title: Enrichment of non-B-form DNA at D. melanogaster centromeres

abstract: Centromeres are essential chromosomal regions that mediate the accurate inheritance of genetic information during eukaryotic cell division. Despite their conserved function, centromeres do not contain conserved DNA sequences and are instead epigenetically marked by the presence of the centromere-specific histone H3 variant CENP-A (centromeric protein A). The functional contribution of centromeric DNA sequences to centromere identity remains elusive. Previous work found that dyad symmetries with a propensity to adopt non-canonical secondary DNA structures are enriched at the centromeres of several species. These findings lead to the proposal that such non-canonical DNA secondary structures may contribute to centromere specification. Here, we analyze the predicted secondary structures of the recently identified centromere DNA sequences from Drosophila melanogaster. Although dyad symmetries are only enriched on the Y centromere, we find that other types of non-canonical DNA structures, including DNA melting and G-quadruplexes, are common features of all D. melanogaster centromeres. Our work is consistent with previous models suggesting that non-canonical DNA secondary structures may be conserved features of centromeres with possible implications for centromere specification.


title: Pentosan polysulfate inhibits attachment and infection by SARS-CoV-2 in vitro: insights into structural requirements for binding.

abstract: Two years since the outbreak of the novel coronavirus SARS-CoV-2 pandemic, there remain few clinically effective drugs to complement vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side-effect. One alternative, with structural similarities to heparin is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights, inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using NMR spectroscopy and LC-MS, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective at inhibiting cell infection than low molecular weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection.

category: biochemistry


title: Control-based continuation: a new approach to prototype synthetic gene networks

abstract: Control-Based Continuation (CBC) is a general and systematic method to carry out the bifurcation analysis of physical experiments. CBC does not rely on a mathematical model and thus overcomes the uncertainty introduced when identifying bifurcation curves indirectly through modelling and parameter estimation. We demonstrate, in silico, CBC applicability to biochemical processes by tracking the equilibrium curve of a toggle switch which includes additive process noise and exhibits bistability. We compare results obtained when CBC uses a model-free and model-based control strategy and show that both can track stable and unstable solutions, revealing bistability. We then demonstrate CBC in conditions more representative of a real experiment using an agent-based simulator describing cells growth and division, cell-to-cell variability, spatial distribution, and diffusion of chemicals. We further show how the identified curves can be used for parameter estimation and discuss how CBC can significantly accelerate the prototyping of synthetic gene regulatory networks.

category: synthetic biology


title: Structural flexibility of the SARS-CoV-2 genome relevant to variation, replication, pathogenicity, and immune evasion.

abstract: The SARS-CoV-2 pandemic continues to be driven by viral variants. Most research has focused on structural proteins and on site-specific mutations. Here, we describe recombination events involving genomic terminal sequences in SARS-CoV-2 and related viruses leading to structural rearrangements in terminal and coding regions and discuss their potential contributions to viral variation, replication, pathogenicity, and immune evasion.

category: microbiology


title: Human gut bifidobacteria inhibit the growth of the opportunistic fungal pathogen Candida albicans

abstract: The human gut microbiota protects the host from invading pathogens and the overgrowth of indigenous opportunistic species via mechanisms such as competition for nutrients and by production of antimicrobial compounds. Here, we investigated the antagonist activity of human gut bacteria towards Candida albicans, an opportunistic fungal pathogen that can cause severe infections and mortality in susceptible patients. Co-culture batch incubations of C. albicans in the presence of faecal microbiota from six different healthy individuals revealed varying levels of inhibitory activity against C. albicans. 16S rRNA gene sequence profiling of these faecal co-culture bacterial communities showed that the Bifidobacteriaceae family, and Bifidobacterium adolescentis in particular, were most correlated with antagonistic activity against C. albicans. Follow up mechanistic studies confirmed that culture supernatants of Bifidobacterium species, particularly B. adolescentis, inhibited C. albicans in vitro under both aerobic and anaerobic conditions. Production of the fermentation acids acetate and lactate, together with the concomitant decrease in pH, were strong drivers of the inhibitory activity. Bifidobacteria may therefore represent attractive targets for the development of probiotics and prebiotic interventions tailored to enhance inhibitory activity against C. albicans in vivo.


title: Intestinal Enteroid Monolayers Model the Human Intestinal Environment for Escherichia coli Infection

abstract:
Enterohemorrhagic Escherichia coli O157:H7 is an enteric pathogen responsible for bloody diarrhea, hemolytic uremic syndrome, and in severe cases even death. The study of O157:H7 is difficult due to the high specificity of the bacteria for the human intestine, along with our lack of sufficiently complex human cell culture models. The recent development of human intestinal enteroids derived from intestinal crypt multipotent stem cells has allowed us to construct 2-dimensional differentiated epithelial monolayers grown in transwells that mimic the human intestine. Unlike previous studies, saline was added to the apical surface, while maintaining culture media in the basolateral well. The monolayers continued to grow and differentiate with apical saline. Apical infection with O157:H7 or commensal E. coli resulted in robust bacterial growth from 105 to over 108 over 24 hours. Despite this robust bacterial growth, commensal E. coli neither adhered to nor damaged the epithelial barrier over 30 hours. However, O157:H7 was almost fully adhered (>90%) by 18 hours with epithelial damage observed by 30 hours. O157:H7 contains the locus of enterocyte effacement (LEE) pathogenicity island responsible for attachment and damage to the intestinal epithelium. Previous studies report the ability of nutrients such as biotin, D-serine, and L-fucose to downregulate LEE gene expression. O157:H7 treated with biotin or L-fucose, but not D-serine displayed both decreased attachment and reduced epithelial damage over 36 hours. These data illustrate enteroid monolayers can serve as a suitable model for the study of O157:H7 pathogenesis, and identification of potential therapeutics.

ImportanceO157:H7 is difficult to study due to its high specificity for the human intestine and the lack of sufficiently complex human cell culture models. The recent development of human intestinal enteroids derived from intestinal crypt multipotent stem cells has allowed us to construct 2-dimensional differentiated epithelial monolayers grown in transwells that mimic the human intestine. Our data illustrates enteroid monolayers can serve as a suitable model for the study of O157:H7 pathogenesis, and allow for identification of potential therapeutics.


title: Replication is the key barrier during the dual-host adaption of mosquito-borne flaviviruses

abstract:
Mosquito-borne flaviviruses (MBFs) adapt to a dual-host transmission circle between mosquitoes and vertebrates. Dual-host affiliated insect-specific flaviviruses (dISFs), discovered from mosquitoes, are phylogenetically similar to MBFs but do not infect vertebrates. Thus, dISF-MBF chimeras could be an ideal model to study the dual-host adaption of MBFs. Using the pseudo-infectious reporter virus particle and reverse genetics systems, we found dISFs entered vertebrate cells as efficiently as the MBFs, but failed to initiate replication. Exchange of the un-translational regions (UTRs) of Donggang virus (DONV), an dISF, with those from Zika virus (ZIKV) rescued DONV replication in vertebrate cells and critical secondary RNA structures were further mapped. Essential UTR-binding host factors were screened for ZIKV replication in vertebrate cells, displaying different binding patterns. Therefore, our data demonstrate a post-entry cross-species transmission mechanism of MBFs, while UTR-host interaction is critical for dual-host adaption.

Significance StatementMost viruses have a relatively narrow host range. In contrast, vector-borne flaviviruses, such as dengue virus and Zika virus, maintain their transmission cycle between arthropods and vertebrates, belonging to different phyla. How do these viruses adapt to the distinct cellular environments of two phyla? By comparing the single-host insect specific flavivirus and dual-host Zika virus, we identified three key molecular factors that determine MBF host tropism. This study will greatly increase the understanding of entry, replication, and cross-species evolution of mosquito-borne flaviviruses.


title: Molecular features underlying Pseudomonas aeruginosa persistence in human plasma

abstract: Persistence of bacterial pathogens is a main cause of treatment failure and establishment of chronic bacterial infection. Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen is a leading cause of bacteremia associated with high mortality. In a previous study, we found that P. aeruginosa forms a persister-like sub-population of evaders in human plasma and blood. However, the molecular mechanisms underlying the emergence and biology of evaders remained unknown. Here, using a gain-of-function genetic screen, we examined the molecular determinants of P. aeruginosa persistence in plasma. In addition to known virulence factors (long O-specific antigens and exopolysaccharides), we found that ATP and biotin availability greatly impact bacterial survival in plasma. Mutants in genes of purine and biotin pathways displayed higher tolerance and persistence, respectively. Moreover, we identified a novel small protein named SrgA whose expression leads to 100-fold higher survival in plasma. The analysis of different steps of complement activity and the use of an outer-membrane impermeable drug, nisin, suggests that the mutants impede MAC activity per se. Through this work we highlight the multifactorial origin of bacterial resilience to plasma and pave the way to a more comprehensive picture of the complex interplay between P. aeruginosa and the complement system in the blood.


