title: A versatile active learning workflow for optimization of genetic and metabolic networks

abstract: The study, engineering and application of biological networks require practical and efficient approaches. Current optimization efforts of these systems are often limited by wet lab labor and cost, as well as the lack of convenient, easily adoptable computational tools. Aimed at democratization and standardization, we describe METIS, a modular and versatile active machine learning workflow with a simple online interface for the optimization of biological target functions with minimal experimental datasets. We demonstrate our workflow for various applications, from simple to complex gene circuits and metabolic networks, including several cell-free transcription and translation systems, a LacI-based multi-level controller and a 27-variable synthetic CO2-fixation cycle (CETCH cycle). Using METIS, we could improve above systems between one and two orders of magnitude compared to their original setup with minimal experimental efforts. For the CETCH cycle, we explored the combinatorial space of [~]1025 conditions with only 1,000 experiments to yield the most efficient CO2-fixation cascade described to date. Beyond optimization, our workflow also quantifies the relative importance of individual factors to the performance of a system. This allows to identify so far unknown interactions and bottlenecks in complex systems, which paves the way for their hypothesis-driven improvement, which we demonstrate for the LacI multi-level controller that we were able to improve by 34-fold after having identified resource competition as limiting factor. Overall, our workflow opens the way for convenient optimization and prototyping of genetic and metabolic networks with customizable adjustments according to user experience, experimental setup, and laboratory facilities.

category: synthetic biology


title: Functional heterogeneity of cell populations increases robustness of pacemaker function in a numerical model of the sinoatrial node tissue

abstract: Each heartbeat is initiated by specialized pacemaker cells operating within the sinoatrial node (SAN). While individual cells within SAN tissue exhibit substantial heterogeneity of their electrophysiological parameters and Ca cycling, the role of this heterogeneity for cardiac pacemaker function remains mainly unknown. Here we investigated the problem numerically in a 25x25 square grid of coupled-clock Maltsev-Lakatta cell models and tested the hypothesis that functional heterogeneity of cell populations increases robustness of SAN function. The tissue models were populated by cells with different degree of heterogeneity of the two key model parameters of the coupled-clock system, maximum L-type Ca current conductance (gCaL) and sarcoplasmic reticulum Ca pumping rate (Pup). Our simulations showed that in the areas of Pup-gCaL parametric space at the edge of the system stability where action potential (AP) firing was absent or dysrhythmic in tissues populated by identical cells, rhythmic AP generation was rescued in tissues populated by cells with uniformly random distributions of gCaL or Pup (but keeping the same average values). This effect to increase robust AP generation was synergistic with respect to heterogeneity in both gCaL and Pup and was further strengthened by clustering of cells with higher gCaL or Pup. The effect of functional heterogeneity was not due to a simple summation of activity of intrinsically firing cells naturally present in SAN; rather AP firing cells locally and critically interacted with non-firing/dormant cells. When firing cells prevailed, they recruited many dormant cells to fire, strongly enhancing overall SAN function. And vice versa, prevailing dormant cells suppressed AP firing in cells with intrinsic automaticity and halted SAN automaticity.

category: systems biology


title: Effects of carbon to nitrogen ratio (C:N) on water quality and growth performance of Litopenaeus vannamei (Boone, 1931) in the biofloc system with a salinity of 5%

abstract: This study aimed to investigate the effects of carbon to nitrogen ratio (C:N) on the water quality and shrimp growth performance during the grow-out culture of Litopenaeus vannamei in the biofloc system under a low salinity condition. Three biofloc treatments with an C:N (contained in the inputted feed and carbon source with the assumption that 75% of the feed nitrogen is excreted) of 8:1 (CN8), 16:1 (CN16) and 24:1 (CN24), respectively, were designed to stocking shrimp juveniles (~ 0.8 g) at a density of 270 individuals m-3, for a 63-days culture experiment at a salinity of about 5{per thousand}. Results showed that in CN8 treatment, the levels of pH (6.9{+/-}0.1), carbonate alkalinity (104.0{+/-}2.8mg L-1 CaCO3), biofloc volume (4.8{+/-}0.9mL L-1) and TSS (327.4{+/-}24.4mg L-1) were significantly lower than those in the other two treatments ([≥]7.6{+/-}0.3, [≥]157.6{+/-}21.6mg L-1 CaCO3, [≥]24.1{+/-}3.7mL L-1 and [≥]508.1{+/-}32.3mg L-1, P<0.05); whereas the levels of TAN (7.1{+/-}0.9mg L-1), nitrite (14.0{+/-}3.6mg L-1) and nitrate (77.0{+/-}5.0mg L-1) were significantly higher than those in the other treatments ([≤]2.0{+/-}0.6mg L-1, [≤]4.9{+/-}3.1mg L-1 and [≤]14.7{+/-}5.9mg L-1, P<0.05). The zootechnical parameters of shrimp were not significantly different between three treatments (P>0.05), except that the survival rates in CN16 treatment (96.8{+/-}2.0%) and CN24 treatment (93.7{+/-}4.2%) were significantly higher than that of CN8 treatment (81.5{+/-}6.4%, P<0.05). The results indicated that an inputted C:N higher than 16:1 was suitable for the biofloc system with a low salinity of 5{per thousand}, with an optimal inferred C:N range of 18.5-21.0:1 for water quality and growth performance.

category: zoology


title: Dendritic integration of sensory and reward information facilitates learning

abstract: Learning goal-directed behaviours requires integration of separate information streams representing context, relevant stimuli and reward. Dendrites of pyramidal neurons are suitable sites for such integration, but it remains elusive how their responses adapt when an animal learns a new task. Here, we identify two distinct classes of dendritic responses that represent either contextual/sensory information or reward information and that differ in their task- and learning-related dynamics. Using longitudinal calcium imaging of apical dendritic tufts of L5 pyramidal neurons in mouse barrel cortex, we tracked dendritic activity across learning and analyzed both local dendritic branch signals and global apical tuft activity. During texture discrimination learning, sensory representations (including contextual and touch information) strengthened and converged on the reward-predicting tactile stimulus when mice became experts. In contrast, reward-associated responses were particularly strong in the naive condition and became less pronounced upon learning. When we blocked the representation of unexpected reward in naive animals with optogenetic inhibition, animals failed to learn until we released the block and learning proceeded normally. Our results suggest that reward signals in dendrites are essential for adjusting neuronal integration of converging inputs to facilitate adaptive behaviour.

