title: Host poly(A) polymerases PAPD5 and PAPD7 provide two layers of protection that ensure the integrity and stability of hepatitis B virus RNA

abstract:
Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize HBV RNA via the interaction with the viral post-transcriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, HBsAg production and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies, therefore it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (EC50 ranged from 1.4 to 6.8 nM) and in vivo by 0.93 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity towards the inhibitory effect of AB-452. Although neither single knock-out (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO could not result in any measurable phenotypic changes, but displays a similar antiviral effect as AB-452 treatment when PAPD5 is depleted simultaneously. PAPD5/7 double KO confers complete resistance to AB-452 treatment. Our results thus indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5 targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication.

ImportanceChronic hepatitis B affects more than 250 million patients and is a major public health concern worldwide. HBsAg plays a central role in maintaining HBV persistence and as such, therapies reducing HBsAg have been extensively investigated. PAPD5/7 targeting inhibitors, with oral bioavailability, represent an opportunity to reduce both HBV RNA and HBsAg. Here we uncover that the SL sequence is required for HBV poly(A) tail integrity and RNA stability, and that the antiviral activity of AB-452 mimics the SL mutants. Although PAPD5 and PAPD7 regulate HBV RNA stability, it remains unclear how they coordinate in stabilizing HBV RNA. Based on our studies, PAPD5 plays a dominant role to stabilize viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a backup protection mechanism. Our studies may point out a direction towards developing PAPD5-selective inhibitors that could be used effectively to treat chronic hepatitis B.


title: Evaluation of the effects of library preparation procedure and sample characteristics on the accuracy of metagenomic profiles

abstract:
Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities, or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera(R) Flex kit, perform when applied to metagenomic DNA. We measured each methods ability to produce metagenomic data that accurately represents the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral-associated, mouse-gut-associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each methods performance, indicating that careful consideration may be needed when selecting between methods, especially for low complexity communities. However, cost-effective preparation methods we assessed are generally comparable to the current gold standard Nextera(R) DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies.

IMPORTANCEMetagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely-accepted standardized library preparation methods can be cost-prohibitive. Newly available approaches may be less expensive, but their efficacy in comparison to standardized methods remains unknown. In this study, we compared five different metagenomic library preparation methods. We evaluated each method across a range of microbial communities varying in complexity and quantity of input DNA. Our findings demonstrate the importance of considering sample properties, including community type, composition, and DNA amount, when choosing the most appropriate metagenomic library preparation method.

category: microbiology


title: eIF2α integrates proteotoxic signals both from ER and cytoplasm: Hsp70-Bag3 module regulates HRI-dependent phosphorylation of eIF2α

abstract: The major heat shock protein Hsp70 has been implicated in many stages of cancer development. These effects are mediated by a scaffold protein Bag3 that binds to Hsp70 and links it to components of multiple cancer-related signaling pathways. Accordingly, the Hsp70-Bag3 complex has been targeted by small molecules, which showed strong anti-cancer effects. Here, our initial question was how JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3, causes cell death. Breast epithelial cells MCF10A transformed with a single oncogene Her2 showed higher sensitivity to JG-98 then parental MCF10A cells. RNA expression analysis showed that this enhanced sensitivity correlated with higher induction of the UPR genes. Indeed, depletion of the pro-apoptotic UPR responsive transcription factor CHOP significantly protected cells from JG-98. Surprisingly, only the eIF2-associated branch of the UPR was activated by JG-98, suggesting that the response was not related to the ER proteotoxicity. Indeed, it was dependent on activation of a distinct cytoplasmic eIF2 kinase HRI. HRI-dependent phosphorylation of eIF2 was also activated by the cytoplasmic proteotoxicity via Hsp70-Bag3 complex, which directly associates with HRI. Dissociation of Hsp70-Bag3 complex led to Bag3-dependent degradation of HRI via autophagy. Therefore, eIF2 integrates proteotoxicity signals from both ER and cytoplasm, and the cytoplasmic response mediates cytotoxicity of the Hsp70-Bag3 inhibitors.

category: cell biology


title: Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm

abstract: During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1{Delta} mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1{Delta} chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic NES or NLS sequences or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.


title: Adipose mitochondrial metabolism controls body growth by modulating cytokine and insulin signaling

abstract: Animals need to adapt their growth to fluctuations in nutrient availability to ensure proper development and survival. These adaptations often rely on specific nutrient-sensing tissues and their control of whole-body physiology through inter-organ communication. While the signaling mechanisms that underlie this communication are well studied, the contributions of metabolic alterations in the nutrient-sensing tissues are less clear. Here, we show how reprogramming of adipose mitochondrial metabolism controls whole-body growth in Drosophila larvae. We find that dietary nutrients alter fat body mitochondrial morphology to lower their bioenergetic activity, which we see can rewire fat body glucose metabolism. Strikingly, we find that genetic reduction of mitochondrial bioenergetics just in the fat body is sufficient to accelerate body growth and development. These growth effects are caused by inhibition of the fat-derived adipokine, TNF/Eiger, which leads to enhanced systemic insulin signaling, the main hormonal stimulator of body growth. Our work reveals how reprogramming of mitochondrial metabolism in one nutrient-sensing tissue is able to couple whole body growth to nutrient availability.

category: developmental biology


title: Phosphoproteomics of ATR Signaling in Prophase I of Mouse Meiosis

abstract: During mammalian meiosis, the ATR kinase plays crucial roles in the coordination of DNA repair, meiotic sex chromosome inactivation and checkpoint signaling. Despite the importance of ATR in meiosis, the meiotic ATR signaling network remains largely unknown. Here we defined ATR signaling during prophase I in mice. Quantitative analysis of phosphoproteomes obtained after genetic ablation of the ATR-activating 9-1-1 complex or chemical inhibition of ATR revealed over 12,000 phosphorylation sites, of which 863 phosphorylation sites were dependent on both 9-1-1 and ATR. ATR and 9-1-1-dependent signaling was enriched for S/T-Q and S/T-X-X-K motifs and included proteins involved in DNA damage signaling, DNA repair, and piRNA and mRNA metabolism. We find that ATR targets the RNA processing factors SETX and RANBP3 and regulate their localization to the sex body. Overall, our analysis establishes a comprehensive map of ATR signaling in spermatocytes and highlights potential meiotic-specific actions of ATR during prophase I.


title: Defense by duplication: The relation between phenotypic glyphosate resistance andEPSPS gene copy number variation in Amaranthus palmeri

abstract: Gene copy number variation (CNV) has been increasingly associated with organismal responses to environmental stress, but we know little about the quantitative relation between CNV and phenotypic variation. In this study we quantify variation in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) copy number using digital drop PCR and variation in phenotypic glyphosate resistance in 22 populations of Amaranthus palmeri (Palmer Amaranth), a range-expanding agricultural weed. Overall, we detected a significant positive relation between population mean copy number and mean resistance. The majority of populations exhibited high glyphosate resistance, yet maintained low-resistance individuals resulting in bimodality in many populations. We investigated linear and threshold models for the relation between copy number and resistance, and found evidence for a threshold of ~15 EPSPS copies: there was a steep increase in resistance before the threshold, followed by a much shallower slope. Moreover, as copy number increases, the range of variation in resistance decreases. This suggests a working hypothesis that as EPSPS copies and dosage increases, negative epistatic interactions may be compensated. We detected a quadratic relation between mean resistance and variation (s.d.) in resistance, consistent with the prediction that as phenotypic resistance increases in populations, stabilizing selection decreases variation in the trait. Finally, patterns of variation across the landscape are consistent with less variation among populations in mean copy number / resistance in Georgia where glyphosate resistance was first detected, and wider variation among populations in resistance and copy number in a more northern state where resistance evolution may be at a younger evolutionary state.