title: Integrated metabolomic, molecular networking and genome mining analyses uncover novel angucyclines from Streptomyces sp. RO-S4 isolated from Bejaia Bay, Algeria

abstract: Multi-omic approaches have recently made big strides towards the effective exploration of microorganisms and accelerating the discovery of new bioactive compounds. We combined metabolomic, molecular networking, and genomic-based approaches to investigate the metabolic potential of the Streptomyces sp. RO-S4 strain isolated from the polluted waters of Bejaia Bay in Algeria. Antagonistic assays against methicillin-resistant Staphylococcus aureus with RO-S4 organic extracts showed an inhibition zone of 20 mm by the agar diffusion method, and its minimum inhibitory concentration was 16 g/mL. A molecular network was created using GNPS and annotated through the comparison of MS/MS spectra against several databases. The predominant compounds in the RO-S4 extract belonged to the angucyclines family. Three compounds were annotated as known metabolites, while all the others were putatively new to Science. Notably, all compounds had fridamycin-like aglycones, and several of them had a lactonized D ring analogous to that of urdamycin L. The whole genome of Streptomyces RO-S4 was sequenced to identify the biosynthetic gene cluster (BGC) encoding for these angucyclines, which yielded a draft genome of 7,497,846 bp with 72.4% G+C content. Subsequently, a genome mining analysis revealed 19 putative biosynthetic gene clusters, including a grincamycin-like BGC with a high similarity to that of Streptomyces sp. CZN-748 previously reported to also produce mostly open fridamycin-like aglycones. As the ring-opening process leading to these compounds is still not defined, we performed comparative analysis with other angucycline BGCs and advanced some hypotheses to explain the ring-opening and lactone formation, possibly linked to the uncoupling between the activity of GcnE and GcnM homologues in the RO-S4 strain. The combination of metabolomic and genomic approaches greatly improved the interpretation of the metabolic potential of the RO-S4 strain.


title: Metabarcoding malaise trap plant components enables monitoring the diversity of plant-insect interactions

abstract: The declines observed in insect abundance and diversity in the past decades has also been observed in plants, and these events are most certainly correlated. Rapid largescale biomonitoring of both plants and insects can help monitor these changes and inform decisions for land management and species protection. Malaise traps have been used for nearly 80 years for passive insect sampling of primarily flying insects, and when they enter these traps, they carry the fragments of the plants they have visited, either as plant fragments and pollen on the body surface, or as digested food material in gut contents. DNA metabarcoding is a potential method to identify these plant traces in the ethanol of the malaise bottles, which is not possible with traditional microscopy. Metabarcoding could offer more insight into what plants insects are directly interacting with at a given time, and allow for the detection of rare plants, and neophyte species visited by insects. This study, to our knowledge, is the first examination of DNA metabarcoding plant traces from Malaise trap samples, we examine 105 samples from 21 sites throughout Germany collected in a 2-week period in May of 2020. Here we report on the feasibility of sequencing these sample types, analysis of the resulting taxa, the usage of cultivated plants by insects near nature conservancy areas, and the detection of rare and neophyte species.

category: molecular biology


title: Soil organic carbon fractionation and metagenomics pipeline to link carbon content and stability with microbial composition - First results investigating fungal endophytes

abstract: Society needs to capture gigatons of carbon dioxide from the atmosphere annually and then store it long-term to limit and ultimately reverse the effects of climate change. Bringing lost carbon back into agricultural soils should be a priority as it brings the added benefit of improving soil properties. Linking soil organic carbon (SOC) fractions of different stability with soil microbial composition can help understand and subsequently manage SOC storage. Here we develop a pipeline for evaluating the effects of microbial management on SOC content using rapid and low-cost SOC fractionation and metagenomics approaches. We tested the methods in a wheat pot trial inoculated with 17 individual endophytic fungal isolates. Two fungi increased total SOC in the area under the plant stem by ~15%. The fractionation assay showed that the medium stability soil aggregate carbon fraction (AggC) was increased by one of these fungi (+21%) and the chemically recalcitrant proportion (bleach oxidation) of AggC by the other (+35%). Both fungi increased mineral-associated organic carbon (MAOC), the long-term SOC storage, by ~10%. We used rapid, portable, low-cost, whole metagenome long read sequencing to detect a shift in the microbial composition for one of the fungi-inoculated treatments. This treatment showed a more diverse microbial community and a higher quantity of DNA in soil. The results emphasise the link between composition and abundance of soil microorganisms with soil carbon formation. Our dual carbon fractional and metagenomic analysis pipeline can be used to further test the effects of microbial management and ultimately to model the soil factors that influence SOC storage, such as nutrient and water availability, starting SOC content, soil texture and aggregation.

category: genomics


title: Deeply hidden genome organization directly mediated by SATB1

abstract:
Mammalian genomes are organized by multi-layered chromatin folding. Whether and how three-dimensional genome organization contributes to cell-type specific transcription remains unclear. Here we uncover genome architecture formed by specialized sequences, base-unpairing regions (BURs), bound to a nuclear architectural protein, SATB1. SATB1 regulates cell-type specific transcription that underlies changes in cellular phenotypes. We developed a modified ChIP-seq protocol that stringently purifies genomic DNA only with its directly-associated proteins and unmasked previously-hidden BURs as direct SATB1 targets genome-wide. These SATB1-bound BURs are mutually exclusive from CTCF binding sites, and SATB1 is dispensable for CTCF/cohesion-mediated topologically associated domains (TADs). Instead, BURs largely overlap with lamina associated domains (LADs), and the fraction of BURs tethered to the SATB1 protein network in the nuclear interior is cell type-dependent. Our results reveal TAD-independent chromatin folding mediated by BUR sequences which serve as genome architecture landmarks for direct targeting by cell type-specific gene regulator, SATB1.

One-Sentence SummaryGenome-wide chromatin folding by direct tethering of base-unpairing regions to SATB1 nuclear architecture is unveiled.


title: The origins and functional effects of postzygotic mutations throughout the human lifespan

abstract:
Postzygotic mutations (PZMs) begin to accrue in the human genome immediately after fertilization, but how and when PZMs affect development and lifetime health remains unclear. To study the origins and functional consequences of PZMs, we generated a multi-tissue atlas of PZMs from 948 donors using the final major release of the Genotype-Tissue Expression (GTEx) project. Nearly half the variation in mutation burden among tissue samples can be explained by measured technical and biological effects, while 9% can be attributed to donor-specific effects. Through phylogenetic reconstruction of PZMs, we find that their type and predicted functional impact varies during prenatal development, across tissues, and the germ cell lifecycle. Remarkably, a class of prenatal mutations was predicted to be more deleterious than any other category of genetic variation investigated and under positive selection as strong as somatic mutations in cancers. In total, the data indicate that PZMs can contribute to phenotypic variation throughout the human lifespan, and, to better understand the relationship between genotype and phenotype, we must broaden the long-held assumption of one genome per individual to multiple, dynamic genomes per individual.

One-Sentence SummaryThe predicted rates, functional effects and selection pressure of postzygotic mutations vary through the human lifecycle.


title: Structural and biophysical characterization of HNF-1A as a tool to study MODY3 diabetes variants

abstract: Hepatocyte nuclear factor 1A (HNF-1A) is a transcription factor expressed in several embryonic and adult tissues, modulating expression of numerous target genes. Pathogenic variants in the HNF1A gene cause maturity-onset diabetes of the young 3 (MODY3 or HNF1A MODY), characterized by dominant inheritance, age of onset before 25-35 years of age, and pancreatic {beta}-cell dysfunction. A precise diagnosis alters management as insulin can be exchanged with sulfonylurea tablets and genetic counselling differs from polygenic forms of diabetes. More knowledge on mechanisms of HNF-1A function and the level of pathogenicity of the numerous HNF1A variants identified by exome sequencing is required for precise diagnostics. Here, we have structurally and biophysically characterized an HNF-1A protein containing both the DNA binding domain and the dimerization domain. We also present a novel approach to characterize HNF-1A variants. The folding and DNA binding capacity of two established MODY3 HNF-1A variant proteins (P112L, R263C) and one variant of unknown significance (N266S) were determined. All three variants showed reduced functionality compared to the wild-type protein. While the R263C and N266S variants displayed reduced binding to an HNF-1A target promoter, the P112L variant was unstable in vitro and in cells. Our results support and mechanistically explain disease causality for all investigated variants and allow for the dissection of structurally unstable and DNA binding defective variants. This points towards structural and biochemical investigation of HNF-1A being a valuable aid in reliable variant classification needed for precision diagnostics and management.