category: neuroscience


title: Traumatic brain injury alters dendritic cell differentiation and distribution in lymphoid and non-lymphoid organs

abstract: Pathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), remains completely unknown. Here, we report that DC-differentiation, maintenance and functions are altered at both early and late phases of TBI. Our studies identify that; 1. frequencies and absolute numbers of DCs in the spleen and BM are altered at both acute and late phases of TBI; 2. surface expression of key molecules involved in antigen presentation of DCs were affected both at early and late phases of TBI; 3. distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; 4. early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors, were deregulated after TBI; and 5. intracellular ROS levels were reduced in DC progenitors and differentiated DCs at both early and late phases of TBI. Our data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs at both early and late phases of TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries.

category: immunology


title: In contrast to TH2-biased approaches, TH1 COVID-19 vaccines protect Syrian hamsters from severe disease in the absence of dexamethasone-treatable vaccine-associated enhanced respiratory pathology

abstract:
Since December 2019, the novel human coronavirus SARS-CoV-2 has spread globally, causing millions of deaths. Unprecedented efforts have enabled development and authorization of a range of vaccines, which reduce transmission rates and confer protection against the associated disease COVID-19. These vaccines are conceptually diverse, including e.g. classical adjuvanted whole-inactivated virus, viral vectors, and mRNA vaccines.

We have analysed two prototypic model vaccines, the strongly TH1-biased measles vaccine-derived candidate MeVvac2-SARS2-S(H) and a TH2-biased Alum-adjuvanted, non-stabilized Spike (S) protein side-by-side, for their ability to protect Syrian hamsters upon challenge with a low-passage SARS-CoV-2 patient isolate. As expected, the MeVvac2-SARS2-S(H) vaccine protected the hamsters safely from severe disease. In contrast, the protein vaccine induced vaccine-associated enhanced respiratory disease (VAERD) with massive infiltration of eosinophils into the lungs. Global RNA-Seq analysis of hamster lungs revealed reduced viral RNA and less host dysregulation in MeVvac2-SARS2-S(H) vaccinated animals, while S protein vaccination triggered enhanced host gene dysregulation compared to unvaccinated control animals. Of note, mRNAs encoding the major eosinophil attractant CCL-11, the TH2 response-driving cytokine IL-19, as well as TH2-cytokines IL-4, IL-5, and IL-13 were exclusively up-regulated in the lungs of S protein vaccinated animals, consistent with previously described VAERD induced by RSV vaccine candidates. IL-4, IL-5, and IL-13 were also up-regulated in S-specific splenocytes after protein vaccination. Using scRNA-Seq, T cells and innate lymphoid cells were identified as the source of these cytokines, while Ccl11 and Il19 mRNAs were expressed in lung macrophages displaying an activated phenotype. Interestingly, the amount of viral reads in this macrophage population correlated with the abundance of Fc-receptor reads. These findings suggest that VAERD is triggered by induction of TH2-type helper cells secreting IL-4, IL-5, and IL-13, together with stimulation of macrophage subsets dependent on non-neutralizing antibodies. Via this mechanism, uncontrolled eosinophil recruitment to the infected tissue occurs, a hallmark of VAERD immunopathogenesis. These effects could effectively be treated using dexamethasone and were not observed in animals vaccinated with MeVvac2-SARS2-S(H).

Taken together, our data validate the potential of TH2-biased COVID-19 vaccines and identify the transcriptional mediators that underlie VAERD, but confirm safety of TH1-biased vaccine concepts such as vector-based or mRNA vaccines. Dexamethasone, which is already in use for treatment of severe COVID-19, may alleviate such VAERD, but in-depth scrutiny of any next-generation protein-based vaccine candidates is required, prior and after their regulatory approval.


title: Aberrant paracrine signalling for bone remodelling underlies the mutant histone-driven giant cell tumour of bone.

abstract:
Oncohistones represent compelling evidence for a causative role of epigenetic perturbations in cancer. Giant cell tumours of bone (GCTs) are characterised by a mutated histone H3.3 as the sole genetic driver present in bone-forming osteoprogenitor cells but absent from abnormally large bone-resorbing osteoclasts which represent the hallmark of these neoplasms. While these striking features imply a pathogenic interaction between mesenchymal and myelomonocytic lineages during GCT development, the underlying mechanisms remain unknown.

We show that the changes in the transcriptome and epigenome in the mesenchymal cells caused by the H3.3-G34W mutation contribute to increase osteoclast recruitment in part via reduced expression of the TGF{beta}-like soluble factor, SCUBE3. In turn, osteoclasts secrete unregulated amounts of SEMA4D enhancing proliferation of mutated osteoprogenitors and arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation upon denosumab treatment, a drug that depletes osteoclasts. In contrast, gain of hTERT activity, commonly found in malignant GCT, makes neoplastic cells insensitive to osteoclasts, predicting the unresponsiveness to denosumab.

We provide a mechanism for GCT initiation and its response to current treatment, the basis of which is dysfunctional cross-talk between bone-forming and bone-resorbing cells, emphasising the importance of tumor/microenvironment bidirectional interactions in tumorigenesis.

category: cancer biology


title: Comparative analysis of Lysine and Arginine biosynthesis pathway in Deinococcus genomes

abstract: The members of the Deinococcaceae family have the ability to survive extreme environmental conditions. Deinococcus species have a complex cell envelope composed of L-ornithine containing peptidoglycan. Anabolism of L-ornithine is intrinsically linked to L-lysine and L-arginine biosynthetic pathways. To understand these two pathways, we analyzed the L-lysine and L-arginine pathways using 23 Deinococcus genomes, including D. indicus. We used BLAST-P based ortholog identification using D. radiodurans genes as the query. We identified some BLAST-P hits that shared the same functional annotation. We analyzed three (class I aminotransferase, acetyl-lysine deacetylase, and acetyl glutamate/acetyl aminoadipate kinase) from L-lysine biosynthesis pathway and three (bifunctional ornithine acetyltransferase or N-acetyl glutamate synthase protein, nitric oxide synthase-like protein, and Acetyl-lysine deacetylase) from L-arginine biosynthesis pathway. Two proteins showed certain structural variations. Specifically, [LysW]-lysine hydrolase proteins sequence and structure level changes indicated changes in oligomeric conformation, which could likely be a result of divergent evolution. And, bifunctional ornithine acetyltransferase or N-acetyl glutamate synthase had its active site pocket positions shifted at the structural level and we hypothesize that it may not perform at the optimal level. Thus, we were able to compare and contrast different Deinococcus species indicating some genes occurring because of divergent evolution.