category: evolutionary biology


title: Recovery of silver fir (Abies alba Mill.) seedlings from ungulate browsing mirrors soil nitrogen availability

abstract: Abies alba has a high potential for mitigating climate change in European mountain forests, yet, its natural regeneration is severely limited by ungulate browsing. Here, we simulated browsing in a common garden experiment to study growth and physiological traits, measured from bulk needles, using a randomized block design with two levels of browsing severity and seedlings originating from 19 populations across Switzerland. Genetic factors explained most variation in growth (on average, 51.5%) and physiological traits (10.2%) under control conditions, while heavy browsing considerably reduced the genetic effects on growth (to 30%), but doubled those on physiological traits related to C storage. While browsing reduced seedling height, it also lowered seedlings water use efficiency (decreased{delta} 13C) and N supply by mycorrhizal fungi as indicated by an increase in{delta} 15N. Different populations reacted differently to browsing stress, and for Height, Starch and{delta} 15N, population differences appeared to be the result of natural selection. We found that the fastest growing populations, originating from the warmest regions, decreased their needle starch level the most as a reaction to heavy browsing, suggesting a potential genetic underpinning for a growth-storage trade-off. Further, we found that seedlings originating from mountain populations growing on steep slopes had a significantly lower N discrimination in the common garden than those originating from flat areas, indicating that they have been selected to grow on N poor, potentially drained, soils. This finding was corroborated by the fact that N concentration in adult needles was lower on steep slopes than on flat ground, strongly indicating that steep slopes are the most N poor environments. Seedlings from these poor environments generally had a low growth rate and high storage, thus might be slower to recover from browsing stress than fast growing provenances from the warm environments with developed soils, such as the Swiss plateau.


title: Structural and mechanistic basis for protein glutamylation by the kinase fold

abstract: The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE-ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.

category: biochemistry


title: Structural basis for the ARF GAP activity and specificity of the C9orf72 complex

abstract: Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. C9orf72 forms a complex with SMCR8 and WDR41, which was reported to have GTPase activating protein activity toward ARF proteins, RAB8A, and RAB11A. We determined the cryo-EM structure of ARF1-GDP-BeF3- bound to C9orf72:SMCR8:WDR41. The SMCR8longin and C9orf72longin domains form the binding pocket for ARF1. One face of the C9orf72longin domain holds ARF1 in place, while the SMCR8longin positions the catalytic finger Arg147 in the ARF1 active site. Mutations in interfacial residues of ARF1 and C9orf72 reduced or eliminated GAP activity. RAB8A GAP required [~]10-fold higher concentrations of the C9orf72 complex than for ARF1. These data support a specific function for the C9orf72 complex as an ARF GAP.


title: Proteome-scale amino-acid resolution footprinting of protein-binding sites in the intrinsically disordered regions of the human proteome

abstract: Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies using a wide range of experimental approaches have identified tens of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional and cell type-specific interactions are likely to be disproportionately under-represented. Moreover, for most known protein-protein interactions the binding regions remain uncharacterized. We previously developed proteomic peptide phage display (ProP-PD), a method for simultaneous proteome-scale identification of short linear motif (SLiM)-mediated interactions and footprinting of the binding region with amino acid resolution. Here, we describe the second-generation human disorderome (HD2), an optimized ProP-PD library that tiles all disordered regions of the human proteome and allows the screening of ~1,000,000 overlapping peptides in a single binding assay. We define guidelines for how to process, filter and rank the results and provide PepTools, a toolkit for annotation and analysis of identified hits. We uncovered 2,161 interaction pairs for 35 known SLiM-binding domains and confirmed a subset of 38 interactions by biophysical or cell-based assays. Finally, we show how the amino acid resolution binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the human proteome. The HD2 ProP-PD library paired with PepTools represents a powerful pipeline for unbiased proteome-wide discovery of SLiM-based interactions.


title: HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA binding domain

abstract: Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its -crystallin domain (CD). These CD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.


title: Iron-sulfur clusters are involved in post-translational arginylation

abstract: Eukaryotic arginylation is an essential post-translational modification that both modulates protein stability and regulates protein half-life through the N-degron pathway. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite its conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we have discovered that ATE1s bind a previously unknown iron-sulfur cluster that is conserved across evolution. We have extensively characterized the nature of this iron-sulfur cluster, and we show that the presence of the iron-sulfur cluster is linked to alterations in arginylation efficacy. Finally, we demonstrate that the ATE1 iron-sulfur cluster is oxygen sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Thus, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.


title: Ultra-fast insulin-pramlintide co-formulation for improved glucose management in diabetic rats

abstract: Dual-hormone replacement therapy with insulin and amylin in patients with type 1 diabetes has the potential to improve glucose management. Unfortunately, currently available formulations require burdensome separate injections at mealtimes and have disparate pharmacokinetics that do not mimic endogenous co-secretion. Here, we use amphiphilic acrylamide copolymers to create a stable co-formulation of monomeric insulin and amylin analogues (lispro and pramlintide) with synchronous pharmacokinetics and ultra-rapid action. The co-formulation is stable for over 16 hours under stressed aging conditions, whereas commercial insulin lispro (Humalog) aggregates in 8 hours. The faster pharmacokinetics of monomeric insulin in this co-formulation resulted in increased insulin-pramlintide overlap of 75 {+/-} 6% compared to only 47 {+/-} 7% for separate injections. The co-formulation resulted in similar delay in gastric emptying compared to pramlintide delivered separately. In a glucose challenge, in rats the co-formulation reduced deviation from baseline glucose compared to insulin only, or separate insulin and pramlintide administrations. Further, comparison of interspecies pharmacokinetics of monomeric pramlintide suggests that pharmacokinetics observed for the co-formulation will be well preserved in future translation to humans. Together these results suggest that the co-formulation has the potential to improve mealtime glucose management and reduce patient burden in the treatment of diabetes.

category: bioengineering


title: Occult polyclonality of preclinical pancreatic cancer models drives in vitro evolution.

abstract: Intratumoral heterogeneity (ITH) is a hallmark of cancer. The advent of single-cell technologies has helped uncover ITH in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal striking ITH in assumptively oligoclonal pancreatic ductal adenocarcinoma (PDAC) cell lines. Our findings reveal a high degree of both genomic and transcriptomic heterogeneity in established and globally utilized PDAC cell lines, custodial variation induced by growing apparently identical PDAC cell lines in different laboratories, and profound transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous "control" lines in experiments. Further, while patient-derived organoid (PDOs) are known to reflect the cognate in vivo biology of the parental tumor, we identify transcriptomic shifts during ex vivo passage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern.

category: cancer biology


title: Estimating the age of poorly dated fossil specimens and deposits using a total-evidence approach and the fossilized birth-death process

abstract: Bayesian total-evidence approaches under the fossilized birth-death model enable biologists to combine fossil and extant data--while accounting for uncertainty in the ages of fossil specimens--in an integrative phylogenetic analysis. Fossil age uncertainty is a key feature of the fossil record as many empirical datasets may contain a mix of precisely dated and poorly dated fossil specimens or deposits. In this study, we explore whether reliable age estimates for fossil specimens can be obtained from Bayesian total-evidence phylogenetic analyses under the fossilized birth-death model. Through simulations based on the example of the Baltic amber deposit, we show that estimates of fossil ages obtained through such an analysis are accurate, particularly when the proportion of poorly dated specimens remains low and the majority of fossil specimens have precise dates. We confirm our results using an empirical dataset of living and fossil penguins by artificially increasing the age uncertainty around some fossil specimens and showing that the resulting age estimates overlap with the recorded age ranges. Our results are applicable to many empirical datasets where classical methods of establishing fossil ages have failed, such as the Baltic amber and the Gobi Desert deposits.

category: paleontology


title: First record of 'tail-belting' in two species of free-ranging rodents (Apodemus flavicollis and Apodemus agrarius): Adaptation to prevent frostbite?

abstract: Rodents are among the most successful mammals because they have the ability to adapt to a broad range of environmental conditions. Here, we present the first record of a hitherto unknown thermal adaptation to low temperatures that repeatedly occurred in two species of non-commensal rodents (Apodemus flavicollis and Apodemus agrarius) between January 16 and February 11, 2021. The classic rodent literature implies that rodents prevent heat loss via a broad range of behavioral adaptations including sheltering, sitting on their tails, curling into a ball, or huddling with conspecifics. Yet, we have repeatedly observed an undescribed behavior which we refer to as "tail-belting". The behavior was performed during the lowest temperatures, whereby animals - which were attracted out of their over-wintering burrows for a highly-palatable food reward - lift and curl the tail medially, before resting it on the dorsal, medial rump while feeding or resting between feeding bouts. We documented 115 instances of the tail-belting behavior; 38 in Apodemus agrarius, and 77 in Apodemus flavicollis. In A. flavicollis, this behavior was only observed below -6.9C, and occurred more often than in A. Agrarius. The latter only demonstrated the behavior below -9.5C. We further detail the environmental conditions under which the behavior is performed, and provide possible functions. We then set several directions for future research in this area.