title: Insight into the mechanism of H+-coupled nucleobase transport

abstract: Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here we report the structure and function PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 [A] resolution by X-ray crystallography. PurTCp forms a homodimer and each protomer has 14 transmembrane segments folded into a substrate-binding domain (core domain) and an interface domain (gate domain) A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines, and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.


title: High-Resolution Structure of the Nuclease Domain of the Human Parvovirus B19 Main Replication Protein NS1

abstract: Two new structures of the N-terminal domain of the main replication protein, NS1, of Human Parvovirus B19 (B19V) are presented. This domain (NS1-nuc) plays an important role in the "rolling hairpin" replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e. nicking) single-stranded DNA at a nearby site known as the trs. One structure of NS1-nuc is solved to 2.4 [A] and shows the positions of two bound phosphate ions. A second structure shows the position of a single divalent cation in the DNA nicking active site. The threedimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain, however shown here at significantly higher resolution. Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented which predict residues important for DNA cleavage and for sequence specific recognition at the viral origin of replication.


title: Porphyrin in prebiotic catalysis: Ascertaining a route for the emergence of early metalloporphyrins

abstract: Metal ions are known to catalyze certain prebiotic reactions. However, the transition from metal ions to extant metalloenzymes remains unclear. Porphyrins are found ubiquitously in the catalytic core of many ancient metalloenzymes. In this study, we evaluated the influence of porphyrin-based organic scaffold, on the catalysis, emergence and putative molecular evolution of prebiotic metalloporphyrins. We studied the effect of porphyrins on the transition metal ion-mediated oxidation of hydroquinone (HQ). We report a change in the catalytic activity of the metal ions in the presence of porphyrin. This was observed to be facilitated by the coordination between metal ions and porphyrins or by formation of non-coordinated complexes. The metal-porphyrin complexes also oxidized NADH, underscoring its versatility at oxidizing more than one substrate. Our study highlights the selective advantage that some of the metal ions would have had in the presence of porphyrin, underscoring their role in shaping the evolution of protometalloenzymes.


title: Popeye Domain-Containing Protein 1 Scaffolds a Complex of Adenylyl Cyclase 9 and the Two-Pore-Domain Potassium Channel TREK-1 in Heart

abstract:
The establishment of macromolecular complexes by scaffolding proteins such as A-kinase anchoring proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for many cardiac functions. We have identified herein a novel AC scaffold, the Popeye domain-containing (POPDC) protein. Unlike other AC scaffolding proteins, POPDC1 binds cAMP with high affinity. The POPDC family of proteins are important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. TREK-1 binds the AC9:POPDC1 complex and co-purifies in a POPDC1-dependent manner with AC9-associated activity in heart. Although the interaction of AC9 and POPDC1 is cAMP independent, TREK-1 association with AC9 and POPDC1 is reduced in an isoproterenol-dependent manner, requiring an intact cAMP binding Popeye domain and AC activity within the complex. We show that deletion of Adcy9 (AC9) gives rise to bradycardia at rest and stress-induced heart rate variability. The phenotype for deletion of Adcy9 is milder than previously observed upon loss of Popdc1, but similar to loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel scaffolding protein for AC9 to regulate heart rate control.

ONE-SENTENCE SUMMARYAdenylyl cyclase type 9 binds in an isoproterenol-dependent manner to the POPDC1:TREK-1 complex regulating heart rate.


title: A deep dive into VDAC1 conformational diversity using all-atom simulations provides new insights into the structural origin of the closed states

abstract: The voltage-dependent anion channel 1 (VDAC1) is a crucial mitochondrial transporter which controls the flow of ions and respiratory metabolites entering or exiting mitochondria. As a voltage-gated channel, VDAC1 can switch between a high conducting "open" state and low conducting "closed" states emerging at high transmembrane potential. Although cell homeostasis depends on channel gating to regulate the transport of ions and metabolites, structural hallmarks characterizing the closed states remain unknown. Here we performed microsecond accelerated molecular dynamics to highlight a vast region of VDAC1 conformational landscape accessible at typical voltage known to promote closure. Conformers exhibiting stable subconducting properties inherent to closed states were identified. In all cases, the low conductance was due to the particular positioning of an unfolded part of the N-terminus which obstructed the channel pore. While the N-terminal tail was found to be sensitive to voltage orientation, our low-conducting models suggest that closed states predominantly take place from disordered events and do not result from the displacement of a voltage sensor or a significant change in the pore. In addition, our results were consistent with conductance jumps observed in experiments and corroborates a recent study describing entropy as a key factor for VDAC gating.

category: biophysics


title: Probing the molecular mechanism of action of Deinococcus radiodurans RecD2 at single-molecule resolution

abstract: Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. Helicases are responsible for resolving double-stranded DNA (dsDNA) into separate single-strands and this activity is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions towards the organisms unusually high tolerance to gamma radiation and hydrogen peroxide. Although previous X-ray Crystallography studies have revealed the structural characteristics of the protein, the direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5   terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.


title: Functional Network Connectivity Imprint In Febrile Seizures

abstract: Complex febrile seizures (CFS), a subset of paediatric febrile seizures (FS), have been studied for their prognosis, epileptogenic potential and neurocognitive outcome. We evaluated their functional connectivity differences with simple febrile seizures (SFS) in children with recent-onset FS. Resting-state fMRI (rs-fMRI) datasets of 24 children with recently diagnosed FS (SFS-n=11; CFS-n=13) were analysed. Functional connectivity (FC) was estimated using time series correlation of seed region-to-whole-brain-voxels and network topology was assessed using graph theory measures. Regional connectivity differences were correlated with clinical characteristics (FDR corrected p < 0.05). CFS patients demonstrated increased FC of the bilateral middle temporal pole (MTP), and bilateral thalami when compared to SFS. Network topology study revealed increased clustering coefficient and decreased participation coefficient in basal ganglia and thalamus suggesting an inefficient-unbalanced network topology in patients with CFS. The number of seizure recurrences negatively correlated with the integration of Left Thalamus (r= -0.58) and FC measures of Left MTP to  Right Supplementary Motor and left Precentral connectivity (r=-0.53). The FC of Right MTP to Left Amygdala, Putamen, Parahippocampal, and Orbital Frontal Cortex (r=0.61) and FC of Left Thalamus to left Putamen, Pallidum, Caudate, Thalamus Hippocampus and Insula (r 0.55) showed a positive correlation to the duration of the longest seizure. The findings of the current study report altered connectivity in children with CFS proportional to the seizure recurrence and duration. Regardless of the causal/consequential nature, such observations demonstrate the imprint of these disease-defining variables of febrile seizures on the developing brain.

category: neuroscience


title: Notch inhibition promotes regeneration and immunosuppression supports cone survival in a zebrafish model of inherited retinal dystrophy

abstract: Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as Retinitis Pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Muller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, MG in zebrafish models of photoreceptor degeneration fail to re-enter the cell cycle and regenerate lost cells. Here, we used the zebrafish cep290 mutant to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to pre-lesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.


title: Multi T1-weighted contrast imaging and T1 mapping with Compressed sensing FLAWS at 3T

abstract:
The Fluid And White matter Suppression (FLAWS) MRI sequence allows for the acquisition of multiple T1-weighted contrasts in a single sequence acquisition. However, its acquisition time is prohibitive for use in clinical practice when the k-space is linearly downsampled and reconstructed using the Generalized Autocalibrating Partially Parallel Acquisition (GRAPPA) technique. This study proposes a FLAWS sequence optimization tailored to allow for the acquisition of FLAWS images with a Cartesian phyllotaxis k-space undersampling and compressed sensing (CS) reconstruction at 3T. The CS FLAWS sequence parameters were determined using a method previously employed to optimize FLAWS imaging at 1.5T and 7T. In-vivo experiments show that the proposed CS FLAWS optimization allows to reduce the FLAWS sequence acquisition time from 8 mins to 6 mins without decreasing the FLAWS image quality. In addition, this study demonstrates for the first time that T1-weighted imaging with low B1 sensitivity and T1 mapping can be performed with the FLAWS sequence at 3T for both GRAPPA and CS reconstructions. The FLAWS T1 mapping was validated using in-silico, in-vitro and in-vivo experiments with comparison against the inversion recovery turbo spin echo and MP2RAGE T1 mappings. These new results suggest that the recent advances in FLAWS imaging allow to combine the MP2RAGE imaging benefits (T1-weigthed imaging with low B1 sensitivity and T1 mapping) and with the previous version of FLAWS imaging benefits (multi T1-weighted contrast imaging) in a single 6 mins sequence acquisition.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: When self comes to a wandering mind: Brain representations and dynamics of self-generated concepts in spontaneous thought

abstract: AO_SCPLOWBSTRACTC_SCPLOWSelf-relevant concepts are major building blocks of spontaneous thought, and their dynamics in a natural stream of thought are likely to reveal ones internal states important for mental health. Here we conducted an fMRI experiment (n = 62) to examine brain representations and dynamics of self-generated concepts in the context of spontaneous thought using a newly developed free association-based thought sampling task. The dynamics of conceptual associations were predictive of individual differences in general negative affectivity, replicating across multiple datasets (n = 196). Reflecting on self-generated concepts strongly engaged brain regions linked to autobiographical memory, conceptual processes, emotion, and autonomic regulation, including the medial prefrontal and medial temporal subcortical structures. Multivariate pattern-based predictive modeling revealed that the neural representations of valence became more person-specific as the level of perceived self-relevance increased. Overall, this study provides a hint of how self-generated concepts in spontaneous thought construct inner affective states and idiosyncrasies.