category: bioinformatics


title: Biomechanical and biochemical assessment of YB-1 expression in melanoma cells

abstract: Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the last five decades. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT) processes. Migratory melanoma cells exhibit two major phenotypes: elongated mesenchymal or rounded amoeboid. The actomyosin cytoskeleton is key in both phenotypes, but intermediate filaments also undergo a significant rearrangement process, switching from cytokeratin-rich to vimentin and nestin-rich network. In this study, we aimed to investigate to what extent YB-1 impacts the biomechanical (cell stiffness) and biochemical aspects of melanoma cells and their cytoskeleton. To this end, we subjected A375 YB-1 knock-out and parental cells to atomic force microscopy investigations (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the corresponding YB-1 knock-out cell line. Proteomic analysis revealed that expression of YB-1 results in a strong co-expression of nestin, vimentin, fascin-1, and septin-9. In the YB-1 knock-out nestin was completely depleted, but zyxin was strongly upregulated. Collectively, our results showed that YB-1 knock-out acquires some characteristics of mesenchymal phenotype but lacks important markers of malignancy and invasiveness such as nestin or vimentin. We posit that there is an association of YB-1 expression with an amoeboid phenotype, which would explain the increased migratory capacity.


title: Interleukin 4 controls the role of macrophages in pulmonary metastatic tumor cell seeding and growth

abstract: Metastasis is the systemic manifestation of cancer and the main cause of death from breast cancer. In mouse models of lung metastases, recruitment of classical monocytes from blood to the lung and their differentiation to metastasis-associated macrophages (MAMs) facilitate cancer cell extravasation, survival, and growth. Ablation of MAMs or their monocytic progenitors inhibits metastasis. We hypothesized that factors controlling macrophage polarization modulate tumor cell extravasation in the lung. We evaluated whether signaling by Th1 or Th2 cytokines in macrophages affected trans-endothelial migration of tumor cells in vitro. Interferon {gamma} and LPS inhibited macrophage-dependent tumor cell extravasation while the Th2 cytokine interleukin-4 (IL4) enhanced this process. We demonstrated that IL4 receptor (IL4r) null mice develop fewer and smaller lung metastasis. Adoptive transfer of wild type monocytes to IL4r deficient mice rescued this phenotype. IL4 signaling in macrophages controls the expression of the chemokine receptor CXCR2, necessary for IL4-mediated tumor cell extravasation in vitro. Furthermore, IL4 signaling in macrophages transcriptionally regulates several other genes already causally associated with lung metastasis including CCL2, CSF1, CCR1, HGF and FLT1. The central role for IL4 signaling in MAMs was confirmed by high-resolution intravital imaging of the lung in mice at the time of metastatic seeding, which showed reduced physical interaction between tumor cells and IL4r-deficient macrophages. This interaction enhances tumor cell survival. These data indicate that IL4 signaling in monocytes and macrophages is key during seeding and growth of breast metastasis in the lung as it regulates pro-tumoral paracrine signaling between cancer cells and macrophages.


title: A high-throughput 3D bioprinted cancer cell migration and invasion model with versatile and broad biological applicability

abstract: Understanding the underlying mechanisms of migration and metastasis is a key focus of cancer research. There is an urgent need to develop in vitro 3D tumor models that can mimic physiological cell-cell and cell-extracellular matrix interactions, with high reproducibility and that are suitable for high throughput (HTP) drug screening. Here, we developed a HTP 3D bioprinted migration model using a bespoke drop-on-demand bioprinting platform. This HTP platform coupled with tunable hydrogel systems enables (i) the rapid encapsulation of cancer cells within in vivo tumor mimicking matrices, (ii) in situ and real-time measurement of cell movement, (iii) detailed molecular analysis for the study of mechanisms underlying cell migration and invasion, and (iv) the identification of novel therapeutic options. This work demonstrates that this HTP 3D bioprinted cell migration platform has broad applications across quantitative cell and cancer biology as well as drug screening.

category: cell biology


title: Precise measurement of nanoscopic septin ring structures in deep learning-assisted quantitative superresolution microscopy

abstract: The combination of image analysis and fluorescence superresolution microscopy methods allows for unprecedented insight into the organization of macromolecular assemblies in cells. Advances in deep learning-based object recognition enables the automated processing of large amounts of data, resulting in high accuracy through averaging. However, while the analysis of highly symmetric structures of constant size allows for a resolution approaching the dimensions of structural biology, deep learning methods are prone to different forms of bias. A biased recognition of structures may prohibit the development of readouts for processes that involve significant changes in size or shape of amorphous macromolecular complexes. What is required to overcome this problem is a detailed investigation of potential sources of bias and the rigorous testing of trained models using real or simulated data covering a wide dynamic range of possible results. Here we combine single molecule localization-based superresolution microscopy of septin ring structures with the training of several different deep learning models for a quantitative investigation of bias resulting from different training approaches and finally quantitative changes in septin ring structures. We find that trade-off exists between measurement accuracy and the dynamic range of recognized phenotypes. Using our trained models, we furthermore find that septin ring size can be explained by the number of subunits they are assembled from alone. Our work provides a new experimental system for the investigation of septin polymerization.


title: The EBNA2-EBF1 complex promotes oncogenic MYC expression levels and metabolic processes required for cell cycle progression of Epstein-Barr virus-infected B cells

abstract:
Epstein-Barr virus (EBV) is a human tumor virus, which preferentially infects resting human B cells. Upon infection in vitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen (EBNA) 2 is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor (EBF) 1, a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. An 1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the 1-helix in EBNA2 can infect and activate B cells efficiently, but the activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F as well as multiple metabolic processes linked to cell cycle progression are impaired in EBV{Delta}1 infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification.

