy (cryo-EM) to visualize a long-lived misfolded state in the folding pathway of the Tetrahymena thermophila group I intron, a paradigmatic RNA structure-function model system. The structure revealed how this state forms native-like secondary structure and tertiary contacts but contains two incorrectly crossed strands, consistent with a previous model. This incorrect topology mispositions a critical catalytic domain and cannot be resolved locally, as extensive refolding is required. This work provides a structural framework for interpreting decades of biochemical and functional studies and demonstrates the power of cryo-EM for the exploration of RNA folding pathways.

category: biochemistry


title: Exploring the effect of mechanical anisotropy of protein structures in the unfoldase mechanism of AAA+ molecular machines

abstract: Essential cellular processes of microtubule disassembly and protein degradation, which span lengths from tens of m to nm, are mediated by specialized molecular machines with similar hexameric structure and function. Our molecular simulations at atomistic and coarse-grained scales show that both the microtubule severing protein spastin and the caseinolytic protease ClpY, accomplish spectacular unfolding of their diverse substrates, a microtubule lattice and dihydrofolate reductase (DHFR), by taking advantage of mechanical anisotropy in these proteins. By considering wild-type and variants of DHFR, we found that optimal ClpY-mediated action probes favorable orientations of the substrate relative to the machine. Unfolding of wild-type DHFR involves strong mechanical interfaces near each terminal and occurs along branched pathways, whereas unfolding of DHFR variants involves softer mechanical interfaces and occurs through single pathways, but translocation hindrance can arise from internal mechanical resistance. For spastin, optimum severing action initiated by pulling on a tubulin subunit is achieved through the orientation of the machine versus the substrate (microtubule lattice). Moreover, changes in the strength of the interactions between spastin and a microtubule filament, which can be driven by the tubulin code, lead to drastically different outcomes for the integrity of the hexameric structure of the machine.

category: biophysics


title: State Dependent Anionic Pore Currents Conducted by Single Countercharge Mutants in a Voltage-Sensing Phosphatase

abstract: Mutating gating charge residues in the S4 segment of voltage-sensing domains (VSDs) can cause ionic leak currents through the VSDs. These leak currents, known as gating pore or omega currents, play important pathophysiological role in many diseases. Here, we show that mutations in a key countercharge residue, D129, in the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) facilitate conduction of unique anionic omega currents. Neutralization of D129 causes a dramatic positive shift of activation, facilitates the formation of a continuous water path through the intermediate state VSD, and creates a positive electrostatic potential landscape inside the VSD leading to anion selectivity. Increasing the population or duration of the conducting state by a high external pH or an engineered Cd2+ bridge markedly increases the current magnitude. Our findings uncover a new role of countercharge residues and could inform on the mechanisms of channelopathies linked to countercharge residue mutations.


title: Advanced transcriptomic analysis reveals the role of efflux pumps and media composition in antibiotic responses of Pseudomonas aeruginosa

abstract:
Pseudomonas aeruginosa is an opportunistic pathogen and major cause of hospital acquired infections. The pathogenicity and virulence of P. aeruginosa is largely determined by its transcriptional regulatory network (TRN). We used 411 transcription profiles of P. aeruginosa from diverse growth conditions to construct a quantitative TRN by identifying independently modulated sets of genes (called iModulons) and their condition-specific activity levels. The current study focused on the use of iModulons to analyze pathogenicity and antibiotic resistance of P. aeruginosa. Our analysis revealed: 1) 116 iModulons, 81 of which show strong association with known regulators; 2) novel roles of two-component systems in regulating antibiotics efflux pumps; 3) substrate-efflux pump associations; 4) differential iModulon activity in response to beta-lactam antibiotics in bacteriological and physiological media; 5) differential activation of  Cell Division iModulon resulting from exposure to different beta-lactam antibiotics; and 6) a role of the PprB iModulon in the stress-induced transition from planktonic to biofilm lifestyle. In light of these results, the construction of an iModulon-based TRN provides a transcriptional regulatory basis for key aspects of P. aeruginosa infection, such as antibiotic stress responses and biofilm formation. Taken together, our results offer a novel mechanistic understanding of P. aeruginosa pathogenicity.