title: Overactive STAT3 drives accumulation of disease-associated CD21low B cells

abstract: Dysregulated STAT3 signalling is correlated with antibody-mediated autoimmunity and B- cell neoplasia, but its effect on B cells is underexplored. Here we address this in children with STAT3 gain-of-function (GOF) syndrome and in mice with STAT3T716M, the most common STAT3 GOF syndrome human mutation, or STAT3K658N, a dimerization interface mutation responsible for STAT3 GOF syndrome in two children. The main B cell consequence of overactive STAT3 was accumulation of CD19high CD21low atypical memory B cells in humans and of CD21low CD23low B cells in mice resembling age-associated B cells expressing T-bet, CD11c and plasma cell differentiation genes. Overactive STAT3 within B cells increased expression of many genes in the B cell receptor and T cell help pathways, increased the tolerogenic receptor CD22, but opposed B cell tolerance checkpoints and increased formation of T-bet+ B cells upon BCR and CD40 stimulation. These results reveal overactive STAT3 as a central driver of a key class of disease- associated B-lymphocytes in humans and mice.

category: immunology


title: Molecular epidemiology of Animal African Trypanosomosis in southwest Burkina Faso

abstract:
BackgroundAnimal African Trypanosomosis (AAT) is a parasitic disease of livestock that has a major socio-economic impact in the affected areas. It is caused by several species of uniflagellate extracellular protists of the genus Trypanosoma mainly transmitted by tsetse flies: T. congolense, T. vivax and T. brucei brucei. In Burkina Faso, AAT hampers the proper economic development of the southwestern part of the country, which is yet the best watered area particularly conducive to agriculture and animal production. It was therefore important to investigate the extend of the infection in order to better control the disease. The objective of the present study was to assess the prevalence of trypanosome infections and collect data on the presence of tsetse flies.

MethodsBuffy coat, Trypanosoma species-specific PCR, Indirect ELISA Trypanosoma sp and trypanolysis techniques were used on 1898 samples collected. An entomological survey was also carried out.

ResultsThe parasitological prevalence of AAT was 1.1%, and all observed parasites were T. vivax. In contrast, the molecular prevalence was 23%, of which T. vivax was predominant (89%) followed by T. congolense (12%) and T. brucei s.l. (7.3%) with a sizable proportion as mixed infections (9.1%). T. brucei gambiense, responsible of sleeping sickness in humans, was not detected. The serological prevalence reached 49%. Once again T. vivax predominated (86.2%), but followed by T. brucei (9.6%) and T. congolense (4.2%), while 34.6% of positive samples tested positive for at least two trypanosome species. Seven samples, from six cattle and one pig, were found positive by trypanolysis. The density per trap of Glossina tachinoides and G. palpalis gambiensis was about three flies.

Conclusions/SignificanceOverall, our study showed a high prevalence of trypanosome infection in the area, pointing out an ongoing inadequacy of control measures.

Authors summaryIn Burkina Faso, like in most countries of sub-Saharan Africa, Animal African Trypanosomosis (AAT) is hampering economic development. It was therefore important to investigate the extent of trypanosome infections after years of control. This study examined circulating trypanosomes in domestic animals using parasitological, molecular and serological tools in southwest Burkina Faso. The prevalence levels observed show that the known epidemiological situation in the region has not really changed. The trypanosome species usually found in the area such as T. congolense, T. vivax and T. brucei s.l. have remained the same, with a stronger presence of T. vivax. The low occurrence of tsetse fly and the predominance of T. vivax attests to the role of the mechanical vectors in maintaining the disease. Although no cases of T. brucei gambiense infection was encountered in the animals examined, trypanolysis tests suggest that there are contact cases in this historical focus of sleeping sickness. Efforts are therefore necessary to reduce or even eliminate the trypanosome burden, and the data provided by this study can assist the decision making.


title: MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

abstract:
Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS-CoV is adept at antagonizing early innate immune pathways - interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L) - generated in response to viral double-stranded (ds)RNA generated during genome replication. This is in contrast to SARS-CoV-2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

ImportanceMiddle East Respiratory Syndrome Coronavirus (MERS-CoV) causes highly lethal respiratory disease. MERS-CoV encodes several innate immune antagonists, accessory proteins NS4a and NS4b unique to the merbeco lineage and the nsp15 protein endoribonuclease (EndoU), conserved among all coronaviruses. While mutation of each antagonist protein alone has little effect on innate immunity, infections with recombinant MERS-CoVs with mutations of EndoU in combination with either NS4a or NS4b, activate innate signaling pathways and are attenuated for replication. Our data indicate that EndoU and accessory proteins NS4a and NS4b together suppress innate immunity during MERS-CoV infection, to optimize viral replication. This is in contrast to SARS-CoV-2 which activates these pathways and consistent with greater mortality observed during MERS-CoV infection compared to SARS-CoV-2.


title: Suicidal chemotaxis in bacteria

abstract: Bacteria commonly live in communities on surfaces where steep gradients of antibiotics and other chemical compounds routinely occur. While many species of bacteria can move on surfaces, we know surprisingly little about how such antibiotic gradients affect cell motility. Here we study the behaviour of the opportunistic pathogen Pseudomonas aeruginosa in stable spatial gradients of a range of antibiotics by tracking thousands of cells in microfluidic devices as they form biofilms. Unexpectedly, these experiments reveal that individual bacteria use pili-based ( twitching) motility to actively navigate towards regions with higher antibiotic concentrations. Our analyses suggest that this biased migration is driven, at least in part, by a direct response to the antibiotics. Migrating cells can reach antibiotic concentrations hundreds of times higher than their minimum inhibitory concentration in a few hours and remain highly motile. However, isolating these cells - using fluid-walled microfluidic devices that can be reconfigured in situ - suggests that these bacteria are terminal and not able to reproduce. In spite of moving towards their death, we show that migrating cells are capable of entering a suicidal program to release bacteriocins that are used to kill other bacteria. Our work suggests that bacteria respond to antibiotics as if they come from a competing colony growing in the neighbourhood, inducing them to invade and attack. As a result, clinical antibiotics have the potential to serve as a bait that lures bacteria to their death.


title: DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

abstract: Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This is a syncytial mode of replication involving asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite replication is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads. Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi. These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors. Comparing them could therefore yield new information about the parameters and limitations of schizogony. We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear replications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi. In both species, gaps become longer as schizogony progresses, whereas each period of active replication grows shorter. In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their replication for many hours while adjacent nuclei continue to replicate. The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.


title: An Atypical RNA Quadruplex Marks RNAs as Vectors for Gene Silencing

abstract: The addition of poly(UG) ("pUG") repeats to 3' termini of mRNAs drives gene silencing and trans-generational epigenetic inheritance in the metazoan C. elegans.1 pUG tails promote silencing by recruiting an RNA-dependent RNA Polymerase (RdRP) that synthesizes small interfering (si)RNAs.1 Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4)2 structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA3,4, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. Its biological importance is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific N7-deaza RNA substitutions that do not adopt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA secondary structure motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in non-coding regions of human RNAs, suggesting the fold likely has biological roles beyond gene silencing.


title: Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring

abstract: Aquatic environmental DNA (eDNA) surveys are transforming how we monitor marine ecosystems. The time-consuming pre-processing step of active filtration, however, remains a bottleneck. Hence, new approaches omitting active filtration are in great demand. One exciting prospect is to use the filtering power of invertebrates to collect eDNA. While proof-of-concept has been achieved, comparative studies between aquatic and filter feeder eDNA signals are lacking. Here, we investigated the differences among four eDNA sources (water; bivalves; sponges; and ethanol in which filter-feeding organisms were stored) along a vertical transect in Doubtful Sound, New Zealand using three metabarcoding primers (fish (16S); MiFish-E/U). While concurrent SCUBA diver observations validated eDNA results, laboratory trials corroborated in-field bivalve eDNA detection results. Combined, eDNA sources detected 59 vertebrates, while divers observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was detected in ethanol, although only a reduced number of species were detected. Bivalves failed to reliably detect eDNA in both field and mesocosm experiments. While additional research into filter feeder eDNA accumulation efficiency is essential, our results provide strong evidence for the potential of incorporating sponges into eDNA surveys.