Significance statementEpstein-Barr virus (EBV) infects primary B cells and establishes life-long latent infection in these cells. EBV nuclear antigen (EBNA) 2 drives early processes of B cell activation and cell cycle entry. The surface of the N-terminal dimerization domain of EBNA2 exposes a five amino acid -helix (1) that recruits EBF1 to activate MYC and downstream targets of both MYC and E2F to support critical metabolic processes in infected B cells and to drive them through S phase in the first cell cycle post-infection. Our study demonstrates how EBNA2 exploits EBF1, a key factor of B cell lineage specification to initiate proliferation and high-lights the 1-helix as a potential Achilles heel of the virus at the stage when latent infection is established.

category: molecular biology


title: Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign

abstract: DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, phosphodiester linkages that create the repeating sugarphosphate backbone of DNA. Can this linkage selectivity observed in nature be overcome by design to produce non-natural nucleic acids? Here, we report that structure-guided redesign of an archaeal DNA polymerase (9{degrees}N) enables a new polymerase activity that is undetectable in the wild type enzyme: catalyzing the formation of N3[->]P5 phosphoramidate linkages in the presence of 3-amino-2,3-dideoxynucleoside 5-triphosphate (3-NH2-ddNTP) building blocks. Replacing a highly conserved metal-binding aspartate in the 9{degrees}N active site (Asp-404) with asparagine was key to the emergence of this unnatural enzyme activity. Molecular dynamics simulations provided insights into how a single substitution could enhance the productive positioning of the 3-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface with substitutions in the finger subdomain led to a quadruple-mutant variant (9{degrees}N-NRQS) that incorporated 3-NH2-ddNTPs into a 3-amino-primer on various DNA templates. This work presents the first example of an active-site substitution of a metal-binding residue that leads to a novel activity in a DNA polymerase, and sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

category: biochemistry


title: Performance and stability of fly-inspired,saccade-based hybrid control

abstract: In a way analogous to human vision, the fruit fly Drosophila melanogaster and many other flying insects generate smooth and saccadic movements to stabilize and shift their gaze in flight, respectively. It has been hypothesized that this combination of continuous and discrete movements benefits both flight stability and performance, particularly at high frequencies or speeds. Here we develop a hybrid control system model to explore the effects of saccades on the yaw stabilization reflex of D. melanogaster. Inspired from experimental data, the model includes a first order plant, a Proportional-Integral (PI) continuous controller, and a saccadic reset system that fires based on the integrated error of the continuous controller. We explore the gain, delay and switching threshold parameter space to quantify the optimum regions for stability and performance. We show that the addition of saccades to a continuous controller provides benefits to both stability and performance across a range of frequencies. Our model suggests that Drosophila operate near its optimal switching threshold for its experimental gain set. We also show that based on experimental data, D. melanogaster operates in a region that trades off performance and stability. This trade-off increases flight robustness to compensate for environmental uncertainties such as wing damage.

category: biophysics


title: The influence of saccades on insect flight control


title: A Novel CRISPR-Engineered, Stem Cell-Derived Cellular Vaccine

abstract: COVID-19 has forced rapid clinical translation of novel vaccine technologies, principally mRNA vaccines, that have resulted in meaningful efficacy and adequate safety in response to the global pandemic. Notwithstanding this success, there remains an opportunity for innovation in vaccine technology to address current limitations and meet the challenges of inevitable future pandemics. We describe a universal vaccine cell (UVC) rationally designed to mimic the natural physiologic immunity induced post viral infection of host cells. Induced pluripotent stem cells were CRISPR engineered to delete MHC-I expression and simultaneously overexpress a NK Ligand adjuvant to increase rapid cellular apoptosis which was hypothesized to enhance viral antigen presentation in the resulting immune microenvironment leading to a protective immune response. Cells were further engineered to express the parental variant WA1/2020 SARS-CoV-2 spike protein as a representative viral antigen prior to irradiation and cryopreservation. The cellular vaccine was then used to immunize non-human primates in a standard 2-dose, IM injected prime + boost vaccination with 1e8 cells per 1 ml dose resulting in robust neutralizing antibody responses (1e3 nAb titers) with decreasing levels at 6 months duration. Similar titers generated in this established NHP model have translated into protective human neutralizing antibody levels in SARS-Cov-2 vaccinated individuals. Animals vaccinated with WA1/2020 spike antigens were subsequently challenged with 1.0 x 105 TCID50 infectious Delta (B.1.617.2) SARS-CoV-2 in a heterologous challenge which resulted in an approximately 3-log order decrease in viral RNA load in the lungs. These heterologous viral challenge results reflect the ongoing real-world experience of original variant WA1/2020 spike antigen vaccinated populations exposed to rapidly emerging variants like Delta and now Omicron. This cellular vaccine is designed to be a rapidly scalable cell line with a modular poly-antigenic payload to allow for practical, large-scale clinical manufacturing and use in an evolving viral variant environment. Human clinical translation of the UVC is being actively explored for this and potential future pandemics.


title: mRNA-1273 and Ad26.COV2.S vaccines protect against the B.1.621 variant of SARS-CoV-2

abstract: Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, viral variants with greater transmissibility or immune evasion properties have arisen, which could jeopardize recently deployed vaccine and antibody-based countermeasures. Here, we evaluated in mice and hamsters the efficacy of preclinical non-GMP Moderna mRNA vaccine (mRNA-1273) and the Johnson & Johnson recombinant adenoviral-vectored vaccine (Ad26.COV2.S) against the B.1.621 (Mu) South American variant of SARS-CoV-2, which contains spike mutations T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N. Immunization of 129S2 and K18-human ACE2 transgenic mice with mRNA-1273 vaccine protected against weight loss, lung infection, and lung pathology after challenge with B.1.621 or WA1/2020 N501Y/D614G SARS-CoV-2 strain. Similarly, immunization of 129S2 mice and Syrian hamsters with a high dose of Ad26.COV2.S reduced lung infection after B.1.621 virus challenge. Thus, immunity induced by mRNA-1273 or Ad26.COV2.S vaccines can protect against the B.1.621 variant of SARS-CoV-2 in multiple animal models.

category: microbiology


title: Functional properties of the spike glycoprotein of the emerging SARS-CoV-2 variant B.1.1.529

abstract: The recently emerged B.1.1.529 (Omicron) SARS-CoV-2 variant has a highly divergent spike (S) glycoprotein. We compared the functional properties of B.1.1.529 S with those of previous globally prevalent SARS-CoV-2 variants, D614G and B.1.617.2. Relative to these variants, B.1.1.529 S exhibits decreased processing, resulting in less efficient syncytium formation and lower S incorporation into virus particles. Nonetheless, B.1.1.529 S supports virus infection equivalently. B.1.1.529 and B.1.617.2 S glycoproteins bind ACE2 with higher affinity than D614G S. The unliganded B.1.1.529 S trimer is less stable at low temperatures than the other SARS-CoV-2 spikes, a property related to spike conformation. Upon ACE2 binding, the B.1.1.529 S trimer sheds S1 at 37 degrees but not at 0 degrees C. B.1.1.529 pseudoviruses are relatively resistant to neutralization by sera from convalescent COVID-19 patients and vaccinees. These properties of the B.1.1.529 spike glycoprotein likely influence the transmission, cytopathic effects and immune evasion of this emerging variant.