category: physiology


title: Sugar Signaling Induces Dynamic Changes during Meristem Development in Arabidopsis

abstract:
Aerial parts of plants originate from pluripotent stem cells in the shoot apical meristem. Their population is maintained via the maintenance regulators WUSCHEL and CLAVATA3 in a negative feed-back loop. Meristem size is dynamic and undergoes a more than 2-fold expansion upon the transition to reproductive growth. The mechanism controlling this doming is largely unknown, but coinciding increased trehalose 6-phosphate and changed meristem size in overexpressing or knockdown lines of TREHALOSE PHOSPHATE SYNTHASE1 suggest a participation of sugar signaling. Here we show that TREHALOSE PHOSPHATE PHOSPHATASEJ is directly regulated by WUSCHEL. Plants with reduced levels of TREHALOSE PHOSPHATE PHOSPHATASEJ in the outer meristem layer are flowering early and its reduction in the late flowering clavata3 mutant, restores wild-type flowering. This is caused by a reduction of mature miR156 and increased expression of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes. We demonstrate that these are important for age pathway-induced flowering, in a negative feed-back loop with WUSCHEL downstream of the trehalose 6-phosphate pathway. In summary, our findings demonstrate a dynamic feed-back regulation between central maintenance and flowering time regulators with sugar signaling.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/439483v2_ufig1.gif" ALT="Figure 1">
View larger version (34K):
org.highwire.dtl.DTLVardef@988d6eorg.highwire.dtl.DTLVardef@16d469borg.highwire.dtl.DTLVardef@1366ce5org.highwire.dtl.DTLVardef@2753f0_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOSynopsis:C_FLOATNO Schematic illustration of dynamic feedback-regulations between sugar signaling, meristem maintenance and the age pathway of the flowering network in the transition SAM as compared to the vegetative SAM.During vegetative growth SAM maintenance is controlled by spatially separated WUS and CLV3 expression in a negative feedback-loop. At floral transition increased activity of the T6P pathway in the SAM uncouples this regulation. This results in an unreported special relocation of WUS expression and involves a transient negative feedback-loop between WUS and SPL4.

C_FIG

category: plant biology


title: Sugar Signaling Induces Dynamic Changes during Meristem Development to Regulate Flowering in Arabidopsis


title: Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

abstract:
The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we characterised two possible motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free swimming bacteria and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations, and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types, and in pathogenic and non-pathogenic strains.

ImportanceHere we characterised two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. Our two compounds were shown to inhibit flagellar motility at 10 M across multiple strains, from non-pathogenic E. coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (EHEC/UPEC) and lastly in sodium-powered Vibrio alginolyticus. Broad anti-motility compounds such as these are important tools in our efforts control virulence of pathogens in health and agricultural settings.


title: Insights into the acquisition of the pks island and production of colibactin in the Escherichia coli population

abstract:
The pks island codes for the enzymes necessary for synthesis of the genotoxin colibactin, which contributes to the virulence of Escherichia coli strains and is suspected of promoting colorectal cancer. From a collection of 785 human and bovine E. coli isolates, we identified 109 strains carrying a highly conserved pks island, mostly from the phylogroup B2, but also from phylogroups A, B1 and D. Different scenarios of pks acquisition were deduced from whole genome sequence and phylogenetic analysis. In the main scenario, pks was introduced and stabilized into certain sequence types (ST) of the B2 phylogroup, such as ST73 and ST95, at the asnW tRNA locus located in the vicinity of the yersiniabactin-encoding High Pathogenicity Island (HPI). In a few B2 strains, pks inserted at the asnU or asnV tRNA loci close to the HPI and occasionally was located next to the remnant of an integrative and conjugative element. In a last scenario specific to B1/A strains, pks was acquired, independently of the HPI, at a non-tRNA locus. All the pks-positive strains except 18 produced colibactin. Sixteen strains contained mutations in clbB or clbD, or a fusion of clbJ and clbK and were no longer genotoxic but most of them still produced low amount of potentially active metabolites associated with the pks island. One strain was fully metabolically inactive without pks alteration, but colibactin production was restored by overexpressing the ClbR regulator. In conclusion, the pks island is not restricted to human pathogenic B2 strains and is more widely distributed in the E. coli population, while preserving its functionality.

IMPACT STATEMENTColibactin, a genotoxin associated with the carcinogenicity of certain strains of E. coli, is encoded by a pathogenicity island called pks. We took advantage of a large collection of non-clinical E. coli strains originating from human and bovine hosts to explore the distribution, conservation and functionality of the pks island. We found that the pks island was not only present in the phylogroup B2 (and more specifically to certain B2 sublineages), but also in other genetic phylogroups, highlighting its capacity to disseminate though horizontal gene transfer. We identified various genetic pks configurations indicative of an introduction of the pks island into E. coli on multiple independent occasions. Despite the existence of various acquisition scenarios, we found that the pks sequences were highly conserved and pks-carrying strains were overwhelmingly capable of producing colibactin, suggesting that the pks island is under selective pressure, through the production of colibactin or other secondary metabolites. Future implications include the identification of such metabolites and their biological activities that could be advantageous to E. coli and enable its adaptation to various ecological niches.

DATA SUMMARYAll sequence data of the 785 E. coli used in this study are freely available from the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under the accession number PRJDB5579. This database was updated to include the sequence data obtained using ONT MinION for the E. coli reference strain SP15 and for E. coli strains ECSC054, JML285, KS-NP019, NS-NP030 and SI-NP020. The sequence data of E. coli strain UPEC129 obtained using PacBio instrument were deposited in the NCBI BioProject database and are available at https://www.ncbi.nlm.nih.gov/Traces/study/ under the accession number PRJNA669570. Hybrid MinION-Illumina and PacBio-Illumina assemblies are available at the NCBI nucleotide database. The genome sequences of 36 other E. coli reference strains and 7 non-E. coli strains were retrieved from NCBI.


title: A simple model explains the cell cycle-dependent assembly of centromeric nucleosomes in holocentric species

abstract: Centromeres are essential for chromosome movement. In independent taxa, species with holocentric chromosomes exist. In contrast to monocentric species, where no obvious dispersion of centromeres occurs during interphase, the organization of holocentromeres differs between condensed and decondensed chromosomes. During interphase, centromeres are dispersed into a large number of CENH3-positive nucleosome clusters in a number of holocentric species. With the onset of chromosome condensation, the centromeric nucleosomes join and form line-like holocentromeres. Using polymer simulations, we propose a mechanism, relying on the interaction between centromeric nucleosomes and Structural Maintenance of Chromosomes (SMC) proteins. All simulations represented a ~20 Mbp-long chromosome, corresponding to ~100,000 nucleosomes. Different sets of molecular dynamic simulations were evaluated by testing four parameters: 1) the concentration of Loop Extruders (LEs) corresponding to SMCs; 2) the distribution and number of centromeric nucleosomes; 3) the effect of centromeric nucleosomes on interacting LEs; and 4) the assembly of kinetochores bound to centromeric nucleosomes. We observed the formation of a line-like holocentromere, due to the aggregation of the centromeric nucleosomes when the chromosome was compacted into loops. A groove-like holocentromere structure formed after a kinetochore complex was simulated along the centromeric line. Similar mechanisms may also organize a monocentric chromosome constriction, and its regulation may cause different centromere types during evolution.

category: molecular biology


title: Conformational landscape of full-length Smad proteins

abstract:
Smad transcription factors, the main effectors of the TGF{beta} (transforming growth factor {beta}) network, have been shaped along the evolution of multicellular animals to regulate essential processes. Smad proteins have a mixed architecture of globular domains and flexible linkers and adopt distinct quaternary structures depending on their activation state and cellular context. Here we studied the structures of full-length Smad4 and Smad2 proteins through an integrative approach combining small-angle X-ray scattering and detailed atomic information obtained from Nuclear Magnetic Resonance spectroscopy, X-ray and molecular dynamic simulations. Both Smad4 and Smad2 populate ensembles of expanded/compact conformations, with the MH1 and MH2 domains tethered by intrinsically disordered linkers that provide conformational freedom to the proteins. In solution, Smad4 is monomeric, whereas Smad2 coexists as monomer-dimer-trimer association states, even without activation. Smad2 dimers, which were previously overlooked, are proposed as key building blocks that define the functional quaternary structures of Smad proteins.