title: Mediator is broadly recruited to gene promoters via a Tail-independent mechanism

abstract: Mediator (MED) is a conserved factor with important roles in both basal and activated transcription. It is believed that MED plays a direct role in transcriptional regulation at most genes by functionally bridging enhancers and promoters. Here, we investigate the genome-wide roles of yeast MED by rapid depletion of its activator-binding domain (Tail) and monitoring changes in nascent transcription. We find that MED Tail and activator-mediated MED recruitment regulate only a small subset of genes. At most genes, MED bypasses the UAS and is directly recruited to promoters to facilitate transcription initiation. Our results define three classes of genes that differ in PIC assembly pathways and the requirements for MED Tail, SAGA, TFIID and BET factors Bdf1/2. We also find that the depletion of the MED middle module subunit Med7 mimics inactivation of Tail, suggesting a functional link. Our combined results have broad implications for the roles of MED, other coactivators, and mechanisms of transcriptional regulation at different gene classes.


title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

abstract:
Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: GRaNIE and GRaNPA: Inference and evaluation of enhancer-mediated gene regulatory networks applied to study macrophages

abstract: Among the biggest challenges in the post-GWAS (genome-wide association studies) era is the interpretation of disease-associated genetic variants in non-coding genomic regions. Enhancers have emerged as key players in mediating the effect of genetic variants on complex traits and diseases. Their activity is regulated by a combination of transcription factors (TFs), epigenetic changes and genetic variants. Several approaches exist to link enhancers to their target genes, and others that infer TF-gene connections. However, we currently lack a framework that systematically integrates enhancers into TF-gene regulatory networks. Furthermore, we lack an unbiased way of assessing whether inferred regulatory interactions are biologically meaningful. Here we present two methods, implemented as user-friendly R-packages, for building and evaluating enhancer-mediated gene regulatory networks (eGRNs) called GRaNIE (Gene Regulatory Network Inference including Enhancers - https://git.embl.de/grp-zaugg/GRaNIE) and GRaNPA (Gene Regulatory Network Performance Analysis - https://git.embl.de/grp-zaugg/GRaNPA), respectively. GRaNIE jointly infers TF-enhancer, enhancer-gene and TF-gene interactions by integrating open chromatin data such as ATAC-Seq or H3K27ac with RNA-seq across a set of samples (e.g. individuals), and optionally also Hi-C data. GRaNPA is a general framework for evaluating the biological relevance of TF-gene GRNs by assessing their performance for predicting cell-type specific differential expression. We demonstrate the power of our tool-suite by investigating gene regulatory mechanisms in macrophages that underlie their response to infection, and their involvement in common genetic diseases including autoimmune diseases.


title: Simultaneous CUT&Tag profiling of the accessible and silenced regulome in single cells

abstract: Cleavage Under Targets & Tagmentation (CUT&Tag) is an antibody-directed transposase tethering strategy for in situ chromatin profiling in small samples and single cells. We describe a modified CUT&Tag protocol using a mixture of an antibody to the initiation form of RNA Polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution to produce high-resolution maps of both the active and repressive regulomes in single cells. The ability to seamlessly map active promoters, enhancers and repressive regulatory elements using a single workflow provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.


title: Δ9-tetrahydrocannabinol (THC) vapor exposure produces conditioned rewarding effects in male and female rats

abstract:
BackgroundVaping of cannabis and cannabis extracts containing {Delta}9-tetrahydrocannabinol (THC, the primary psychoactive constituent of cannabis) is on the rise. Development of animal models using vapor exposure is important for increasing our understanding of the rewarding and aversive effects of vaped cannabinoids. Currently there are limited data on the conditioned rewarding effects of THC vapor in rats, and no studies to date examining sex differences.

MethodsMale and female Sprague-Dawley rats (N=96; 12 per sex/group) underwent place conditioning after exposure to vaporized THC or vehicle (propylene glycol, PG). THC vapor-conditioned rats received one of three THC vapor exposure amounts (THC Group 1: 5 puffs of 100 mg/ml THC, THC Group 2: 5 puffs of 200 mg/ml THC, or THC Group 3: 10 puffs of 200 mg/ml THC) and matched vehicle vapor (PG) exposure and on alternate days for two sets of 8 daily sessions (16 days total). Vehicle-conditioned rats (Veh Group 0) received only PG vapor exposure each day. Rats were passively administered vapor for 30-min immediately before daily, 30-min conditioning sessions. Untreated rats completed place preference tests after the 8th and 16th conditioning sessions and then testing continued daily until extinction occurred. Following extinction, rats underwent a THC vapor-primed reinstatement session.

ResultsMale and female rats showed an exposure-dependent preference for the THC vapor-paired chamber, though sex differences were observed. The lowest THC vapor exposure group tested (THC Group 1) did not produce CPP in males or females. Exposure to the middle condition tested (THC Group 2) resulted in preference for the THC vapor-paired chamber in males, but not females. The highest THC vapor exposure condition tested (THC Group 3) produced place preference in both males and females. Preference for the THC-paired chamber extinguished more quickly in males than in females. Following extinction, THC vapor re-exposure (i.e., drug-prime) did not result in reinstatement of place preference for either sex.

ConclusionThis study demonstrated conditioned rewarding effects of THC vapor in both male and female rats, and provides evidence for sex differences in doses of THC vapor that produce CPP and in time to extinction. Conditioned place aversion was not observed at any of the THC vapor exposure amounts tested. These data provide a foundation for future exploration of the conditioned rewarding effects vaporized THC, cannabis and its constituents in preclinical models.

category: animal behavior and cognition


title: De Novo Design of Immunoglobulin-like Domains

abstract: Antibodies and antibody derivatives such as nanobodies contain immunoglobulin-like (Ig) {beta}-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here we develop design rules for the central feature of the Ig fold architecture - the non-local cross-{beta} structure connecting the two {beta}-sheets - and use these to de novo design highly stable seven-stranded Ig domains, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of a new class of antibody-like scaffolds with tailored structures and superior biophysical properties.


title: Delivery of CAR-T Cells in a Transient Injectable Stimulatory Hydrogel Niche Improves Treatment of Solid Tumors

abstract: Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers such as B cell malignancies, but traditional approaches to ACT are poorly effective in treating the multifarious solid tumors observed clinically. Locoregional cell delivery methods have shown promising results in treating solid tumors compared to standard intravenous delivery methods, but the approaches that have been described to date have several critical drawbacks ranging from complex manufacturing and poor modularity to challenging adminstration. In this work, we develop a simple-to-implement self-assembled and injectable hydrogel material for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improves treatment of solid tumors. We evaluate a range of hydrogel formulations to optimize the creation of a transient inflammatory niche that affords sustained exposure of CAR-T cells and cytokines. This facile approach yields increased CAR-T cell expansion, induces a more tumor-reactive CAR-T phenotype, and improves efficacy in treating solid tumors in mice.

category: bioengineering


title: Process engineering of biopharmaceutical production in moss bioreactors via model-based description and evaluation of phytohormone impact

abstract: The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 {micro}M) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-Kynurenine (100 {micro}M) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 hours after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first two hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/ g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.


title: Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

abstract: Thermal engineering at microscale such as the control and measurement of temperature is a key technology in basic biological research and biomaterials development, which remains challenge yet. Here, we engineered the polymeric nanoparticle, in which a fluorescent temperature sensory dye and a photothermal dye were embedded in its polymer matrices, termed nanoHT. When a near infrared laser at 808 nm is illuminated to the particle, it enables to create the subcellular-sized heat spot in a live cell, where fluorescence thermometry allows the read out of the temperature increment concurrently at individual heat spots. Owing to the controlled local heating, we found that the cell death of HeLa cells was induced at the certain temperature at rate of a few seconds. It should be also noted that the cell death was triggered from the very local heat spot at subcellular level. Furthermore, nanoHT was applied for the induction of muscle contraction of the C2C12 myotube by heat. We successfully showed that the heat-induced contraction took place at the limited area of a single myotube according to the alteration of protein-protein interactions related to the contraction event. These studies demonstrated that even a single heat spot provided by a photothermal material could be very effective in altering cellular functions, paving the way for novel photothermal therapies.