title: A Combined Immunophenotypic And Transcriptional Single-Cell Map Of First Trimester Human Fetal Liver Hematopoiesis

abstract:
Knowledge of human fetal blood development and how it differs from adult is highly relevant for our understanding of congenital blood and immune disorders as well as childhood leukemia, the latter known to originate in utero. Blood production during development occurs in waves that overlap in time and space adding to heterogeneity, which necessitates single cell approaches. Here, a combined single cell immunophenotypic and transcriptional map of first trimester primitive blood development is presented. Using CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) the molecular profile of established immunophenotypic gated progenitors was analyzed in the fetal liver (FL). Classical markers for hematopoietic stem cells (HSCs) such as CD90 and CD49F were largely preserved, whereas CD135 (FLT3) and CD123 (IL3R) had a ubiquitous expression pattern capturing heterogenous populations. Direct molecular comparison with an adult bone marrow (BM) data set revealed that HSC-like cells were less frequent in FL, whereas cells with a lympho-myeloid signature were more abundant. Furthermore, an erythro-myeloid primed multipotent progenitor cluster was identified, potentially representing a transient, FL-specific progenitor. Based on the projection performed, up- and downregulated genes between fetal and adult cells were analyzed. In general, cell cycle pathways, including MYC targets were shown to be upregulated in fetal cells, whereas gene sets involved in inflammation and human leukocyte antigen (HLA) complex were downregulated. Importantly, a fetal core molecular signature was identified that could discriminate certain types of infant and childhood leukemia from adult counterparts.

Our detailed single cell map presented herein emphasizes molecular as well as immunophenotypic differences between fetal and adult primitive blood cells, of significance for future studies of pediatric leukemia and blood development in general.

category: developmental biology


title: A transient apical extracellular matrix relays cytoskeletal patterns to shape permanent acellular ridges on the surface of adult C. elegans

abstract: Apical extracellular matrices can form protruding structures such as denticles, ridges, scales, or teeth on the surfaces of epithelia. The mechanisms that shape these structures remain poorly understood. Here, we show how the actin cytoskeleton and a provisional matrix work together to sculpt acellular longitudinal alae ridges in the cuticle of adult C. elegans. Transient actomyosin-dependent constriction of the underlying lateral epidermis accompanies deposition of the provisional matrix at the earliest stages of alae formation. Actin is required to pattern the provisional matrix into longitudinal bands that are initially offset from the pattern of longitudinal actin filaments. These bands appear ultrastructurally as alternating regions of adhesion and separation within laminated provisional matrix layers. The provisional matrix is required to establish these demarcated zones of adhesion and separation, which ultimately give rise to alae ridges and their intervening valleys, respectively. Provisional matrix proteins shape the alae ridges and valleys but are not present within the final structure. We propose a morphogenetic mechanism wherein cortical actin patterns are relayed mechanically to the laminated provisional matrix to set up distinct zones of matrix layer separation and accretion that shape a permanent and acellular matrix structure.


title: Ret loss-of-function decreases enteric neural crest progenitor proliferation and restricts developmental fate potential during enteric nervous system development

abstract:
The receptor tyrosine kinase gene RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. Pathogenic RET loss of function (LoF) alleles are associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. ENCDCs invade the developing GI tract, proliferate, migrate caudally, and differentiate into all of the major ENS cell types. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, its cell type and state-specific effects are unknown. Consequently, we performed single- cell RNA sequencing (scRNA-seq) on an enriched population of ENCDCs isolated from the developing GI tract of Ret null heterozygous and homozygous mouse embryos at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: (1) Ret-expressing ENCDCs are a heterogeneous population composed of ENS progenitors as well as glial and neuronal committed cells; (2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; (3) HSCR-associated and Ret gene regulatory network genes exhibit distinct expression patterns across Ret-expressing ENCDC with their expression impacted by Ret LoF; and (4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes associated with the proper development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.

Summary StatementRet LoF affects proper development of the mouse ENS through multiple distinct cellular phenotypes including restriction of neuronal fate potential, disruption of ENCDC migration, and modulation of progenitor proliferation rate.


title: A single cell transcriptional roadmap for human pacemaker cell differentiation

abstract: Each heartbeat is triggered by the sinoatrial node, the natural pacemaker of the heart. Animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into "transitional", "tail" and "head" subtypes. However, the underlying molecular mechanisms are poorly understood. Here, we studied the differentiation of human induced pluripotent stem cells into pacemaker cardiomyocytes. Single cell RNA sequencing identified the presence of myocardial populations resembling subtypes present in the formed sinoatrial node, and in addition revealed a side population of (pro)epicardial cells. Time-course trajectory analysis uncovered a role for WNT signaling in determining myocardial versus proepicardial cell fate. We experimentally demonstrate that presence of WNT signaling prior to the branching point of a common progenitor enhances proepicardial cell differentiation at the expense of myocardial pacemaker cells. Furthermore, we uncover a role for TGF{beta} and WNT signaling in differentiation towards transitional and head pacemaker subtypes, respectively. Our findings provide new biological insights into human pacemaker differentiation, open avenues for complex disease modeling and inform regenerative approaches.


title: Dynamic Expedition of Leading Mutations in SARS-CoV-2 Spike Glycoproteins

abstract: During the ongoing CoVID-19 epidemic, the continuous genomic evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been generating new variants with enhanced transmissibility and immune escape. Being one key target of antibodies, mutations of the spike glycoprotein play a vital role in the trajectory of virus evasion. Here, we present a time-resolved statistical method, dynamic expedition of leading mutations (deLemus), to analyze the evolution dynamics of the spike protein. Together with analysis on single amino-acid polymorphism (SAP), we proposed one L-index to quantify the mutation strength of each amino acid for unravelling mutation pattern of spike glycoprotein. The sites of interest (SOI) with high L-index hold great promise to detect potential signal of emergent variants.

category: evolutionary biology


title: Efficient CRISPR/Cas12a-based genome editing toolbox for metabolic engineering in Methanococcus maripaludis

abstract: The rapid-growing and genetically tractable methanogen Methanococcus maripaludis is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits to the primary metabolism that affect cell growth. Here, we have designed a genome editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in M. maripaludis. Remarkably, this toolbox can knock out target genes with a success rate of up to 95%, despite the hyper-polyploidy of M. maripaludis. For the purposes of demonstrating a large-sized deletion, we have replaced the flagellum operon (ca. 8.9 kbp) by the {beta}-glucuronidase gene. To facilitate metabolic engineering and flux balancing in M. maripaludis, the relative strength of 15 different promoters were quantified in the presence of the two common growth substrates, formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and fast method for genome editing in a methanogen.