SignificanceLarge data sets and machine learning are impacting a growing number of areas of research in the life sciences. Once the compendia of bacterial transcriptomes reached a critical size, we could use source signal extraction algorithms to find lists of co-regulated genes (called iModulons) associated with a transcription factor (TF) to them. The gene composition of iModulons and their condition-dependent activity levels constitute a quantitative description of the composition of bacterial transcriptomes. This study shows how this approach can be used to reveal the responses of P. aeruginosa to antibiotics and thus yield a deep regulatory understanding of pathogenicity properties. This study motivates the execution of similar studies for the other ESKAPEEs to yield a broad understanding of the role of TRNs in antibiotic responses to these urgent threat bacterial pathogens.


title: An Ultralong Bovine CDRH3 that Targets a Conserved, Cryptic Epitope on SARS-CoV and SARS-CoV-2

abstract: The ability of broadly neutralising antibodies to target conserved epitopes gives them huge potential as antibody-based therapeutics, particularly in the face of constant viral antigen evolution. Certain bovine antibodies are highly adept at binding conserved, glycosylated epitopes, courtesy of their ultralong complementarity determining region (CDR)H3. Here, we used a SARS-naive, bovine ultralong CDRH3 library and mammalian cell display, to isolate a bovine paratope that engages the SARS-CoV and SARS-CoV-2 receptor-binding domain (RBD). This neutralises viruses pseudo-typed with SARS-CoV Spike protein but not by competition with RBD binding to ACE2. Instead, using differential hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis, we demonstrate that this ultralong CDRH3 recognises a rarely identified, conserved, cryptic epitope that overlaps the target of pan-sarbecovirus antibodies (7D6/6D6). The epitope is glycan-shielded and becomes accessible only transiently via inter-domain movements. This represents the first bovine anti-sarbecovirus paratope and highlights the power of this approach in identifying novel tools to combat emerging pathogens.


title: Impact of energy limitations on function and resilience in long-wavelength Photosystem II

abstract: Photosystem II (PSII) uses the energy from red light to split water and reduce quinone, an energy-demanding process based on chlorophyll a (Chl-a) photochemistry. Two kinds of cyanobacterial PSII can use Chl-d and Chl-f to perform the same reactions using lower energy, far-red light. PSII from Acaryochloris marina has Chl-d replacing all but one of its 35 Chl-a, while PSII from Chroococcidiopsis thermalis, a facultative far-red species, has just 4 Chl-f and 1 Chl-d and 30 Chl-a. From bioenergetic considerations, the far-red PSII were predicted to lose photochemical efficiency and/or resilience to photodamage. Here, we compare enzyme turnover efficiency, forward electron transfer, back-reactions and photodamage in Chl-f-PSII, Chl-d-PSII and Chl-a-PSII. We show that: i) all types of PSII have a comparable efficiency in enzyme turnover; ii) the modified energy gaps on the acceptor side of Chl-d-PSII favor recombination via PD1+Phe- repopulation, leading to increased singlet oxygen production and greater sensitivity to high-light damage compared to Chl-a-PSII and Chl-f-PSII; ii) the acceptor-side energy gaps in Chl-f-PSII are tuned to avoid harmful back reactions, favoring resilience to photodamage over efficiency of light usage. The results are explained by the differences in the redox tuning of the electron transfer cofactors Phe and QA and in the number and layout of the chlorophylls that share the excitation energy with the primary electron donor. PSII has adapted to lower energy in two distinct ways, each appropriate for its specific environment but with different functional penalties.


category: plant biology


title: Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation, and ssDNA binding kinetics

abstract: SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), enabling the enzyme to localize to stalled replication forks (RFs) and promote their restart. Since localization of a potent dNTPase at stalled RFs is not harmonious with DNA replication, we used a series of kinetic and thermodynamic measurements to explore a hypothesis where the combined effects of T592 phosphorylation and ssDNA binding serves as a dual switch to turn-off SAMHD1 dNTPase activity. We report that phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1. However, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (~15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial baseline dynamics in the C-terminal domain (which contains T592) which may be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.


title: Deep Local Analysis evaluates protein docking conformations with locally oriented cubes

abstract:
AO_SCPLOWBSTRACTC_SCPLOWWith the recent advances in protein 3D structure prediction, protein interactions are becoming more central than ever before. Here, we address the problem of determining how proteins interact with one another. More specifically, we investigate the possibility of discriminating near-native protein complex conformations from incorrect ones by exploiting local environments around interfacial residues. Deep Local Analysis (DLA)-Ranker is a deep learning framework applying 3D convolutions to a set of locally oriented cubes representing the protein interface. It explicitly considers the local geometry of the interfacial residues along with their neighboring atoms and the regions of the interface with different solvent accessibility. We assessed its performance on three docking benchmarks made of half a million acceptable and incorrect conformations. We show that DLA-Ranker successfully identifies near-native conformations from ensembles generated by molecular docking. It surpasses or competes with other deep learning-based scoring functions. We also showcase its usefulness to discover alternative interfaces.