title: Reversable deformation of artificial cell colony for muscle behavior mimicry triggered by actin polymerization

abstract: The mimicry of living tissues from artificial cells is beneficial to understanding the interaction mechanism among cells, as well as holding great potentials in the tissue engineering field. Self-powered artificial cells capable of reversible deformation are developed by encapsulating living mitochondria, actin proteins, and methylcellulose. Upon the addition of pyruvate molecules, the mitochondria produce ATP molecules as energy sources to trigger the polymerization of actin. ATP molecules were produced by mitochondria (2.76x1010/ml) with the concentrations of 35.8{+/-}3.2 {micro}M, 158.2{+/-}19.3 {micro}M and 200.7{+/-}20.1 M by adding pyruvate molecules with the concentration of 3 M, 12 M and 21 M, respectively. The reversible deformation of artificial cells is experienced with spindle shape resulting from the polymerization of actins to form filaments adjacent to the lipid bilayer, subsequently back to spherical shape resulting from the depolymerization of actin filaments upon laser irradiations. The linear colonies composed of these artificial cells exhibit collective contraction and relaxation behavior to mimic muscle tissues. At the stage of maximum contraction, the long axis of each GUV is in parallel to each other. All colonies are synchronized in the contraction phase. The deformation of each GUV in the colonies is influenced by its adjacent GUVs. The muscle-like artificial cell colonies paved the path to develop sustainably self-powered artificial tissues in the field of tissue engineering.


title: Therapeutic potential of methylxanthine drug on alpha synuclein protein in Parkinson's disease

abstract: Parkinsons disease (PD) is the second most common neurodegenerative disorder after Alzheimer. It exists in sporadic (90 to 95%) and familial (5 to 10%) form. Its pathogenesis is due to oxidative stress, glutamate excitotoxicity, protein aggregation, neuroinflammation and neurodegeneration. There is currently no cure for this disease. The protein-protein interaction and gene ontology/functional enrichment analysis have been performed to find out the prominent interactor protein and shared common biological pathways, especially PD pathway. Further in silico docking analysis was performed on target protein to investigate the prominent drug molecule for PD. Through computational molecular virtual screening of small molecules from selected twelve natural compounds, and among these compounds methylxanthine was shown to be prominent inhibitor to SNCA protein that ultimately prevent PD. The interaction of methylxanthine compound with the target protein SNCA suggested that, it interacted with prominent binding site with good docking score and might be involved in blocking the binding of neuroinducing substances like: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to SNCA protein. Thus methylxanthine compounds can be explored as promising drugs for the prevention of Parkinsons disease.

category: bioinformatics


title: Explore synergistic and competitive miRNA regulation mechanisms in the miRNA-mRNA regulatory network from the information decomposition perspective

abstract: Since multiple microRNAs can target 3 untranslated regions of the same mRNA transcript, it is likely that these endogenous microRNAs may form synergistic alliances, or compete for the same mRNA harbouring overlapping binding site matches. Synergistic and competitive microRNA regulation is an intriguing yet poorly elucidated mechanism. We here introduce a computational method based on the multivariate information measurement to quantify such implicit interaction effects between microRNAs. Our informatics method of integrating sequence and expression data is designed to establish the functional correlation between microRNAs. To demonstrate our method, we exploited TargetScan and The Cancer Genome Atlas data. As a result, we indeed observed that the microRNA pair with neighbouring binding site(s) on the mRNA is likely to trigger synergistic events, while the microRNA pair with overlapping binding site(s) on the mRNA is likely to cause competitive events, provided that the pair of microRNAs has a high functional similarity and the corresponding triplet presents a positive/negative  synergy-redundancy score.


title: Identification of upstream transcription factor binding sites in orthologous genes using mixed Student's t-test statistics

abstract:
BackgroundTranscription factor (TF) regulates the transcription of DNA to messenger RNA by binding to upstream sequence motifs. Identifying the locations of known motifs in whole genomes is computationally intensive.

Methodology/Principal FindingsThis study presents a computational tool, named "Grit", for screening TF-binding sites (TFBS) by coordinating transcription factors to their promoter sequences in orthologous genes. This tool employs a newly developed mixed Students t-test statistical method that detects high-scoring conserved and non-conserved binding sites among species. The program performs sequence scanning at a rate of 3.2 Mb/s on a quad-core Amazon server and has been benchmarked by the well-established ChIP-Seq datasets, putting Grit amongst the top-ranked TFBS predictors. It marginally outperforms the well-known transcription factor motif scanning tools, Pscan (4.8%) and FIMO (17.8%), in analyzing well-documented ChIP-Atlas human genome Chip-Seq datasets.

SignificanceGrit is a good alternative to current available motif scanning tools and is publicly available at http://www.thua45.cn/grit under an academic free license.

Author SummaryLocating transcription factor-binding (TF-binding) site in the genome and identification their function is fundamental in understanding various biological processes. Improve the performance of the prediction tools is important because accurate TF-binding site prediction can save cost and time for wet-lab experiments. Also, genome wide TF-binding site prediction can provide new insights for transcriptome regulation in system biology perspective. This study developed a new TF-binding site prediction tool based on mixed Students t-test statistical method. The tool is amongst the top-ranked TF-binding site predictors, as such, it can help the researchers in TF-binding site identification and transcriptional regulation mechanism interpretation of genes.


title: Scellseg: a style-aware cell instance segmentation tool with pre-training and contrastive fine-tuning

abstract: Deep learning-based cell segmentation is increasingly utilized in cell biology and molecular pathology, due to massive accumulation of diverse large-scale datasets and excellent progress in cell representation. However, the development of specialized algorithms has long been hampered by a paucity of annotated training data, whereas the performance of generalist algorithm was limited without experiment-specific calibration. Here, we present a deep learning-based tool called Scellseg consisted of novel pre-trained network architecture and contrastive fine-tuning strategy. In comparison to four commonly used algorithms, Scellseg outperformed others in average precision and Aggregated Jaccard Index on three disparate datasets. Interestingly, we found that eight images are sufficient for model tuning to achieve satisfied performance based on a shot data scale experiment. We also developed a graphical user interface integrated with functions of annotation, fine-tuning and inference, that allows biologists to easily specialize their self-adaptive segmentation model for analyzing images at the single-cell level.


title: rareSurvival: rare variant association analysis for time-to-event outcomes.

abstract:
SummaryRare variants have been proposed as contributing to the "missing heritability" of complex human traits. There has been much recent development of methodology to investigate association of complex traits with multiple rare variants within pre-defined "units" from sequence and array-based studies of the exome or genome. However, software for modelling time to event outcomes for rare variant associations has been under developed in comparison with binary and quantitative traits. We introduce a new command line application, rareSurvival, used for the analysis of rare variants with time to event outcomes. The program is compatible with high performance computing (HPC) clusters for batch processing. rareSurvival implements statistical methodology, which are a combination of widely used survival and gene-based analysis techniques such as the Cox proportional hazards model and the burden test. We introduce a novel piece of software that will be at the forefront of efforts to discover rare variants associated with a variety of complex diseases with survival endpoints.

Availability & ImplementationrareSurvival is implemented in C#, available on Linux, Windows and Mac OS X operating systems. It is freely available (GNU General Public License, version 3) to download from https://www.liverpool.ac.uk/translational-medicine/research/statistical-genetics/software/. Download Mono for Linux or Mac OS X to run software.

Contacthamzah.syed@liverpool.ac.uk

Supplementary informationLinks to additional figures and tables are available at Bioinformatics online.


title: MOPower: an R-shiny application for the simulation and power calculation of multi-omics studies.

abstract:
BackgroundMulti-omics studies are increasingly used to help understand the underlying mechanisms of clinical phenotypes, integrating information from the genome, transcriptome, epigenome, metabolome, proteome and microbiome. This integration of data is of particular use in rare disease studies where the sample sizes are often relatively small. Methods development for multi-omics studies is in its early stages due to the complexity of the different individual data types. There is a need for software to perform data simulation and power calculation for multi-omics studies to test these different methodologies and help calculate sample size before the initiation of a study. This software, in turn, will optimise the success of a study.

ResultsThe interactive R shiny application MOPower described below simulates data based on three different omics using statistical distributions. It calculates the power to detect an association with the phenotype through analysis of n number of replicates using a variety of the latest multi-omics analysis models and packages. The simulation study confirms the efficiency of the software when handling thousands of simulations over ten different sample sizes. The average time elapsed for a power calculation run between integration models was approximately 500 seconds. Additionally, for the given study design model, power varied with the increase in the number of features affecting each method differently. For example, using MOFA had an increase in power to detect an association when the study sample size equally matched the number of features.

ConclusionsMOPower addresses the need for flexible and user-friendly software that undertakes power calculations for multi-omics studies. MOPower offers users a wide variety of integration methods to test and full customisation of omics features to cover a range of study designs.


title: Intracranial Electric Field Recording During Multichannel Transcranial Electrical Stimulation

abstract:
BackgroundMultichannel transcranial electrical stimulation (tES) modeling and optimization have been widely studied in recent years. Its theoretical bases include quasi-static assumption and linear superposition. However, there is still a lack of direct in vivo evidence to validate the simulation model and theoretical assumptions.