category: bioengineering


title: Capturing a crucial 'disorder-to-order transition' at the heart of the coronavirus molecular pathology - triggered by highly persistent, interchangeable salt-bridges

abstract: The COVID-19 origin debate has greatly been influenced by Genome comparison studies of late, revealing the seemingly sudden emergence of the Furin-Like Cleavage Site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCSSpike) containing its 681PRRAR685 motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a disorder-to-order transition of the FLCSSpike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike-Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCSSpike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seems the other key characteristic features of the Spike-Furin binding in SARS-CoV-2 allowing the system to breathe - a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike-Furin binding events generally in the coronavirus lineage seems to have a preference for ionic bond formation, even with lesser number of cationic residues at their potentially polybasic FLCSSpike patches. The interaction energies are suggestive of a characteristic metastabilities attributed to Spike-Furin interactions generally to the coronavirus lineage - which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. T he current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution but also add substantially to the existing theories of proteolytic cleavages.


title: All-atom molecular dynamics simulations of synaptic vesicle fusion I: a glimpse at the primed state

abstract: Synaptic vesicles are primed into a state that is ready for fast neurotransmitter release upon Ca2+-binding to synaptotagmin-1. This state likely includes trans-SNARE complexes between the vesicle and plasma membranes that are bound to synaptotagmin-1 and complexins. However, the nature of this state and the steps leading to membrane fusion are unclear, in part because of the difficulty of studying this dynamic process experimentally. To shed light into these questions, we performed all-atom molecular dynamics simulations of systems containing trans-SNARE complexes between two flat bilayers or a vesicle and a flat bilayer with or without fragments of synaptotagmin-1 and/or complexin-1. Our results help visualize potential states of the release machinery en route to fusion, and suggest mechanistic features that may control the speed of release. In particular, the simulations suggest that the primed state contains almost fully assembled trans-SNARE complexes bound to the synaptotagmin-1 C2B domain and complexin-1 in a spring-loaded configuration where interactions of the C2B domain with the plasma membrane orient complexin-1 toward the vesicle, avoiding premature membrane merger but keeping the system ready for fast fusion upon Ca2+ influx.


title: Transcription Profile And Pathway Analysis Of The Endocannabinoid Receptor Inverse Agonist AM630 In The Core And Infiltrative Boundary Of Human Glioblastoma Cells

abstract:
BackgroundWe have previously reported that the endocannabinoid receptor inverse agonist AM630 is a potent inhibitor of isocitrade dehydrogenase-1 wild-type glioblastoma (GBM) core tumor cell proliferation. To uncover the mechanism behind the anti-tumour effects we have performed a transcriptional analysis of AM630 activity both in the tumour core cells (U87) and the invasive margin cells (GIN-8), the latter representing a better proxy of post-surgical residual disease.

ResultsThe core and invasive margin cells exhibited markedly different gene expression profiles and only the core cells had high expression of a potential AM630 target, the CB1 receptor. Both cell types had moderate expression of the HTR2B serotonin receptor, a reported AM630 target. We found that the AM630 driven transcriptional response was substantially higher in the central cells than in the invasive margin cells, with the former driving the up regulation of immune response and the down regulation of cell cycle and metastatic pathways and correlating with transcriptional responses driven by established anti-neoplastics as well as serotonin receptor antagonists.

ConclusionOur results highlight the different responsiveness of the core and invasive margin cells. Taken together, whilst our findings identify AM630 as an anti-neoplastic drug, showing a high correlation with known anti-proliferative drugs, we find distinct drug sensitivies of the infiltrative margin relative to contrast-enhanced core regions of GBM upon which failed molecular targeted therapies to date have been predicated.


title: Deciphering sex-specific miRNAs as heat-recorders in zebrafish

abstract: MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression in a wide variety of physiological processes, including those related to the reproductive system. Although in the last decade a plethora of miRNAs has been reported, the miRNA alterations occurred by environmental cues and their biological functions have not yet been elucidated. With the aim to identify epigenetic regulations mediated by miRNAs in the gonads in a climate change scenario, zebrafish (Danio rerio) were subjected to high temperatures during sex differentiation (18-32 days post fertilization, dpf), a treatment that results in male-skewed sex ratios. Once the fish reached adulthood (90 dpf), ovaries and testes were sequenced by high-throughput technologies. About 101 million high-quality reads were obtained from gonadal samples. Analyses of the expression levels of the miRNAs identified a total of 23 and 1 differentially expressed (DE) miRNAs in ovaries and testes, respectively, two months after the heat treatment. Most of the identified miRNAs were involved in human sex-related cancer. After retrieving 3 UTR regions, ~400 predicted targets of the 24 DE miRNAs were obtained, some with reproduction-related functions. Their synteny in the zebrafish genome was, for more than half of them, in the chromosomes 7, 2, 4, 3 and 11 in the ovaries, chromosome 4 being the place where the predicted sex-associated-region (sar) is localized in wild zebrafish. Further, spatial localization in the gonads of two selected miRNAs (miR-122-5p and miR-146-5p) showed exclusive expression in the ovarian germ cells. The present study expands the catalog of sex-specific miRNAs and deciphers, for the first time, thermosensitive miRNAs in the zebrafish gonads that might be used as potential epimarkers to predict environmental past events.