HighlightsO_LISolution ensembles of full-length Smad2 and Smad4 proteins
C_LIO_LISmad4 populates dynamic extended and compact monomeric conformations
C_LIO_LISmad2 dimers may be the intermediate state to form heterotrimers with Smad4
C_LIO_LINew integrative methodological strategy for examining multi-domain and flexible proteins
C_LI


title: Resistance training rejuvenates the mitochondrial methylome in aged human skeletal muscle

abstract: Resistance training (RT) alters skeletal muscle nuclear DNA methylation patterns (or the methylome). However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older untrained males (65{+/-}7 years old) performed six weeks of full-body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 hours following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16-kilobase mtDNA methylome (254 CpG sites). Various biochemical assays were also performed, and older male data were compared to younger trained males (22{+/-}2 years old, n=7). RT increased whole-body lean tissue mass (p=0.017), VL thickness (p=0.012), and knee extensor torque (p=0.029) in older males. RT also profoundly affected the mtDNA methylome in older males, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p<0.05). Notably, several mtDNA sites presented a more "youthful" signature after RT in older males when comparisons were made to younger males. The 1.12 kilobase D-loop/control region on mtDNA, which regulates mtDNA replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H- and L-strand genes and increases in mitochondrial complex III and IV protein levels were also observed (p<0.05). This is the first study to show RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT remarkably evokes mitochondrial methylome profiles to mimic a more youthful signature in older males.


title: Adipsin promotes bone marrow adiposity by priming mesenchymal stem cells

abstract:
BackgroundBone marrow (BM) adipose tissue (BMAT) has been shown to be vital for regulating metabolism and maintaining skeletal homeostasis in the marrow niche. As a reflection of BM remodeling, BMAT is highly responsive to nutrient fluctuations, hormonal changes and metabolic disturbances such as obesity and diabetes mellitus. Expansion of BMAT has also been strongly associated with bone loss in mice and humans. However, the regulation of BM plasticity remains poorly understood, as does the mechanism that links changes in marrow adiposity with bone remodeling.

MethodsUsing C57BL/6 mice as a model, we employed the bone-protected PPAR{gamma} constitutive deacetylation (2KR), Adipsin, and its downstream effector, C3, knockout mice. These mice were challenged to thiazolidinedione treatment, calorie restriction, or aging in order to induce bone loss and MAT expansion. Analysis of bone density and marrow adiposity was performed using a CT scanner and by RNA analysis to assess adipocyte and osteoblast markers. For in vitro studies, primary bone marrow stromal cells (BMSCs) were isolated and subjected to osteoblastogenic or adipogenic differentiation or chemical treatment followed by morphological and molecular analyses. Clinical data was obtained from samples of a previous clinical trial of fasting and high calorie diet in healthy human volunteers.

ResultsWe have shown that Adipsin is the most up-regulated adipokine during BMAT expansion in mice and humans, in a PPAR{gamma} acetylation-dependent manner. Ablation of Adipsin in mice specifically inhibited BMAT expansion but not peripheral adipose depots, and improved bone mass during calorie restriction, thiazolidinedione treatment, and aging. These effects were mediated through its downstream effector, complement component C3, to prime common progenitor cells toward adipogenesis rather than osteoblastogenesis through inhibiting Wnt/{beta}- catenin signaling.

ConclusionsAdipsin promotes new adipocyte formation and affects skeletal remodeling in the BM niche. Our study reveals a novel mechanism whereby the BM sustains its own plasticity through paracrine and endocrine actions of a unique adipokine.

FundingThis work was supported by the National Institutes of Health T32DK007328 (NA), F31DK124926 (NA), R01DK121140 (JCL), R01AR068970 (BZ), R01AR071463 (BZ), R01DK112943 (LQ), and P01HL087123 (LQ).


title: One gene - two proteins: The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria

abstract:
Muscarinic acetylcholine receptors are G protein-coupled receptors activated by the endogenous neurotransmitter acetylcholine. We show here that the carboxyl terminal fragment of the muscarinic M2 receptor, containing the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site. Radioligand binding, biochemical and molecular biology experiments, combined to advanced fluorescence microscopy reveal that the M2tail, whose expression is upregulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here it controls oxygen consumption, cell proliferation and the formation of ROS by reducing oxidative phosphorylation. Genetic editing of the key methionine where cap-independent translation occurs, reveals the physiological role of this process in influencing cell proliferation and oxygen consumption. The expression of the C-terminal of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably unrelated to the canonical signalling function of the GPCR at the plasma membrane.

TeaserA G protein-coupled receptor fragment localizing in the mitochondria unexpectedly links two hitherto unrelated aspects of cell biology.


title: P5A-ATPases control the ER translocation of Wnt for neuronal migration

abstract: Wnt family are conserved secretory proteins required for developmental patterning and tissue homeostasis. The mechanisms underlying intracellular maturation and intercellular signal transduction of Wnt proteins have been extensively studied. However, how Wnt is targeted to the endoplasmic reticulum (ER) for processing and secretion remains elusive. Here we report that CATP-8/P5A-ATPase directs neuronal migration non-cell autonomously in C. elegans by regulating EGL-20/Wnt biogenesis. CATP-8 functions as a translocase to translocate EGL-20/Wnt nascent polypeptide into the ER by interacting with the hydrophobic core region of EGL-20 signal sequence. Such regulation of Wnt biogenesis by P5A-ATPase is conserved in human cells. These findings reveal physiological roles of P5A-ATPase in neural development and identify Wnt proteins as direct substrates of P5A-ATPase for ER translocation.


title: P5A-ATPase controls the ER translocation of Wnt in neuronal migration


title: To buffer or to be labile? A framework to disentangle demographic patterns and evolutionary processes

abstract: Until recently, natural selection was assumed to reduce temporal fluctuation in vital rates due to its negative effects on population dynamics - the so-called Demographic Buffering Hypothesis (DBH). After several failures to support the DBH in the two decades since it was first posited, an alternative hypothesis was suggested; the Demographic Lability Hypothesis (DLH), where population vital rates should track rather than buffer the environmental conditions. Despite the huge contribution of both hypotheses to comprehend the demographic strategies to cope the environmental stochasticity, it remains unclear if they represent two competing patterns or the extreme ends of a continuum encompassing all demographic strategies. To solve this historical debate, we unify several methods with an integrative theoretical approach where: i) using the sum of stochastic elasticity with respect to mean and variance - a first-order derivative approach - we rank species on a Buffering-Lability (DB-DL) continuum and ii) using the second-order derivative, we examine how vital rates are shaped by natural selection. Our framework, applied to 40 populations of 34 mammals, successfully placed the species on the DB-DL continuum. We could also link the species' position on the DB-DL continuum to their generation time and time to recovery. Moreover, the second-order derivative unveiled that vital rates with lower temporal variation are not necessarily under a strong pressure of stabilizing selection, as predicted by DBH and DLH. Our framework provides an important step towards unifying the different perspectives of DBH and DLH with key evolutionary concepts.

category: ecology


title: Filling gaps: fishing, genetics, and conservation of groupers, especially the comb grouper (badejo) (Mycteroperca acutirostris), in SE Brazil (2013-2020)

abstract: There are large gaps in our knowledge of the biology of important fish consumed by people in tropical countries, which makes conservation difficult. Small-scale fisheries are difficult to study and regulate, especially in countries with no systematic species monitoring. It is even more difficult to estimate the influence of these fisheries on vulnerable fish species and to diagnose possible damage to local fish populations. In this study, 490 individuals of badejo, or comb grouper (Mycteropeca acutirostris), were observed at the Posto 6 fishery in Copacabana, Rio de Janeiro, for the periods of 2013-2014 and 2018-2020. A pattern of decreasing catches was observed for comb grouper. Therefore, provided that the fishing gear and the number of fish have remained the same, the apparent decrease in comb grouper needs to be further investigated. The results provide information regarding the reproduction of comb grouper, with major spawning season around spring (September-December) and additional spawning during April in SE Brazil. Samples from 96 groupers along the coast of Brazil were obtained, and genetic analyses were conducted. The genetic information obtained for grouper species enabled us to determine the relative genetic proximity of M. acutirostris and Mycteroperca bonaci and to obtain information that can be useful for aquaculture and conservation.


title: Are Antarctic and sub-Antarctic marine food webs different?