Availabilityhttp://gitlab.lcqb.upmc.fr/dla-ranker/DLA-Ranker.git


title: Architecture of the chikungunya virus replication organelle

abstract: Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.


title: Copper binding leads to increased dynamics in the regulatory N-terminal domain of full-length human copper transporter ATP7B

abstract: ATP7B is a human copper-transporting P1B-type ATPase that is involved in copper homeostasis and resistance to platinum drugs in cancer cells. ATP7B consists of a copper-transporting core and a regulatory N-terminal tail that contains six metal-binding domains (MBD1-6) connected by linker regions. The MBDs can bind copper, which changes the dynamics of the regulatory domain and activates the protein, but the underlying mechanism remains unknown. To identify possible copper-specific structural dynamics involved in transport regulation, we constructed a model of ATP7B spanning the N-terminal tail and core catalytic domains and performed molecular dynamics (MD) simulations with (holo) and without (apo) copper ions bound to the MBDs. In the holo protein, MBD2, MBD3 and MBD5 showed enhanced mobilities, which resulted in a more extended N-terminal regulatory region. The observed separation of MBD2 and MBD3 from the core protein supports a mechanism where copper binding activates the ATP7B protein by reducing interactions among MBD1-3 and between MBD1-3 and the core protein. Instead, an increased interaction between MBD5 and the core protein was observed that brought the copper-binding site of MBD5 closer to the high-affinity internal copper-binding site in the core protein. The simulation results assign specific, mechanistic roles to the metal-binding domains involved in ATP7B regulation that are testable in experimental settings.


title: Overexpression of TopBP1 leads to transformation with a TP53 mutation of non-tumorigenic breast epithelial cells

abstract:
DNA topoisomerase II{beta} - binding protein 1 (TopBP1) is a mediator protein that regulates the cell cycle checkpoint signaling pathway. A plethora of studies suggests high TopBP1 levels are positively associated with various cancers. Although TopBP1 transcript, as well as protein expression levels, are high in breast cancers, its role in breast tumorigenesis is not yet explored. In our studies, we observed that TopBP1 levels are high in premalignant and malignant cells of the MCF10A cancer progression series compared to the non-tumorigenic MCF10A cells. In order to establish the role of TopBP1 in tumorigenesis, TopBP1 overexpression in non-tumorigenic MCF10A, and stable knock-down in malignant MCF10CA1a cells were performed and grown in Matrigel as breast spheroids.

Overexpression of TopBP1 in MCF10A spheroids induced hyperproliferation, disruption of polarity and cell-cell junctions. Moreover, TopBP1 overexpressing 3D dissociated cells exhibited EMT-like phenotype and tumorigenic properties such as increased cell migration, invasion, colony formation capabilitiy and anchorage-independent growth, indicating acquisition of cellular transformation. Finally, we demonstrated TopBP1 overexpressing cells to form tumors in athymic mice thereby confirming their tumorigenic potential. We also confirmed that overexpression of TopBP1 led to a mutation in TP53 and other genomic insults. To summarise, we observed that ectopic expression of TopBP1 transforms MCF10A breast epithelial cells. These transformed cells harbour phenotypic and genotypic characteristics similar to that of malignant cells.


title: Nitroimidazopyrazinones with oral activity against tuberculosis and Chagas disease in mouse models of infection

abstract:
Tuberculosis remains one of the leading causes of death from a single infectious agent, surpassing both AIDS and malaria. In recent years, two bicyclic nitroimidazole drugs, delamanid and pretomanid have been approved to treat this airborne infection. This has spurred a renewed interest in developing new and improved nitroimidazole analogs. We have previously identified a new bicyclic heteroaromatic subclass, the nitroimidazopyrazinones, with substituted analogs showing promising activity against Mycobacterium tuberculosis under both aerobic and hypoxic environments. A second generation of nitroimidazopyrazinones with extended biaryl side chain also possessed good antiparasitic activity against Trypanosoma brucei brucei and Trypanosoma cruzi, suggesting the utility of this new scaffold for development into potential candidates against both tuberculosis and the kinetoplastid parasites which cause neglected tropical diseases. In this study, we further evaluated the properties of nitroimidazopyrazinone derivatives by assessing their selectivity against different mycobacterial species, measuring their reduction potential, and determining the kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn), which is the activating enzyme of delamanid and pretomanid in M. tuberculosis. We also conducted an in vivo evaluation of a lead compound, MCC8967 that demonstrated a favorable pharmacokinetic profile, with good oral bioavailability and efficacy in an acute M. tuberculosis infection model. Two other promising compounds MCC9481 and MCC9482, with good in vitro activity (IC50 = 0.016 and 0.10 {micro}M, respectively) against T. cruzi, the causative agent for Chagas diseases, were similarly tested for in vivo activity. These compounds also exhibited good oral bioavailability, and transiently reduced the acute-stage parasite burden by >98-99% at doses of 50 mg/kg once or twice daily, similar to benznidazole at 100 mg/kg once daily. Overall, we have demonstrated that active nitroimidazopyrazinones have potential to be developed as clinical candidates against both tuberculosis and Chagas disease.