category: genomics


title: The P2Y2 Nucleotide Receptor Mediates Monocyte Tissue Factor Expression and Endotoxemia Death in Mice

abstract: Recently we reported that in human coronary artery endothelial cells, activation of the P2Y2 receptor (P2Y2R) induces up-regulation of tissue factor (TF), a vital initiator of the coagulation cascade. However, others have shown that monocyte TF is more critical than endothelial TF in provoking a pro-thrombotic state. Thus, we aimed to study whether monocytes express the P2Y2R, its role in controlling TF expression, and its relevance in vivo. RT-PCR and receptor activity assays revealed that among the eight P2Y nucleotide receptors, the P2Y2 subtype was selectively and functionally expressed in human monocytic THP-1 cells and primary monocytes. Stimulation of the cells by ATP or UTP dramatically increased TF protein expression, which was abolished by AR-C118925, a selective P2Y2R antagonist, or by siRNA silencing the P2Y2R. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA preceded with an increased TF pre-mRNA, indicating enhanced TF gene transcription. In addition, stimulation of the monocyte P2Y2R significantly activated ERK1/2, JNK, p38, and Akt, along with their downstream transcription factors including c-Jun, c-Fos, and ATF-2, whereas blocking these pathways respectively, all significantly suppressed P2Y2R-mediated TF expression. Furthermore, we found that LPS triggered ATP release and TF expression, the latter of which was suppressed by apyrase or P2Y2R blockage. Importantly, P2Y2R-null mice were more resistant than wild-type mice in response to a lethal dose of LPS, accompanied by much less TF expression in bone marrow cells. These findings demonstrate for the first time that the P2Y2R mediates TF expression in human monocytes through mechanisms involving ERK1/2, JNK, p38, and AKT, and that P2Y2R deletion protects the mice from endotoxemia-induced TF expression and death, highlighting monocyte P2Y2R may be a new drug target for the prevention and/or treatment of relevant thrombotic disease.

category: pharmacology and toxicology


title: AtERF60 negatively regulates ABR1 to modulate salt, drought, and basal resistance in Arabidopsis

abstract: ABSCISIC ACID REPRESSOR-1 (ABR1), an APETALA2 (AP2) domain containing transcription factor (TF) contribute important function against variety of external cues. Here, we report an AP2/ERF TF, AtERF60 that serves as an important regulator of ABR1 gene. AtERF60 is induced in response to drought, salt, abscisic acid (ABA), salicylic acid (SA), and bacterial pathogen PstDC3000 infection. AtERF60 interacts with DEHYDRATION RESPONSE ELEMENTS (DRE1/2) and GCC box indicating its ability to regulate multiple responses. Overexpression of AtERF60 results in the drought and salt stress tolerant phenotype in both seedling and mature Arabidopsis plants in comparison with the wild type (WT-Col). However, mutation in AtERF60 showed hyperactive response against drought and salt stress in comparison with its overexpression and WT. Microarray and qRT-PCR analysis of overexpression and mutant lines indicated that AtERF60 regulates both abiotic and biotic stress inducible genes. One of the differentially expressing transcripts was ABR1 and we found that AtERF60 interacts with the DRE cis-elements present in the ABR1 promoter. The mutation in AtERF60 showed ABA hypersensitive response, increased ABA content, and reduced susceptibility to PstDC3000. Altogether, we conclude that AtERF60 represses ABR1 transcript by binding with the DRE cis-elements and modulates both abiotic and biotic stress responses in Arabidopsis.

category: plant biology


title: Inside-out Signalling From Aminopeptidase N (CD13) To Complement Receptor 3 (CR3, CD11b/CD18)

abstract:
Upon ligand engagement, certain receptors can activate an integrin through a mechanism called inside-out signalling. This phenomenon prepares the cell for the next steps of the process it will perform. CR3 (Complement receptor 3), the most abundant {beta}2 integrin in monocytes and macrophages, and CD13 (aminopeptidase N) are two immune receptors with overlapping activities: adhesion, phagocytosis of opsonized particles, and respiratory burst induction. They can be found together in functional signalling microdomains, or lipid rafts, on the surface of human leukocytes. Thus, given their common functions, shared physical location and the fact that some phagocytic and adhesion receptors activate a selection of integrins, we hypothesized that CD13 could activate CR3 through an inside-out signalling mechanism. To test this hypothesis, we first ascertained the activation of CR3 after CD13 crosslinking in human monocyte-derived macrophages. We used an integrated analysis of bioinformatics and experimental data to suggest two possible signalling cascades that could explain the phenomenon. Finally, we show that the non-receptor tyrosine kinase Syk is a key attenuator of this pathway. Our results demonstrated that, even in the absence of canonical signalling motifs, and despite having a noticeably short cytoplasmic tail (7-10 amino acids), CD13 was capable of triggering an inside-out signalling cascade, adding a new function to those already known for this moonlighting protein.

One Sentence SummaryStimulation of CD13 activated the integrin CR3 via an inside-out signalling pathway, a mechanistic model is proposed.


title: Shrimp draft genome contains independent clusters of ancient and current endogenous viral elements (EVE) of the parvovirus IHHNV

abstract:
BackgroundShrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a draft genome of the giant tiger shrimp (Penaeus monodon)(GenBank record JABERT000000000) for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV).

ResultsThe shrimp draft genome contained 3 piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in linkage group 35 (LG35). Both LG35 clusters contained multiple DNA fragments with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called " non-infectious IHHNV Type A" (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two LG35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in linkage group 7 (LG7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against infectious IHHNV.

ConclusionsOur results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them.

category: genetics


title: Genome assembly, transcriptome and SNP database for chum salmon (Oncorhynchus keta)

abstract: Chum salmon (Oncorhynchus keta) is the species with the widest geographic range of the anadromous Pacific salmonids. Chum salmon is the second largest of the Pacific salmon, behind Chinook salmon, and considered the most plentiful Pacific salmon by overall biomass. This species is of significant commercial and economic importance: on average the commercial chum salmon fishery has the second highest processed value of the Pacific salmon within British Columbia. The aim of this work was to establish genomic baseline resources for this species. Our first step to accomplish this goal was to generate a chum salmon reference genome assembly from a doubled-haploid chum salmon. Gene annotation of this genome was facilitated by an extensive RNA-seq database we were able to create from multiple tissues. Range-wide resequencing of chum salmon genomes allowed us to categorize genome-wide geographic variation, which in turn reinforced the idea that genetic differentiation was best described on a regional, rather than at a stock-specific, level. Within British Columbia, chum salmon regional groupings were described at the conservation unit (CU) level, and there may be substructure within particular CUs. Genome wide associations of phenotypic sex to SNP genetic markers identified two clear peaks, a very strong peak on Linkage Group 15, and another on Linkage Group 3. With these new resources, we were better able to characterize the sex-determining region and gain further insights into sex determination in chum salmon and the general biology of this species.


title: An automated microfluidic platform integrating functional vascularized organoids-on-chip

abstract: The development of vascular networks on-chip is crucial for the long-term culture of three-dimensional cell aggregates such as organoids, spheroids, tumoroids, and tissue explants. Despite the rapid advancement of microvascular network systems and organoid technology, vascularizing organoids-on-chips remains a challenge in tissue engineering. Moreover, most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical settings to operate. Considering these constraints, we developed an innovative platform to establish and monitor the formation of endothelial networks around model spheroids of mesenchymal and endothelial cells as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 15 days on-chip. Importantly, these networks were functional, demonstrating intravascular perfusion within the spheroids or vascular organoids connected to neighbouring endothelial beds. This microphysiological system thus represents a viable organ-on-chip model to vascularize biological tissues and should allow to establish perfusion into organoids using advanced microfluidics.