abstract: Food web structure plays an important role in determining ecosystem stability against perturbations. High-latitude marine ecosystems are being affected by environmental stressors and biological invasions. In the West Antarctic Peninsula these transformations are mainly driven by climate change, while in the sub-Antarctic region by anthropogenic activities. Understanding the differences between these areas is necessary to monitor the changes that are expected to occur in the upcoming decades. Here, we compared the structure and stability of Antarctic (Potter Cove) and sub-Antarctic (Beagle Channel) marine food webs. We compiled species trophic interactions (predator-prey) and calculated complexity, structure and stability metrics. Even if both food webs presented the same connectance, we found important differences between them. The Beagle Channel food web is more complex, but less stable and sensitive to the loss of its most connected species, while the Potter Cove food web presented lower complexity and greater stability against perturbations.


title: Marine food webs are more complex but less stablein sub-Antarctic than in Antarctic regions


title: Intergenic ORFs as elementary structural modules of de novo gene birth and protein evolution

abstract: The noncoding genome plays an important role in de novo gene birth and in the emergence of genetic novelty. Nevertheless, how noncoding sequences properties could promote the birth of novel genes and shape the evolution and the structural diversity of proteins remains unclear. Therefore, by combining different bioinformatic approaches, we characterized the fold potential diversity of the amino acid sequences encoded by all intergenic ORFs (Open Reading Frames) of S. cerevisiae with the aim of (i) exploring whether the large structural diversity observed in proteomes is already present in noncoding sequences, and (ii) estimating the potential of the noncoding genome to produce novel protein bricks that can either give rise to novel genes or be integrated into pre-existing proteins, thus participating in protein structure diversity and evolution. We showed that amino acid sequences encoded by most yeast intergenic ORFs contain the elementary building blocks of protein structures. Moreover, they encompass the large structural diversity of canonical proteins with strikingly the majority predicted as foldable. Then, we investigated the early stages of de novo gene birth by identifying intergenic ORFs with a strong translation signal in ribosome profiling experiments and by reconstructing the ancestral sequences of 70 yeast de novo genes. This enabled us to highlight sequence and structural factors determining de novo gene emergence. Finally, we showed a strong correlation between the fold potential of de novo proteins and the one of their ancestral amino acid sequences, reflecting the relationship between the noncoding genome and the protein structure universe.


title: Target enrichment improves phylogenetic resolution in the genus Zanthoxylum (Rutaceae) and indicates both incomplete lineage sorting and hybridization events

abstract:
Background and aimsZanthoxylum L. is the only pantropical genus within Rutaceae, with a few species native to temperate eastern Asia and North America. Efforts using Sanger sequencing failed to resolve the backbone phylogeny of Zanthoxylum. In this study, we employed target enrichment high-throughput sequencing to improve resolution. Gene trees were examined for concordance and sectional classifications of Zanthoxylum were evaluated. Off-target reads were investigated to identify putative single-copy markers for bait refinement, and low-copy markers for evidence of putative hybridization events.

MethodsWe developed a custom bait set for target enrichment of 745 exons in Zanthoxylum and applied it to 45 Zanthoxylum species and one Tetradium species as the outgroup. Illumina reads were processed via the HybPhyloMaker pipeline. Phylogenetic inferences were conducted using coalescent and concatenated methods. Concordance was assessed using quartet sampling. Off-target reads were assembled and putative single- and low-copy genes were extracted. Additional phylogenetic analyses were performed based on these alignments.

Key resultsFour major clades are supported within Zanthoxylum: the African clade, the Z. asiaticum clade, the Asian-Pacific-Australian clade, and the American-eastern Asian clade. While overall support has improved, regions of conflict are similar to those previously observed. Gene tree discordances indicate a hybridization event in the ancestor of the Hawaiian lineage, and incomplete lineage sorting for the American backbone. Off-target putative single-copy genes largely confirm on-target results, and putative low-copy genes provide additional evidence for hybridization in the Hawaiian lineage. Only two of the five sections of Zanthoxylum are resolved as monophyletic.

ConclusionTarget enrichment is suitable to assess phylogenetic relationships in Zanthoxylum. Our phylogenetic analyses reveal that current sectional classifications need revision. Quartet tree concordance indicates several instances of reticulate evolution. Off-target reads are proven useful to identify additional phylogenetically informative regions for bait refinement or gene tree based approaches.


title: Genomic patterns of transcription-replication interactions in mouse primary B cells

abstract: Conflicts between transcription and replication machinery are a potent source of replication stress and genome stability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1,009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. While TRIs themselves map to early replicating regions, they exhibit enhanced Replication Protein A association and replication fork termination, marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.

category: genomics


title: A phased genome assembly for allele-specific analysis in Trypanosoma brucei

abstract:
Many eukaryotic organisms are diploid or even polyploid, i.e. they harbour two or more independent copies of each chromosome. Yet, to date most reference genome assemblies represent a mosaic consensus sequence in which the homologous chromosomes have been collapsed into one sequence. This procedure generates sequence artefacts and impedes analyses of allele-specific mechanisms. Here, we report the allele-specific genome assembly of the diploid unicellular protozoan parasite Trypanosoma brucei.

As a first step, we called variants on the allele-collapsed assembly of the T. brucei Lister 427 isolate using short-read error-corrected PacBio reads. We identified 96 thousand heterozygote variants across the genome (average of 4.2 variants / kb), and observed that the variant density along the chromosomes was highly uneven. Several long (>100 kb) regions of loss-of-heterozigosity (LOH) were identified, suggesting recent recombination events between the alleles. By analysing available genomic sequencing data of multiple Lister 427 derived clones, we found that most LOH regions were conserved, except for some that were specific to clones adapted to the insect lifecycle stage. Surprisingly, we also found that some Lister 427 clones were aneuploid. We found evidence of trisomy in chromosome five (chr 5), chr 2, chr 6 and chr 7. Moreover, by analysing RNA-seq data, we showed that the transcript level is proportional to the ploidy, evidencing the lack of a general expression control at the transcript level in T. brucei.

As a second step, to generate an allele-specific genome assembly, we used two powerful datatypes for haplotype reconstruction: raw long reads (PacBio) and chromosome conformation (Hi-C) data. With this approach, we were able to assign 99.5% of all heterozygote variants to a specific homologous chromosome, building a 66 Mb long T. brucei Lister 427 allele-specific genome assembly. Hereby, we identified genes with allele-specific premature termination codons and showed that differences in allele-specific expression at the level of transcription and translation can be accurately monitored with the fully phased genome assembly.

The obtained reference-grade allele-specific genome assembly of T. brucei will enable the analysis of allele-specific phenomena, as well as the better understanding of recombination and evolutionary processes. Furthermore, it will serve as a standard to  benchmark much needed automatic genome assembly pipelines for highly heterozygous wild species isolates.


title: Selection for or against Escape from Nonsense Mediated Decay is a Novel Signature for the Detection of Cancer Genes

abstract: Escape from nonsense mediated decay (NMD-) can produce activated or inactivated gene products, and bias in rates of escape can identify functionally important genes in germline disease. We hypothesized that the same would be true of cancer genes, and tested for NMD- bias within The Cancer Genome Atlas pan-cancer somatic mutation dataset. We identify 29 genes that show significantly elevated or suppressed rates of NMD-. This novel approach to cancer gene discovery reveals genes not previously cataloged as potentially tumorigenic, and identifies many potential driver mutations in known cancer genes for functional characterization.


title: Rapid auxin-mediated phosphorylation of Myosin regulates trafficking and polarity in Arabidopsis

abstract: The signaling molecule auxin controls plant development through a well-known transcriptional mechanism that regulates many genes. However, auxin also triggers cellular responses within seconds or minutes, and mechanisms mediating such fast responses have remained elusive. Here, we identified an ultrafast auxin-mediated protein phosphorylation response in Arabidopsis roots that is largely independent of the canonical TIR1/AFB receptors. Among targets of this novel response are Myosin XI and its adaptor protein MadB2. We show that their auxin-mediated phosphorylation regulates trafficking and polar, subcellular distribution of PIN auxin transporters. This phosphorylation-based auxin signaling module is indispensable during developmental processes that rely on auxin-mediated PIN repolarization, such as termination of shoot gravitropic bending or vasculature formation and regeneration. Hence, we identified a fast, non-canonical auxin response targeting multiple cellular processes and revealed auxin-triggered phosphorylation of a myosin complex as the mechanism for feedback regulation of directional auxin transport, a central component of auxin canalization, which underlies self-organizing plant development.


title: Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveal Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation

abstract:
Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504, which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing non-preferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, {beta}-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation and degradation in biomass hydrolysates.

IMPORTANCEYarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates. Our investigation into the genetic diversity of undomesticated Y. lipolytica strains, coupled with detailed strain characterization and proteomic analysis, revealed metabolic processes and regulatory elements conferring desirable phenotypes for growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates by these natural variants. This study provides a better understanding of the robust metabolism of Y. lipolytica and suggests potential metabolic engineering strategies to enhance its performance.

category: systems biology


title: Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

abstract: The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter (AOTF), up to 6 subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low ([~]1%) crosstalks and high temporal resolutions (down to [~]10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and FRET (Forster resonance energy transfer) fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.


title: The effect of different exercise training modes on dentate gyrus neurodegeneration and synaptic plasticity in morphine-dependent rats

abstract: Various impacts of exercise on brain performance have been documented following morphine dependence induction; however, the underlying neuronal mechanisms remain unclear. The present research was done to investigate the impact of different exercise training modes on neuronal maturation, and synaptic plasticity in the perforant pathway (PP)-dentate gyrus (DG) synapse in the morphine-dependent rats. Five groups, including a control group (Con, ten healthy rats) and forty morphine-dependent rats were considered as follows (n=10/group): 1) sedentary-dependent (Sed-D); 2) endurance exercise-dependent (En-D); 3) strength exercise-dependent (St-D); and 4) concurrent exercise-dependent (Co-D). The exercise training groups were subjected to endurance, strength, and concurrent training 5 days per week for 10 weeks. After training sessions, the field excitatory postsynaptic potential (fEPSP) slope and population spike (PS) amplitude in DG were determined in response to high-frequency stimulation (HFS) of the PP. For assessing neurogenesis NeuroD level was evaluated after performing all experiments. Concurrent training increased PS amplitude and EPSP than the control group. NeuroD in the morphine-dependent rats significantly decreased, but concurrent training returned the NeuroD to the healthy rat level. Concurrent training can ameliorate synaptic plasticity impairment in morphine-dependent rats through neurogenesis promotion. According to the results, concurrent training can be an appropriate novel candidate for treating opioid addiction.

category: animal behavior and cognition


title: Accelerated electron transfer in nanostructured electrodes improves the sensitivity of electrochemical biosensors

abstract: Electrochemical biosensors hold the exciting potential to integrate molecular detection with signal processing and wireless communication in a miniaturized, low-cost system. However, as electrochemical biosensors are miniaturized to the micron scale, their detection sensitivity degrades precipitously, thereby greatly reducing their utility in the context of molecular diagnostic applications. Studies have reported that nanostructured electrodes can greatly improve electrochemical biosensor sensitivity, but the underlying mechanism remains poorly understood, thus making it difficult to fully exploit this phenomenon to improve biosensor performance. In this work, we propose and experimentally validate a novel mechanism in which electron transfer is physically accelerated within nanostructured electrodes due to reduced charge screening, resulting in enhanced sensitivity. We show that this mechanism can be exploited to achieve up to 24-fold increase in signal and nearly four-fold lower limit-of-detection relative conventional planar electrodes. This accelerated electron transfer mechanism should prove broadly applicable for improving the performance of electrochemical biosensors.


title: Relationing of Labellum and Glossae Biomorphological characteristics of honeybee workers as breeding tool on honey collection potential of Apis mellifera L. honeybee colonies

abstract: This study focused on the correlation of honey collection Potential and the length and width of labellum and glossae in worker honey bees (Apis mellifera Ligustica). Sixty honeybee A. mellifera L. colonies were selected, among these 60 colonies, 3 worker bees were sampled from each colony total numbers of samples collected were 180 adult worker foraging bees. Fifteen colonies for each group were used to check the correlation of honey production with length of labellum, width of labellum, length of glossae and the width of glossae respectively. These worker bees were bought to the laboratory frozen, boiled, dissected and mounted on the slides. Measurements of the labellum length, labellum width, glossae length and glossae width were taken by the stereomicroscope with ocular micrometer at 0.8X magnification. Correlation values for the honey collectionand length and width of labellum and glossae were high and positive. These Results support the perception that worker bees with larger labellum and glossae have more ability for honey collection Potential. It is concluded that Biomorphological characters of labellum and glossae are significantly correlated with the honeycollection Potential in A. mellifera L.

category: zoology


title: Clinical Efficacy of Prebiotics and Glycosaminoglycans versus Placebo In Dogs with Food Responsive Enteropathy Receiving a Hydrolyzed Diet: A Pilot Study

abstract:
Induction of remission is easily achieved with dietary treatment in dogs diagnosed with Food Responsive Chronic Enteropathy (FRD). Administration of prebiotics and glycosaminoglycans (GAGs) may improve epithelial cell integrity and therefore be useful as adjunct treatment. This study evaluated whether the relapse rate of FRD dogs that are switched back to a normal diet can be influenced using supplemental treatment with prebiotics and GAGs.

A randomized, controlled clinical trial (RCCT) was performed in dogs diagnosed with FRD. Dogs were diagnosed based on clinical exclusion diagnosis, endoscopic biopsies showing predominantly lymphoplasmacytic infiltration, and response to dietary treatment. Dogs were randomized to be fed a combination of prebiotics and GAGs (group 1) or placebo (group 2) in addition to a hydrolyzed diet. At week 10, a second endoscopy was performed and dogs were switched back to normal diet. Relapse rate was monitored every 2 weeks after that until week 18.

Statistical analysis was performed for each outcome (Canine Chronic Enteropathy Clinical Activity Index (CCECAI), clinicopathological data, endoscopic scoring, mWSAVA histological scoring index (mWSAVA), and number of relapses following switch to normal diet) using a linear mixed effects model for group comparison. Time, group, and their interactions were included as a fixed effect, whereas each dog was treated as a random effect.

Of the 35 dogs enrolled into the clinical trial, 10 in each group reached the point of second endoscopy. A total of 13 dogs (n=8 in group 1 and n=5 in group 2) reached the trial endpoint of 18 weeks. After switching back to normal diet, none of the dogs in either group relapsed. No significant differences were found over time or between groups for CCECAI, endoscopy scoring and histological scoring. Although there was a clinical worsening in the placebo group after switching back to the original diet, this was not statistically significant (CCECAI p=0.58). Post-hoc power calculation revealed that 63 dogs per group would have been needed to detect statistically significant differences in CIBDAI between treatment groups. Standard dietary treatment induced rapid clinical response in all cases, however, additional supplementation with prebiotics and GAGs did not significantly improve clinical outcome within 4 months after switching back to normal diet. Since there are very few RCCT published in CE in dogs, this pilot study provides important power analyses for planning of further studies.

category: immunology


title: Interaction of the Causal Agent of Apricot bud gall Acalitus phloeocoptes (Nalepa) with Apricot: Implications in Infested Tissues

abstract: Apricot bud gall mite, Acalitus phloeocoptes (Nalepa), is a destructive arthropod pest that causes significant economic losses to apricot trees worldwide. Infested bud examination revealed that the starch granules in the bud axon were extended at the onset of the attack. During the later stages of the attack, the cytoplasm was distributed in apricot. The results also demonstrated that the accumulation of large amounts of cytokinin (zeatin, ZT) and auxin (indoleacetic acid, IAA) led to rapid bud proliferation during the rapid growth period. Abscisic acid (ABA) controls the development of gall buds and plays a vital role in gall bud maturity. The reduction of gibberellic acid (GA3) content led to rapid lignification at the later phase of bud development. Our results reveal the mechanism underlying the interaction of apricot bud gall with its parasite and provide reliable information for designing valuable breeding programs. This study will be quite useful for pest management and will provide a comprehensive evaluation of ecology-based cost-effective control, life history and demographic parameters of A. phloeocoptes.

category: microbiology


title: Berberine as siderophore from Talaromyces trachyspermus: augmentation and characterization".