Author SummaryTuberculosis and parasitic infections continue to impose a significant threat to public health and economic growth worldwide. Most of the efforts to control these diseases still rely on drug treatments with limited effectiveness and significant side effects. There is now an urgent need to develop new treatments to combat these infections. Here, we report the in vitro and in vivo profile of a new bicyclic nitroimidazole subclass, namely nitroimidazopyrazinones, against mycobacteria and Trypanosoma cruzi. We found that derivatives with monocyclic side chains are selective against Mycobacterium tuberculosis, the causative agent of tuberculosis, but not active against other nontuberculosis mycobacteria. In an acute mouse model, they were able to reduce the bacterial load in lungs via oral administration. From a biochemistry perspective, we demonstrated that deazaflavin-dependent nitroreductase (Ddn) could act effectively on nitroimidazopyrazinones, indicating the potential of Ddn as an activating enzyme for these new compounds in M. tuberculosis. We also showed that derivatives with extended biaryl side chain were effective in suppressing infection in an acute T. cruzi infected murine model, with satisfactory oral bioavailability. These findings improve the understanding of the biological profile of nitroimidazopyrazinones for further development as potential antitubercular and antiparasitic agents.

category: pharmacology and toxicology


title: PEP7 is a ligand for receptor kinase SIRK1 to regulate aquaporins and root growth

abstract: Plant receptor kinases constitute a large protein family that regulate various aspects of development and responses to external biotic and abiotic cues. Functional characterization of this protein family and particularly the identification of their ligands remains a major challenge in plant biology. Previously, we identified plasma membrane-intrinsic SUCROSE INDUCED RECEPTOR KINASE 1 (SIRK1) and QIAN SHOU KINASE 1 (QSK1) as a receptor / co-receptor pair involved in regulation of aquaporins in response to osmotic conditions induced by sucrose. Here, we identified a member of the Elicitor Peptide (PEP) family, namely PEP7, as the specific ligand of receptor kinase SIRK1. PEP7 binds to the extracellular domain of SIRK1 with a binding constant of 1.44{+/-}0.79 {micro}M and is secreted to the apoplasm specifically in response to sucrose treatment. Stabilization of a signaling complex involving SIRK1, QSK1 and aquaporins as substrates is mediated by alterations in the external sucrose concentration or by PEP7 application. Moreover, the presence of PEP7 induces the phosphorylation of aquaporins in vivo and enhance water influx into protoplasts. The loss-of-function mutant of SIRK1 is not responsive to external PEP7 treatment regarding kinase activity, aquaporin phosphorylation and water influx activity. Our data indicate that the PEP7/SIRK1/QSK1 complex represents a crucial perception and response module mediating sucrose-controlled water flux in plants.


title: The potyviral protein 6K2 from Turnip mosaic virus increases plant resilience to drought

abstract: Drought is a major cause of yield loss for crops worldwide. Climate change is predicted to increase global crop losses due to drought through rising temperature and decreased water availability. Virus infection can increase drought tolerance of infected plants compared to non-infected plants; however, the mechanisms mediating virus-induced drought tolerance remain unclear. In this study, we demonstrate Turnip mosaic virus (TuMV) infection increases Arabidopsis thaliana survival under drought compared to uninfected plants. To determine if specific TuMV proteins mediate drought tolerance, we cloned the coding sequence for each of the major viral proteins and generated transgenic A. thaliana that constitutively express each protein. Three TuMV proteins, 6K1, 6K2, and NIa-Pro, enhanced drought tolerance of A. thaliana when expressed constitutively in plants compared to controls. Expression of 6K2 also increased plant biomass relative to controls, but had no impact on root biomass, trichome numbers, or on the number of stomata. While drought induced transcripts related to abscisic acid (ABA) biosynthesis and ABA levels in control plants, compared to under well-watered conditions, there were no changes in ABA or related transcripts in plants expressing 6K2 under drought conditions compared to well-watered. 6K2 expression also conveyed drought tolerance in another host plant, Nicotiana benthamiana, when expressed using a virus over expression construct derived from Foxtail mosaic virus (FoMV). Although the exact mechanisms are still unknown, these results suggest 6K2-induced drought tolerance is ABA-independent and that plant viruses may represent novel sources of drought tolerance for crop plants.