MethodsWe directly measured the multichannel tES-induced voltage changes with implanted stereotactic-electroencephalographic (sEEG) electrodes in 12 epilepsy subjects. By combining these measured data, we investigate the linear superposition and prediction accuracy of simulation models for multi-electrode stimulation and further compare the induced EF differences between transcranial direct current stimulation (tDCS) and transcranial alternating current stimulation (tACS).

ResultsOur in vivo measurements demonstrated that the multi-electrode tES-induced voltages were almost equal to the sum of the voltages generated independently by bipolar stimulation. Both measured voltages and electric fields obtained in vivo were highly correlated with the predicted values in our cohort (Voltages: r = 0.92, p < 0.001; electric fields: r = 0.74, p < 0.001). Under the same stimulation intensity, the tDCS-induced peak-zero voltages were highly correlated with the values of tACS (r = 0.99, p < 0.001; s = 0.99).

ConclusionsThe in vivo measurements provides confirmatory results for linear superposition and quasi-static assumption within the human brain. Furthermore, we found that the individualized simulation model reliably predicted the multi-electrode tES-induced electric fields.


title: Genetic and compound screens uncover factors modulating cancer cell response to indisulam

abstract: Discovering biomarkers of drug response and finding powerful drug combinations can support the reuse of previously abandoned cancer drugs in the clinic. Indisulam is an abandoned drug that acts as a molecular glue, inducing degradation of splicing factor RBM39 through interaction with CRL4DCAF15. Here, we performed genetic and compound screens to uncover factors mediating indisulam sensitivity and resistance. First, a dropout CRISPR screen identified SRPK1 loss as a synthetic lethal interaction with indisulam that can be exploited therapeutically by the SRPK1 inhibitor SPHINX31. Moreover, a CRISPR resistance screen identified components of the degradation complex that mediate resistance to indisulam: DCAF15, DDA1, and CAND1. Lastly, we show that cancer cells readily acquire spontaneous resistance to indisulam. Upon acquiring indisulam resistance, pancreatic cancer (Panc10.05) cells still degrade RBM39 and are vulnerable to BCL-xL inhibition. The better understanding of the factors that influence the response to indisulam can assist rational reuse of this drug in the clinic.

category: cancer biology


title: Antibodies to ILT3 abrogate myeloid immunosuppression and enable tumor killing

abstract: Tumor myeloid suppressor cells impede response to T cell checkpoint immunotherapy. Immunoglobulin-like transcript 3 (ILT3, gene name, LILRB4) expressed on dendritic cells (DCs) promotes antigen-specific tolerance. Circulating monocytic MDSCs that express ILT3 have been linked to clinical outcomes and a soluble form of ILT3 is elevated in certain cancers. We find that LILRB4 expression is correlated with Gene Expression Profile of T-cell inflamed tumor microenvironment shown to be significantly associated with response to the anti-PD1 antibody pembrolizumab across several tumor types. A potent and selective anti-ILT3 mAb effectively antagonized IL-10 polarization of DCs and enabled T cell priming. In an MLR assay anti-ILT3 combined with pembrolizumab afforded greater CD8+ T cell activation compared to either agent alone. Anti-ILT3 antibodies impaired the acquisition of a suppressive phenotype of monocytes co-cultured with SK-MEL-5 cancer cells, accompanied by a reduction in surface detection of peptidase inhibitor 16, a cis interaction candidate for ILT3. Growth of myeloid cell-abundant SK-MEL-5 tumors was abrogated by ILT3 blockade and remodeling of the immune tumor microenvironment was evident by CyTOF. These data support the testing of anti-ILT3 antibodies for the treatment of a wide range of solid tumors replete with myeloid cells.


title: Quantifying the patterns of metabolic plasticity and heterogeneity along the epithelial-hybrid-mesenchymal spectrum in cancer

abstract: Cancer metastasis is the leading cause of cancer-related mortality and the process of Epithelial to Mesenchymal Transition (EMT) is crucial for cancer metastasis. Either a partial or complete EMT have been reported to influence the metabolic plasticity of cancer cells in terms of switching among oxidative phosphorylation, fatty acid oxidation and glycolysis pathways. However, a comprehensive analysis of these major metabolic pathways their associations with EMT across different cancers is lacking. Here, we analyse more than 180 cancer cell datasets and show diverse associations of these metabolic pathways with the EMT status of cancer cells. Our bulk data analysis shows that EMT generally positively correlates with glycolysis but negatively with oxidative phosphorylation and fatty acid metabolism. These correlations are also consistent at the level of their molecular master regulators, namely AMPK and HIF1. Yet, these associations are shown to not be universal. Analysis of singlecell data of EMT induction shows dynamic changes along the different axes of metabolic pathways, consistent with general trends seen in bulk samples. Together, our results reveal underlying patterns of metabolic plasticity and heterogeneity as cancer cells traverse through the epithelial-hybrid-mesenchymal spectrum of states.


title: Endothelial Rbpj is essential for the education of tumour-associated macrophages

abstract: Epithelial ovarian cancer (EOC) is one of the most lethal gynaecological cancers worldwide. EOC cells educate tumour-associated macrophages (TAMs) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumour microenvironment (TME). In addition, tumour cells frequently activate Notch1 receptors on endothelial cells (ECs) to facilitate metastasis. However, little is known whether the endothelium would also influence the education of recruited monocytes. Here, we report that canonical Notch signalling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature. This was associated with stronger cytotoxic activity of T cells and decreased tumour burden. Mechanistically, we identified CXCL2 as a novel Notch/RBPJ target gene. This angiocrine factor regulates the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. As such, EOC cells employ the tumour endothelium to secrete CXCL2 in order to facilitate an immunosuppressive microenvironment.


title: Oxytocin Signaling Regulates the Homeostatic Response to Cold Stress in Poikilothermic Vertebrates

abstract: When exposed to low temperature, homeothermic vertebrates maintain internal body temperature by activating thermogenesis and by altered metabolism, synchronized by neuroendocrine responses. Although such physiological responses also occur in poikilothermic vertebrates, the prevailing notion is that their reactions are passive. Here, we explored molecular hypothalamic and physiological responses to cold stress in the tropical poikilotherm Nile tilapia (Oreochromis niloticus). We show that cold exposed tilapia exhibit complex homeostatic responses, including increased hypothalamic oxytocin, plasma glucose and cortisol concomitant with reduced plasma lactate and metabolic rate. Pharmacological or genetic blockage of oxytocin signaling further affected metabolic rate in two cold-exposed poikilothermic models. This indicates that oxytocin, a known thermoregulator in homeotherms, actively regulates temperature-related homeostasis in poikilotherms. Overall, our findings show that the brain of poikilotherms actively responds to cold temperature by regulating metabolic physiology. Moreover, we identify oxytocin signaling as an adaptive and evolutionarily conserved metabolic regulator of temperature-related homeostasis.

category: physiology


title: MIAAIM: Multi-omics image integration and tissue state mapping using topological data analysis and cobordism learning

abstract: High-parameter tissue imaging enables detailed molecular analysis of single cells in their spatial environment. However, the comprehensive characterization and mapping of tissue states through multimodal imaging across different physiological and pathological conditions requires data integration across multiple imaging systems. Here, we introduce MIAAIM (Multi-omics Image Alignment and Analysis by Information Manifolds) a modular, reproducible computational framework for aligning data across bioimaging technologies, modeling continuities in tissue states, and translating multimodal measures across tissue types. We demonstrate MIAAIMs workflows across diverse imaging platforms, including histological stains, imaging mass cytometry, and mass spectrometry imaging, to link cellular phenotypic states with molecular microenvironments in clinical biopsies from multiple tissue types with high cellular complexity. MIAAIM provides a robust foundation for the development of computational methods to integrate multimodal, high-parameter tissue imaging data and enable downstream computational and statistical interrogation of tissue states.

category: systems biology


title: Integrated Proteomic and Metabolomic Analyses of the Mitochondrial Neurodegenerative Disease MELAS

abstract:
MELAS (mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes) is a progressive neurodegenerative disease caused by pathogenic mitochondrial DNA variants. The pathogenic mechanism of MELAS remains enigmatic due to the exceptional clinical heterogeneity and the obscure genotype-phenotype correlation among MELAS patients. To gain insights into the pathogenic signature of MELAS, we designed a comprehensive strategy integrating proteomics and metabolomics in patient-derived dermal fibroblasts harboring the ultra-rare MELAS pathogenic variant m.14453G>A, specifically affecting the mitochondrial respiratory Complex I. Global proteomics was achieved by data-dependent acquisition (DDA) and verified by data-independent acquisition (DIA) using both Spectronaut and the recently launched MaxDIA platforms. Comprehensive metabolite coverage was achieved for both polar and nonpolar metabolites in both reverse phase and HILIC LC-MS/MS analyses. Our proof-of-principle MELAS study with multi-omics integration revealed OXPHOS dysregulation with a predominant deficiency of Complex I subunits, as well as alterations in key bioenergetic pathways, glycolysis, tricarboxylic acid cycle, and fatty acid {beta}-oxidation. The most clinically relevant discovery is the downregulation of the arginine biosynthesis pathway, likely due to blocked argininosuccinate synthase, which is congruent with the MELAS cardinal symptom of stroke-like episodes and its current treatment by arginine infusion. In conclusion, we demonstrated an integrated proteomic and metabolomic strategy for patient-derived fibroblasts, which has great clinical potential to discover therapeutic targets and design personalized interventions after validation with a larger patient cohort in the future.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/473301v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1f59135org.highwire.dtl.DTLVardef@110451forg.highwire.dtl.DTLVardef@129f9d8org.highwire.dtl.DTLVardef@d1f347_HPS_FORMAT_FIGEXP  M_FIG Graphic Abstract:

Integrated proteomics and metabolomics of patient fibroblasts revealed dysregulations in arginine biosynthesis, OXPHOS complexes, and bioenergetic pathways in MELAS, a mitochondrial neurodegenerative disease caused by mitochondrial DNA mutations.