abstract: In the present study, a siderophore compound produced by an endophytic fungus, Talaromyces trachyspermus was optimized for maximum production, 88.9 % SU by applying Plackett-Burman design and Response Surface Methodology through Central Composite Design that showed the succinic acid (1.141 g/L), sucrose (31.028 g/L) and temperature (27.475 {degrees}C) as significant factors. On scale up, a further increase in siderophore yield was obtained (by 3%) The compound was extracted, purified and detected chemically as catecholate siderophore showing max. {lambda} absorbance at 279nm. Contained of hydroxy benzene as shown by GC-MS analysis and further identified as berberine by HRLC-MS studies. The compound berberine is clinically a very important drug with several ethnobotanical properties. This is rare to report fungal catecholate and first to report the production of berberine from Talaromyces species.


title: The ubiquitin ligase HOIL-1L regulates immune responses by interacting with linear ubiquitin chains

abstract: The Linear Ubiquitin Assembly Complex (LUBAC), composed of HOIP, HOIL-1L and SHARPIN, promotes Tumor Necrosis Factor (TNF)-dependent NF-{kappa}B signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological role in vivo has remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice (Hoil-1lT20;A;R208A/T201A;R208A) expressing a HOIL-1L NZF mutant, and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production and liver damage markers. Cells derived from the HOIL-1L knockin mice show reduced TNF-dependent NF-{kappa}B activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that the HOIL-1L-NZF domain cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammation in vivo.

category: cell biology


title: Identification of the combining abilit o f grain yield and its components in hybrid barley populations based on topcross analysis

abstract: The topcross method for assessing combining abilityis more economical and less laborious compared to diallel analysis, and also allows the breeder to obtain quite valuable information about the inbred material. In this research, the determination of the general (GCA) and specific (SCA) combining ability of barley (Hordeum vulgare L.) hybridsin two regions of Kazakhstan contrasting in soil and climatic conditions,the role of additive and non-additive genes in the determination of thetraits understudyhas beenrevealed. It is concluded thatthe predominance of additive gene interactions in the control of the traits understudyin theconditions of the Aral Sea region indicates the possibility of effective selection already in the F2 generation, and in the piedmontzone of the Almaty region, due to the high determination of these traits by dominant genes, it is necessary to differentiate the populations of hybrids, starting from the first generation and further selection shall be carried out in several cycles until the achievement of homozygosity of loci carrying dominant genes. Consequently, the genetic contribution of the additive and non-additive effects of genes to the determination and inheritance of the studied traits significantly depends on the conditions of growing the genotypes of spring barley. Of greatest practical interest are the varieties Rihane, WI2291/Roho/WI2269 from the International Center ICARDA and the variety-tester Odessa 100 (Odessa Selection and Genetic Institute, Ukraine) with high GCA and SCA effects, little dependent on growing conditions, which are recommended to be used as reliable donors in breeding programs.

category: genetics


title: Ancient DNA reveals that few GWAS loci have been strongly selected during recent human history

abstract: Genetic data from ancient humans has provided new evidence in the study of loci thought to be under historic selection, and thus is a powerful tool for identifying instances of selection that might be missed by methods that use present-day samples alone. Using a curated set of disease-associated variants from the NHGRI-EBI GWAS Catalog, we provide an analysis to identify disease-associated variants that bear signatures of selection over time. After accounting for the fact that not every ancient individual contributed equally to modern genomes, a Bayesian inference method was used to infer allele frequency trajectories over time and determine which disease-associated loci exhibit signatures of natural selection. Of the 2,709 variants analyzed in this study, 895 show at least a weak signature of selection (|s| > 0.001), including multiple variants that are introgressed from Neanderthals. However, only nine disease-associated variants show a signature of strong selection (|s| > 0.01). Additionally, we find that many risk-associated alleles have increased in frequency during the past 10,000 years. Overall, we find that disease-associated variants from GWAS are governed by nearly neutral evolution. Exceptions to this broad pattern include GWAS loci that protect against asthma and variants in MHC genes. Ancient samples allow us an unprecedented look at how our species has changed over time, and our results represent an important early step in using this new source of data to better understand the evolution of hereditary disease risks.

category: evolutionary biology


title: Selective prefrontal-amygdala circuit interactions underlie social and nonsocial valuation in rhesus macaques

abstract:
Lesion studies in macaques suggest dissociable functions of the orbitofrontal cortex (OFC) and medial frontal cortex (MFC), with OFC being essential for goal-directed decision making and MFC supporting social cognition. Bilateral amygdala damage results in impairments in both of these domains. There are extensive reciprocal connections between these prefrontal areas and the amygdala; however, it is not known whether the dissociable roles of OFC and MFC depend on functional interactions with the amygdala. To test this possibility, we compared the performance of male rhesus macaques (Macaca mulatta) with crossed surgical disconnection of the amygdala and either MFC (MFC x AMY, n=4) or OFC (OFC x AMY, n=4) to a group of unoperated controls (CON, n=5). All monkeys were assessed for their performance on two tasks to measure: (1) food-retrieval latencies while viewing videos of social and nonsocial stimuli in a test of social interest, and (2) object choices based on current food value using reinforcer devaluation in a test of goal-directed decision making. Compared to the CON group, the MFC x AMY group, but not the OFC x AMY group, showed significantly reduced food-retrieval latencies while viewing videos of conspecifics, indicating reduced social valuation and/or interest. By contrast, on the devaluation task, group OFC x AMY, but not group MFC x AMY, displayed deficits on object choices following changes in food value. These data indicate that the MFC and OFC must functionally interact with the amygdala to support normative social and nonsocial valuation, respectively.

Significance StatementAscribing value to conspecifics (social) vs. objects (nonsocial) may be supported by distinct but overlapping brain networks. Here we test whether two nonoverlapping regions of the prefrontal cortex, the medial frontal cortex and the orbitofrontal cortex, must causally interact with the amygdala to sustain social valuation and goal-directed decision making, respectively. We found that these prefrontal-amygdala circuits are functionally dissociable, lending support for the idea that medial frontal and orbital frontal cortex make independent contributions to cognitive appraisals of the environment. These data provide a neural framework for distinct value assignment processes and may enhance our understanding of the cognitive deficits observed following brain injury or in the development of mental health disorders.


title: Inhibition of gibberellin accumulation by water deficiency promotes fast and long-term 'drought avoidance' responses in tomato

abstract: Plants reduce transpiration to avoid dehydration during drought episodes by stomatal closure and inhibition of canopy growth. While abscisic acid (ABA) has a primary role in  drought avoidance, previous studies suggest that gibberellin (GA), might also be involved. Here we show in tomato (Solanum lycopersicum) that shortage of water inhibited the expression of the GA biosynthesis genes GA20 oxidase1 (GA20ox1) and GA20ox2 and induced the GA-deactivating gene GA2ox7 in leaves and guard cells, resulting in reduced bioactive GA levels. Drought regulation of GA metabolism was mediated by ABA-dependent and independent pathways, and by the transcription factor DEHYDRATION RESPONSIVE ELEMENT BINDING (DREB), TINY1. Mutations in GA20ox1 and GA20ox2 reduced water loss due to the smaller canopy area. On the other hand, loss of GA2ox7 did not affect leaf size, but attenuated stomatal response to water deficiency; during soil dehydration, ga2ox7 plants closed their stomata and reduced transpiration later than WT, suggesting that ga2ox7 stomata are hyposensitive to soil dehydration. Together, the results suggest that drought-induced GA deactivation in guard cells contributes to stomatal closure at the early stages of soil dehydration, whereas inhibition of GA synthesis in leaves promotes mainly the long-term reduction in canopy growth to reduce transpiration area.


title: Epigenomic profiling of stem cells within the pilosebaceous unit identifies PRDM16 as a regulator of sebaceous gland homeostasis

abstract: The epidermis consists of different compartments such as the hair follicle (HF), sebaceous gland (SG) and interfollicular epidermis (IFE), each containing distinct stem cell (SC) populations. However, with the exception of the SCs residing within the HF bulge, other epidermal SC populations remain less well understood. Here we used an epigenomic strategy that combines H3K27me3 ChIP-seq and RNA-seq profiling to identify major regulators of pilosebaceous unit (PSU) SC located outside the bulge. When applied to the bulk of PSU SC isolated from mouse skin our approach identified both previously known and potentially novel non-bulge PSU SC regulators. Among the latter, we found that PRDM16 was predominantly enriched within the Junctional Zone (JZ), which harbors SC that contribute to renewal of the upper HF and the SG. To investigate PRDM16 function in the PSU SC, we generated an epidermal-specific Prdm16 Knock-out mouse model (K14-Cre-Prdm16fl/fl). Notably, SG homeostasis was disturbed upon loss of PRDM16 resulting in enlarged SGs, and excessive sebum production, resembling some of the features associated with human acne and sebaceous hyperplasia. Importantly, PRDM16 is essential to shut down proliferation in differentiating sebocytes. Overall, our study provides a list of putative novel regulators of PSU SC outside the bulge and identifies PRDM16 as a major regulator of SG homeostasis.