title: Screening of the optimal CpG-oligodeoxynucleotide for anti-inflammatory responses in avian macrophage cell line HD11

abstract: CpG-oligodeoxynucleotides (CpG-ODN) has been shown to possess immunostimulatory features in both mammals and birds. However, compared to its pro-inflammatory effects, little is known about the anti-inflammatory responses triggered by CpG-ODN in avian cells. Hence, in this study, we aimed to characterize the anti-inflammatory response in the chick macrophage cell line HD11 under the stimulation of five kinds of CpG-ODN: CpG-A1585, CpG-AD35, CpG-B1555, CpG-BK3, and CpG-C2395. Single-stimulus CpG-B1555, CpG-BK3, and CpG-C2395 induced the interleukin (IL)-10 expression without causing cellular injury. The effects of pretreatment with each CpG-ODN before subsequent lipopolysaccharide stimulation were also evaluated. Interestingly, only CpG-C2395 maintained a high expression level in this situation. Finally, expression analysis of inflammation-related genes, such as the tumor necrosis factor-, IL-1{beta}, IL-6, and Toll-like receptor 4, was conducted, and pretreatment with CpG-C2395 significantly reduced their expression. Overall, our results shed light on the anti-inflammatory responses triggered by CpG-C2395 stimulation using a comparative analysis of three major classes of CpG-ODN in chick macrophages.


title: Evidence for complex interplay between quorum sensing and antibiotic resistance in Pseudomonas aeruginosa

abstract:
Quorum sensing (QS) is a cell-density-dependent, intercellular communication system mediated by small diffusible signaling molecules. QS regulates a range of bacterial behaviors, including biofilm formation, virulence, drug resistance mechanisms, and antibiotic tolerance. Enzymes capable of degrading signaling molecules can interfere in QS - a process termed quorum quenching (QQ). Remarkably, previous work reported some cases where enzymatic interference in QS was synergistic to antibiotics against Pseudomonas aeruginosa. The premise of combination therapy is attractive to fight against multidrug-resistant bacteria, yet comprehensive studies are lacking. Here we evaluate the effects of QS signal disruption on the antibiotic resistance profile of P. aeruginosa by testing 222 antibiotics and antibacterial compounds from 15 different classes. We found compelling evidence that QS signal disruption does indeed affect antibiotic resistance (40% of all tested compounds; 89/222), albeit not always synergistically (not synergistic for 48% of compounds with an effect (43/89)). For some tested antibiotics, like sulfathiazole and trimethoprim, we were able to relate the changes in resistance caused by QS signal disruption to the modulation of the expression of key genes of the folate biosynthetic pathway. Moreover, using a P. aeruginosa-based Caenorhabditis elegans killing model, we confirm that enzymatic QQ modulates the effects of antibiotics on P. aeruginosas pathogenicity in vivo. Altogether, these results show that signal disruption has profound and complex effects on the antibiotic resistance profile of P. aeruginosa. This work suggests that combination therapy including QQ and antibiotics should not be discussed globally but rather in case-by-case studies.

IMPORTANCEInterference in bacterial Quorum Sensing (QS) is a promising approach to control microbial behavior. Of particular interest is the potential of this strategy to reduce biofilms and virulence of antibiotic resistant strains. Interestingly, several studies report synergistic interactions between antibiotic treatments and interference in QS. However, it is unclear whether this is a generality, let alone the molecular mechanisms underlying the observed synergies. Here, we provide a comprehensive description of combination treatment in the model organism, opportunistic human pathogen P. aeruginosa. Screening > 200 antimicrobials, and combining them to QS signals and disruption strategies, we show that there is no systematic synergy between these approaches in vitro, as well as in vivo, in a C. elegans infection model. Altogether, this work show that QS has complex connections to the antibiotic resistance profile of P. aeruginosa, and that combination treatment should not be discussed globally, but rather in case-by-case studies.