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title: Top-down contribution to motor network reorganization during action preparation

abstract:
BACKGROUNDIt is unclear whether the Bereitschaftspotential (BP) recorded in humans during action preparation mirrors motor areas activation escalation, or if its early and late phases reflect the engagement of different functional networks.

OBJECTIVEHere, we aimed at recording the TMS evoked-potentials (TEP) stimulating the supplementary motor area (SMA) to assess whether and how cortical excitability and functional connectivity of this region change as the BP increases. We hypothesize that, at later stages, the SMA functional network should become more connected to regions relevant for the implementation of the final motor plan.

METHODSWe performed TMS-EEG recordings on fourteen healthy subjects during the performance of a visuomotor Go/No-go task, eliciting and recording cortical activity and functional connectivity at -700 ms and -300 ms before the onset of visual stimuli over the SMA.

RESULTSWhen approaching stimulus onset, and thus BP peak, the SMA increased its functional connectivity with movement-related structures in the gamma and alpha bands, indicating a regional top-down preparation to implement the motor act. Beta-band connectivity, instead, was maintained constant for the whole BP time-course, being potentially related to sustained attention required by the experimental task.

CONCLUSIONThese findings reveal that the BP is not a mere result of increased activation of the SMA, but the functional networks in which this region is involved qualitatively changes over time, becoming more related to the execution of the motor act.


title: Gustatory cortex is involved in evidence accumulation during food choice

abstract: Food choice is one of the most fundamental and most frequent value-based decisions for all animals including humans. However, the neural circuitry involved in food-based decisions is only recently being addressed. Given the relatively fast dynamics of decision formation, EEG-informed fMRI analysis is highly beneficial for localizing this circuitry in humans. Here by using the EEG correlates of evidence accumulation in a simultaneously recorded EEG-fMRI dataset, we found a significant role for the right temporal-parietal operculum (PO) and medial insula including gustatory cortex (GC) in binary choice between food items. These activations were uncovered by using the "EEG energy" (power 2) as the BOLD regressor and were missed if conventional analysis with the EEG signal itself were to be used, in agreement with theoretical predictions for EEG and BOLD relations. No significant positive correlations were found with higher powers of EEG (powers 3 or 4) pointing to specificity and sufficiency of EEG energy as the main correlate of the BOLD response. This finding extends the role of cortical areas traditionally involved in palatability processing to value-based decision making and offers the "EEG energy" as a key regressor of BOLD response in simultaneous EEG-fMRI designs.


title: Cortical and thalamic influences on striatal involvement in instructed, serial reversal learning; implications for the organisation of flexible behaviour.

abstract: Cognitive flexibility is essential for enabling an individual to respond adaptively to changes in their environment. Evidence from human and animal research suggests that the control of cognitive flexibility is dependent on an array of neural architecture. Cortico-basal ganglia circuits have long been implicated in cognitive flexibility. In particular, the role of the striatum is pivotal, acting as an integrative hub for inputs from the prefrontal cortex and thalamus, and modulation by dopamine and acetylcholine. Striatal cholinergic modulation has been implicated in the flexible control of behaviour, driven by input from the centromedian-parafascicular nuclei of the thalamus. However, the role of this system in humans is not clearly defined as much of the current literature is based on animal work. Here, we aim to investigate the roles corticostriatal and thalamostriatal connectivity in serial reversal learning. Functional connectivity between the left centromedian-parafascicular nuclei and the associative dorsal striatum was significantly increased for negative feedback compared to positive feedback. Similar differences in functional connectivity were observed for the right lateral orbitofrontal cortex, but these were localised to when participants switched to using an alternate response strategy following reversal. These findings suggest that connectivity between the centromedian-parafascicular nuclei and the striatum may be used to generally identify potential changes in context based on negative outcomes, and the effect of this signal on striatal output may be influenced by connectivity between the lateral orbitofrontal cortex and the striatum.


title: Aging impairs primary task resumption and attentional control processes following interruptions

abstract: Attentional selection of working memory content is impaired after an interruption. This effect was shown to increase with age. Here we investigate how electrophysiological mechanisms underlying attentional selection within working memory differ during primary task resumption between younger and older adults. Participants performed a working memory task, while being frequently interrupted with either a cognitively low- or high-demanding arithmetic task. Afterwards, a retrospective cue (retro-cue) indicated the working memory content required for later report. The detrimental effect of the interruption was evident in both age groups, but while younger adults were more strongly affected by a high-than by a low-demanding interruption, the performance deficit appeared independently of the cognitive requirements of the interruption task in older adults. A similar pattern was found regarding frontal-posterior connectivity in the theta frequency range, suggesting that aging decreases the ability to selectively maintain relevant information within working memory. The power of mid-frontal theta oscillations (4-7 Hz) featured a comparable effect of interruptions in both age groups. How-ever, posterior alpha power (8-14 Hz) following the retro-cue was more diminished by a preceding interruption in older adults. These results suggest an age-related deficit in the atten-tional selection and maintenance of primary task information following an interruption that appeared independent from the cognitive requirements of the interrupting task.


title: Even well-practiced movements benefit from repetition

abstract:
Professional golf players spend years practicing, but will still perform one or two practice swings without a ball before executing the actual swing. Why do they do this? In this study we tested the hypothesis that repeating a well-practiced movement leads to a reduction of movement variability. To operationalize this hypothesis, participants were tested in a center- out reaching task with four different targets, on four different days. To probe the effect of repetition they performed random sequences from one to six movements to the same target. Our findings show that, with repetition, movements are not only initiated earlier but their variability is reduced across the entire movement trajectory. Furthermore, this effect is present within and across the four sessions. Together, our results suggest that movement repetition changes the tradeoff between movement initiation and movement precision.

New & NoteworthyProfessional athletes practice movements that they have performed thousands of times in training just before it is their turn in a game. Why do they do this? Our results indicate that both initial and endpoint variability reduce with repetition in a short sequence of reaching movements. This means that even well-practiced movements benefit from practice.


title: Effectiveness and relationship between biased and unbiased measures of dopamine release and clearance

abstract: Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine (DA) release and clearance throughout the brain, including the ventral and dorsal striatum. Striatal DA terminals are abundant with signals heavily regulated by release machinery and the dopamine transporter (DAT). Peak height is a common method for measuring release but can be affected by changes in clearance. The Michaelis-Menten model has been a standard in measuring DA clearance, but requires experimenter fitted modeling subject to experimenter bias. The current study presents the use of the first derivative (velocity) of evoked DA signals as an alternative approach for measuring dopamine release and clearance and can be used to distinguish the two measures. Maximal upwards velocity predicts reductions in DA peak height due to D2 and GABAB receptor stimulation and by alterations in calcium concentrations. The Michaelis-Menten maximal velocity (Vmax) measure, an approximation for DAT numbers, predicted maximal downward velocity in slices and in vivo. Dopamine peak height and upward velocity were similar between wildtype C57 (WT) and DAT knock out (DATKO) mice. In contrast, downward velocity was considerably reduced and exponential decay (tau) was increased in DATKO mice, supporting use of both measures for changes in DAT activity. In slices, the competitive DAT inhibitors cocaine, PTT and WF23 increased peak height and upward velocity differentially across increasing concentrations, with PTT and cocaine reducing these measures at high concentrations. Downward velocity and tau values decreased and increased respectively across concentrations, with greater potency and efficacy observed with WF23 and PTT. In vivo recordings demonstrated similar effects of WF23 and PTT on measures of release and clearance. Tau was a more sensitive measure at low concentrations, supporting its use as a surrogate for the Michaelis-Menten measure of apparent affinity (Km). Together, these results inform on the use of these measures for DA release and clearance.