title: The extracellular matrix controls stem cell specification and tissue morphology in the developing and adult gut

abstract: The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. In recent years, the signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. Here, we set out to systematically compare the effect of two major ECM classes, fibrillar collagen type I and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all differentiated and undifferentiated cell types present in both cultures, albeit at different ratios. Specific to the collagen culture was a subset of cells with a fetal-like gene expression program. In contrast, laminin, but not collagen IV, increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture, resembles the gut development in vivo. Here, the ECM is dramatically remodelled by mesenchymal cells, which is accompanied by a specific and local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.


title: The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult gut


title: Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

abstract:
Stem cell research is emerging both as a scientifically and clinically relevant area. One of the current challenges in stem cell research and regenerative medicine is assessment of the pluripotency state of induced pluripotent stem (iPS) cells. Once a stem cell differentiation process is initiated the challenge is how to assess the state of differentiation, and the purity of the differentiated cell population. Stem cell potency and differentiation states are determined by tightly coordinated activity of developmental signaling pathways, such as the Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B pathways. Source of the stem cells and culture protocols may influence stem cell phenotype, with potential consequences for pluripotency and in general for experimental reproducibility. Human pluripotent embryonic (hES) and iPS stem cell lines under different culture conditions, organ derived multipotent stem cells, and differentiated cell types, were phenotyped with respect to functional activity of developmental signaling pathways.

MethodsWe previously reported on the development and validation of a novel assay platform for quantitative measurement of activity of multiple signal transduction pathways (STP) simultaneously in a single sample, based on interpreting a preselected set of target mRNA expression levels. Assays were used to calculate Notch, Hedgehog, TGF{beta}, Wnt, PI3K, MAPK-AP1, and NF{kappa}B signal transduction pathway activity scores for individual cell samples, using publicly available Affymetrix expression microarray data.

ResultsCulture conditions (e.g. mouse versus human feeder) influenced pluripotent stem cell pathway activity profiles. hES and iPS stem cell lines cultured in the same lab under similar conditions showed minimal variation in pathway activity profile despite different genetic backgrounds, while across different labs larger variations were measured, even for the same stem cell line. Pathway activity scores for PI3K, MAPK, Hedgehog, Notch, TGF{beta}, and NF{kappa}B pathways rapidly decreased upon pluripotent stem cell differentiation, while increasing for the Wnt pathway. Further differentiation to intestinal progenitor cells resulted in higher PI3K, Wnt and Notch pathway activity. In multipotent intestinal crypt stem cells obtained from intestinal mucosa samples, similar Notch and even higher Wnt pathway activity were measured, which disappeared upon differentiation to mucosal cells.

ConclusionResults support the validity of using these STP assays for quantitative phenotyping of stem cells and differentiated derivatives, and enabled definition of a pluripotency profile with high PI3K, MAPK, Hedgehog, TGF{beta}, Notch, and NF{kappa}B, and low Wnt pathway activity scores.

Measurement of combined signaling pathway activity scores is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture and differentiation. It enables controlled manipulation of signaling pathway activity using pathway targeting compounds. An envisioned additional utility may lie in quality control for regenerative medicine purposes.

category: developmental biology


title: Multipotent progenitors and hematopoietic stem cells arise independently during the endothelial to hematopoietic transition in the early mouse embryo

abstract: During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSC). Although previous studies have shown that yolk sac-derived erythromyeloid progenitors and HSC emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSC originate from common HE. Here we demonstrate by clonal assays and single cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSC. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSC arise independently from HE with distinct phenotypic and transcriptional properties.


title: Functional analysis of deoxyhexose sugar utilization in Escherichia coli reveals fermentative metabolism under aerobic conditions

abstract:
L-rhamnose and L-fucose are the two main 6-deoxyhexoses Escherichia coli can use as carbon and energy sources. Deoxyhexose metabolism leads to the formation of lactaldehyde whose fate depends on oxygen availability. Under anaerobic conditions, lactaldehyde is reduced to 1,2-propanediol whereas under aerobic condition, it should be oxidised into lactate and then channelled into the central metabolism. However, although this all-or-nothing view is accepted in the literature, it seems overly simplistic since propanediol is also reported to be present in the culture medium during aerobic growth on L-fucose. To clarify the functioning of 6-deoxyhexose sugar metabolism, a quantitative metabolic analysis was performed to determine extra- and intracellular fluxes in E. coli K-12 MG1655 (a laboratory strain) and in E. coli Nissle 1917 (a human commensal strain) during anaerobic and aerobic growth on L-rhamnose and L-fucose. As expected, lactaldehyde is fully reduced to 1,2-propanediol in anoxic conditions allowing complete reoxidation of the NADH produced by glyceraldehyde-3-phosphate-dehydrogenase. We also found that net ATP synthesis is ensured by acetate production. More surprisingly, lactaldehyde is also primarily reduced into 1,2-propanediol under aerobic conditions. For growth on L-fucose, 13C-metabolic flux analysis revealed a large excess of available energy, highlighting the need to better characterize ATP utilization processes. The probiotic E. coli Nissle 1917 strain exhibits similar metabolic traits, indicating that they are not the result of the K-12 strains prolonged laboratory use.

IMPORTANCEE. colis ability to survive, grow and colonize the gastrointestinal tract stems from its use of partially digested food and hydrolysed glycosylated proteins (mucins) from the intestinal mucus layer as substrates. These include L-fucose and L-rhamnose, two 6-deoxyhexose sugars, whose catabolic pathways have been established by genetic and biochemical studies. However, the functioning of these pathways has only partially been elucidated. Our quantitative metabolic analysis provides a comprehensive picture of 6-deoxyhexose sugar metabolism in E. coli under anaerobic and aerobic conditions. We found that 1,2-propanediol is a major by-product under both conditions, revealing the key role of fermentative pathways in 6-deoxyhexose sugar metabolism. This metabolic trait is shared by both E. coli strains studied here, a laboratory strain and a probiotic strain. Our findings add to our understanding of E. colis metabolism and of its functioning in the bacteriums natural environment.


title: HCV spread kinetics reveal varying contributions of transmission modes to infection dynamics

abstract:
Hepatitis C virus (HCV) is capable of spreading within a host by two different transmission modes: cell-free and cell-to-cell. Although viral dissemination and diffusion of viral particles facilitates the infection of distant cells, direct cell-to-cell transmission to uninfected neighboring cells is thought to shield the virus from immune recognition. However, the contribution of each of these transmission mechanisms to HCV spread is unknown. To dissect the contribution of these different transmission modes to HCV spread, we measured HCV lifecycle kinetics and used an in vitro spread assay to monitor HCV spread kinetics after low multiplicity of infection in the absence and presence of a neutralizing antibody that blocks cell-free spread. By analyzing these data with a spatially-explicit mathematical model that describes viral spread on a single-cell level, we quantified the contribution of cell-free and cell-to-cell spread to the overall infection dynamics and show that both transmission modes act synergistically to enhance the spread of infection. Thus, the simultaneous occurrence of both transmission modes likely represents an advantage for HCV that may contribute to the efficient establishment of chronic infection. Notably, the relative contribution of each viral transmission mode appeared to vary dependent on different experimental conditions and suggests that viral spread is optimized according to the environment. Together, our analyses provide insight into the transmission dynamics of HCV and reveal how different transmission modes impact each other.

ImportanceHepatitis C Virus can spread within a host by diffusing viral particles or direct cell-to-cell transfer of viral material between infected and uninfected cells. To which extend these cell-free and cell-to-cell transmission modes contribute to HCV spread, establishment of chronicity and antiviral escape is still unknown. By combining in vitro experimental HCV spread data with a multi-scale mathematical model we have disentangled the contribution and interplay of cell-free and cell-to-cell transmission modes during HCV infection. Our analysis revealed synergistic effects between the two transmission modes, with the relative contribution of each transmission mode varying dependent on the experimental conditions. This highlights the adaptability of the virus and suggests that transmission modes might be optimized dependent on the environment, which could contribute to viral persistence.