title: Extended genomic analyses of the broad-host-range phages vB_KmiM-2Di and vB_KmiM-4Dii reveal slopekviruses have highly conserved genomes

abstract: High levels of antimicrobial resistance among members of the Klebsiella oxytoca complex (KoC) have led to renewed interest in the use of bacteriophage (phage) therapy to tackle infections caused by these bacteria. In this study we characterized two lytic phages, vB_KmiM-2Di and vB_KmiM-4Dii, that were isolated from sewage water against two GES-5-positive Klebsiella michiganensis strains (PS_Koxy2 and PS_Koxy4, respectively). ViPTree analysis showed both phages belonged to the genus Slopekvirus. rpoB gene-based sequence analysis of 108 presumptive K. oxytoca isolates (n=59 clinical, n=49 veterinary) found K. michiganensis to be more prevalent (46 % clinical and 43 % veterinary, respectively) than K. oxytoca (40 % clinical and 6 % veterinary, respectively). Host range analysis against these 108 isolates found both vB_KmiM-2Di and vB_KmiM-4Dii showed broad lytic activity against KoC species. Several putative homing endonuclease genes were encoded within the genomes of both phages, which may contribute to their broad host range. Pangenome analysis of 24 slopekviruses found that genomes within this genus are highly conserved, with more than 50 % of all predicted coding sequences representing core genes at [≥]95 % identity and [≥]70 % coverage. Given their broad host ranges, our results suggest vB_KmiM-2Di and vB_KmiM-4Dii represent attractive potential therapeutics. In addition, current recommendations for phage-based pangenome analyses may require revision.


title: A positive feedback loop controls Toxoplasma chronic differentiation

abstract: Successful infection strategies must balance pathogen amplification and persistence. In Toxoplasma gondii, this is accomplished through differentiation into dedicated cyst-forming chronic stages that avoid clearance by the host immune system. The transcription factor BFD1 is both necessary and sufficient for stage conversion; however, its regulation is not understood. We examine five factors transcriptionally activated by BFD1. One of these is a cytosolic RNA-binding protein of the CCCH-type zinc finger family, which we name BFD2. Parasites lacking BFD2 fail to induce BFD1 and are consequently unable to fully differentiate in culture or in mice. BFD2 interacts with the BFD1 transcript in a stress-dependent manner. Deletion of BFD2 reduces BFD1 protein levels, but not mRNA abundance. The reciprocal effects on BFD2 transcription and BFD1 translation outline a positive feedback loop that enforces commitment to differentiation. BFD2 helps explain how parasites commit to the chronic gene-expression program and elucidates how the balance between proliferation and persistence is achieved over the course of infection.


title: Dental pulp metagenomics reveals ancient human Herpesviridae.

abstract: Following a recent report on ancient human Herpesviridae (HHV), we adapted a standard protocol used for the high-throughput sequencing of modern DNA to recover ancient DNA (aDNA) from dental pulp specimens collected at two historical burial sites. Investigation of 36 soldiers buried in Sevastopol, Crimea, during the 1853-1856 Crimean War yielded genomic sequences covering 8.5% of the herpes simplex virus (HSV)-1 genome, 15.7% of the HSV-2 genome, 1.9% of the Epstein-Barr virus (EBV) genome and 3.9% of the cytomegalovirus genome. Further investigation of 16 civilians buried in the 17th century in Amiens, France, yielded genomic sequences covering 9.8% of the HHV-1 genome and 2.2% of the HHV-6A genome; all data obtained with parallel negative controls was appropriately negative. Specific qPCRs confirmed the presence of four HSV-1, two HSV-2 and one EBV sequences in Sevastopol and two HSV-1 and one HHV-6 sequences in Amiens. HHV-6 was further detected by fluorescent in situ hybridization. These results demonstrated the usefulness of modern metagenomic protocols for high-throughput sequencing of aDNA in dental pulp.


title: Culexarchaeia, a novel archaeal class of anaerobic generalists inhabiting geothermal environments

abstract: Geothermal environments, including terrestrial hot springs and deep-sea hydrothermal sediments, often contain many poorly understood lineages of archaea. Here, we recovered ten metagenome-assembled genomes (MAGs) from geothermal sediments and propose that they constitute a new archaeal class within the TACK superphylum, "Candidatus Culexarchaeia", named after the Culex Basin in Yellowstone National Park. Culexarchaeia harbor distinct sets of proteins involved in key cellular processes that are either phylogenetically divergent or are absent from other closely related TACK lineages, with a particular divergence in cell division and cytoskeletal proteins. Metabolic reconstruction revealed that Culexarchaeia have the capacity to metabolize a wide variety of organic and inorganic substrates. Notably, Culexarchaeia encode a unique modular, membrane associated, and energy conserving [NiFe]-hydrogenase complex that potentially interacts with heterodisulfide reductase (Hdr) subunits. Comparison of this [NiFe]-hydrogenase complex with similar complexes from other archaea suggests that interactions between membrane associated [NiFe]-hydrogenases and Hdr may be more widespread than previously appreciated in both methanogenic and non-methanogenic lifestyles. The analysis of Culexarchaeia further expands our understanding of the phylogenetic and functional diversity of lineages within the TACK superphylum and the ecology, physiology, and evolution of these organisms in extreme environments.


title: Using Culture-Enriched Phenotypic Metagenomics for Targeted High-Throughput Monitoring of Clinically-Important Fraction of Beta-Lactam Resistome

abstract: The high bacterial community complexity greatly hinders the monitoring of clinically prevalent extended-spectrum beta-lactam resistant bacteria, which are usually present as rare but key populations involving in the environmental dissemination of clinical resistance. Here, we introduce culture-enriched phenotypic metagenomics that integrates culture enrichment, phenotypic screening and metagenomic analyses as an emerging methodology to profile beta-lactam resistome in a municipal wastewater treatment plant (WWTP) and its receiving river. The results showed that clinically prevalent carbapenemase genes (e.g., NDM and KPC family) and extended-spectrum beta-lactamase genes (e.g., CTX-M, TEM and OXA family) considerably existed in the WWTP and showed prominent potential in horizontal dissemination. Strikingly, carbapenem and polymyxin resistance genes co-occurred in highly virulent Enterobacter kobei (MCR-1 and NDM-6) and Citrobacter freundii (ArnA and NDM-16) genomes. Overall, this study exemplifies phenotypic metagenomics for high-throughput surveillance of targeted fraction of clinically-important resistome and substantially expands current knowledge on extended-spectrum beta-lactam resistance in WWTPs.


title: Unexpected Complexity of the Ammonia Monooxygenase in Archaea

abstract: Ammonia oxidation as the first step of nitrification constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase is lacking, in particular for the environmentally abundant ammonia oxidizing archaea (AOA). Here, the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Beside the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modelling and in gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase indicating that AmoY and AmoZ serve an important structural and functional role. These data open avenues for further structure-function studies of this ecologically important key nitrification complex.


title: Barcoding genetically distinct Plasmodium falciparum strains for comparative assessment of fitness and antimalarial drug resistance

abstract: The repeated emergence of antimalarial drug resistance in Plasmodium falciparum, including to the current frontline antimalarial artemisinin, is a perennial problem for malaria control. Nextgeneration sequencing has greatly accelerated the identification of polymorphisms in resistance-associated genes, but has also highlighted the need for more sensitive and accurate laboratory tools to profile current and future antimalarials, and to quantify the impact of drug resistance acquisition on parasite fitness. The interplay of fitness and drug response is of fundamental importance in understanding why particular genetic backgrounds are better at driving the evolution of drug resistance in natural populations, but the impact of parasite fitness landscapes on the epidemiology of drug resistance has typically been laborious to accurately quantify in the lab, with assays being limited in accuracy and throughput. Here we present a scalable method to profile fitness and drug response of genetically distinct P. falciparum strains with well-described sensitivities to several antimalarials. We leverage CRISPR/Cas9 genome-editing and barcode sequencing to track unique barcodes integrated into a non-essential gene (pfrh3). We validate this approach in multiplex competitive growth assays of three strains with distinct geographical origins. Furthermore, we demonstrate that this method can be a powerful approach for tracking artemisinin response as it can identify an artemisinin resistant strain within a mix of multiple parasite lines, suggesting an approach for scaling the laborious ring-stage survival assay (RSA) across libraries of barcoded parasite lines. Overall, we present a novel high-throughput method for multiplexed competitive growth assays to evaluate parasite fitness and drug response


title: Disentangling leaf-microbiome interactions in Arabidopsis thaliana by network mapping

abstract:
The leaf microbiota plays a key role in plant development, but a detailed mechanism of microbe-plant relationships remains elusive. Many genome-wide association studies (GWAS) have begun to map leaf microbes, but few has systematically characterized the genetics of how microbes act and interact. Previously, we integrated behavioral ecology and game theory to define four types of microbial interactions - mutualism, antagonism, aggression, and altruism, in a microbial community assembly. Here, we apply network mapping to identify specific plant genes that mediate the topological architecture of microbial networks. Analyzing leaf microbiome data from an Arabidopsis GWAS, we identify several heritable hub microbes for leaf microbial communities and detect 140-728 SNPs responsible for emergent properties of microbial network. We reconstruct Bayesian genetic networks from which to identify 22-43 hub genes found to code molecular pathways related to leaf growth, abiotic stress responses, disease resistance and nutrition uptake. A further path analysis visualizes how genetic variants of Arabidopsis affect its fecundity through the internal workings of the leaf microbiome. We find that microbial networks and their genetic control vary along spatiotemporal gradients. Our study provides a new avenue to reveal the "endophenotype" role of microbial networks in linking genotype to end-point phenotypes in plants. Our integrative theory model provides a powerful tool to understand the mechanistic basis of structural-functional relationships within the leaf microbiome and supports the need for future research on plant breeding and synthetic microbial consortia with a specific function.

IMPORTANCEIt is found that plant genes act as microbiome gatekeepers to select which microbes get to live inside the leaves for health. Many genome-wide association studies (GWAS) have begun to map leaf microbes, but few has systematically characterized the genetics of how microbes act and interact. This work illustrates a more comprehensive picture of the genetic architecture underlying the leaf microbiome by network mapping. This study also dissects how genetic variants affect its fecundity by direct path and indirect path through microbial network, revealing the "endophenotype" role of microbial networks in linking genotype to end-point phenotypes. Future studies could benefit from this work to improve understanding the underlying genetic mechanisms that govern the relationships between plants and their microbiomes, and to manipulate plant genetic system to reconfigure microbiome. Plants could become more efficient at selecting their microbial partners to improve their health, resilience, and productivity.


title: Structural studies of hepatitis C virus non-structural protein-5b of genotype 4a

abstract:
Hepatitis C virus genotype 4a non-structural protein-5b is an RNA-dependent RNA-polymerase responsible for the efficient virus genome replication. It has been of great interest as a drug target. The availability of the crystal structure of HCV genotype 4a NS5b would facilitate the structure-based drug design. Here we report the X-ray structure of NS5b solved at 3.1 [A] resolution, setting the groundwork for the above-described therapeutic strategy.

SynopsisX-ray structure of hepatitis C virus genotype 4a non-structural protein-5b, RNA-dependent RNA-polymerase responsible for the efficient virus genome replication.


title: New system for serial crystallography data collection at SBC APS

abstract: Serial synchrotron crystallography enables studies of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed by using a high-performance computing pipeline. Here, the system was applied to two {beta}-lactamases: a class D serine {beta}-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-{beta} -lactamase from Stenotrophomonas maltophilia K279a.


title: Multiple congenital malformations arise from somatic mosaicism for constitutively active Pik3ca signalling

abstract: 1Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a range of rare and common cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumours and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of skin, brain, adipose, connective, musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD due to cell-autonomous activation of the PI3K signaling pathway within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Effects in cells having once expressed Wnt1, including the brain roofplate and most neural crest, were most dramatic in the head. Outcomes included megalencephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of either mesodermal or neural crest origin was associated with widespread vascular anomalies, leading us to incidentally discover previously undescribed lineages that had expressed the transcription factor Egr2 (Krox20) and that may be co-opted in pathogenesis. Schwann cell precursors having transcribed either Krox20 or Sox10 also gave rise to adult-onset vascular tumors and cancers, including melanoma, after Pik3ca activation. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.

category: developmental biology


title: Overlapping and distinct cis-acting requirements for oskar mRNA localization pathways

abstract: Localization of oskar mRNA to the posterior of the Drosophila oocyte is essential for abdominal patterning and germline development. oskar localization is a multi-step process involving temporally and mechanistically distinct transport modes. Numerous cis-acting elements and trans-acting factors have been identified that mediate earlier motor-dependent transport steps leading to accumulation of oskar at the posterior. Little is known, however, about what features of oskar are required for the later localization phase, that occurs by diffusion with local entrapment and results in the accumulation of large oskar ribonucleoprotein granules by the end of oogenesis. Here we test whether cis- and trans-acting requirements for kinesin-dependent oskar transport, the structured oskar spliced localization element (SOLE) and the exon junction complex (EJC), are also required for late-phase localization. In contrast to kinesin-dependent motility, late-phase localization is independent of the EJC and depends not only on the structure but also the sequence of the SOLE. Late-phase localization also requires the oskar 3 UTR and we show that the 3 UTR is sufficient for ribonucleoprotein granule selectivity.


title: The {varphi}PA3 Phage Nucleus is Enclosed by a Self-Assembling, 2D Crystalline Lattice

abstract: A growing number of jumbo bacteriophages, with genomes exceeding 200 kb, have been found to establish a Phage Nucleus--a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the Phage Nucleus while nucleotide synthesis, translation, and numerous other host and exogenous proteins are effectively excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that fragments of the Phage Nucleus isolated from {phi}PA3 infected Pseudomonas aeruginosa cells form a square lattice and demonstrate that the recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into sheets also constructed from a square lattice which we resolve to 3.8 [A] by cryo-EM. Our structure reveals that the flexible termini and large loops mediate adaptable inter-tetramer contacts that drive shell assembly into a C2-symmetric lattice. While the interfaces between subunits are mostly well packed, two of the interfaces are open, forming clear channels that likely have important functional implications for the transport of proteins, mRNA, and small molecules.