title: Stable m7G Cap-Distal 5'UTR Hairpin Structure Mediates Distinct 40S and 60S Binding Dynamics

abstract: The 5' untranslated region (UTR) of diverse mRNAs contains secondary structures that can influence protein synthesis by modulating the initiation step of translation. Studies support the ability of these structures to inhibit 40S subunit recruitment and scanning, but the dynamics of ribosomal subunit interactions with mRNA remain poorly understood. Here, we developed a reconstituted Saccharomyces cerevisiae cell-free translation system with fluorescently labeled ribosomal subunits. We applied this extract and single-molecule fluorescence microscopy to monitor, in real time, individual 40S and 60S interactions with mRNAs containing 5 UTR hairpin structures with varying thermostability. In comparison to mRNAs containing no or weak 5'UTR hairpins ({Delta}G >= -5.4 kcal/mol), mRNAs with stable hairpins ({Delta}G <= -16.5 kcal/mol) showed reduced numbers of 60S recruitment to mRNA, consistent with the expectation of reduced translation efficiency for such mRNAs. Interestingly, such mRNAs showed increased numbers of 40S recruitment events to individual mRNAs but with shortened duration on mRNA. Correlation analysis showed that these unstable 40S binding events were nonproductive for 60S recruitment. Furthermore, although the mRNA sequence is long enough to accommodate multiple 40S, individual mRNAs are predominantly observed to engage with a single 40S at a time, indicating the sequestering of mRNA 5 end by initiating 40S. Altogether, these observations suggest that stable cap-distal hairpins in 5 UTR reduce initiation and translation efficiency by destabilizing 40S-mRNA interactions and promoting 40S dissociation from mRNA. The premature 40S dissociation frees mRNA 5'-end accessibility for new initiation events, but the increased rate of 40S recruitment is insufficient to compensate for the reduction of initiation efficiency due to premature 40S dissociation. This study provides the first single-molecule kinetic characterization of 40S/60S interactions with mRNA during cap-dependent initiation and the modulation of such interactions by cap-distal 5 UTR hairpin structures.


title: Copper imbalance linked to oxidative stress and cell death during Zika virus infection in human astrocytes

abstract: The Zika virus (ZIKV) caused neurological abnormalities in more than 3500 Brazilian newborns between 2015 and 2020. Data have pointed to oxidative stress in astrocytes as well as to dysregulations in neural cell proliferation and cell cycle as important events accounting for the cell death and neurological complications observed in Congenital Zika Syndrome. Copper imbalance has been shown to induce similar alterations in other pathologies, and disturbances in copper homeostasis have already been described in viral infections. For this reason, we investigated copper homeostasis imbalance as a factor that could contribute to the cytotoxic effects of ZIKV infection in iPSC-derived human astrocytes. Our results show that ZIKV infection leads to a downregulation of one of the transporters mediating copper release, ATP7B protein. We also observed the activation of mechanisms that counteract high copper levels, including the synthesis of copper chaperones and the reduction of the copper importer protein CTR1. Finally, we show that chelator-mediated copper sequestration in ZIKV-infected astrocytes reduces the levels of reactive oxygen species and improves cell viability, but does not change the overall percentage of infected cells. In summary, our results show that copper homeostasis imbalance plays a role in the pathology of ZIKV in astrocytes, indicating that it may also be a factor accounting for the developmental abnormalities in the central nervous system following viral infection. Evaluating micronutrient levels and the use of copper chelators in pregnant women susceptible to ZIKV infection may be promising strategies to manage novel cases of congenital ZIKV syndrome.


title: The GluN2A subunit of the NMDA receptor modulates the rate of functional maturation in parvalbumin-positive interneurons

abstract: N-methyl-D-aspartate receptors (NMDARs) are excitatory glutamate-gated ion channels that are expressed throughout the central nervous system. NMDARs mediate calcium entry into cells, and are involved in a host of neurological functions, including neuronal development and maturation. The GluN2A subunit, encoded by the GRIN2A gene, has a slightly delayed expression pattern, with low transcript levels during embryonic development that peak in the early neonatal period. Given its unique expression pattern and ability to speed up the synaptic time course after incorporation into the postsynaptic density compared to other GluN2 subunits, the GluN2A subunit is well positioned to participate in synaptic maturation and circuit refinement. By using Grin2a knockout mice, we show that the loss of GluN2A signaling impacts parvalbumin-positive GABAergic interneuron development in the hippocampal CA1 subfield. Specifically, Grin2a knockout mice have 33% more parvalbumin-positive cells in CA1 compared to wild type controls, with no impact on cholecystokinin-positive cell density. By using immunohistochemical colocalization staining and electrophysiological recordings, we demonstrate that these excess parvalbumin cells do eventually incorporate into the hippocampal network and participate in phasic inhibition, although their presynaptic release probability may be dampened. Moreover, we show that although the morphology of Grin2a knockout parvalbumin-positive cells is unaffected, key measures of intrinsic excitability and action-potential firing properties show age-dependent alterations. Preadolescent (P20-25) parvalbumin-positive cells have an increased input resistance, longer membrane time constant, longer action-potential half-width, a lower current threshold for depolarization-induced block of action-potential firing, and a decrease in peak action-potential firing rate. Each of these electrophysiological measures becomes corrected in adulthood, reaching wild type levels, suggesting a delay of electrophysiological maturation. The circuit and behavioral implications of delayed parvalbumin-positive interneuron maturation are not known; however, we find that neonatal Grin2a knockout mice are more susceptible to lipopolysaccharide and febrile-induced seizures, consistent with a critical role for early GluN2A signaling in neuronal development and maintenance of excitatory-inhibitory balance. These results could provide insights into how loss-of-function GRIN2A human variants can generate an epileptic phenotype.


title: Single Nuclei Transcriptome Reveals Perturbed Brain Vascular Molecules in Alzheimer's Disease

abstract:
Blood-brain barrier (BBB) dysfunction is well-known in Alzheimers disease (AD), but the precise molecular changes contributing to its pathophysiology are unclear. To understand the transcriptional changes in brain vascular cells, we performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in 24 AD and control brains resulting in 79,751 nuclei, 4,604 of which formed three distinct vascular clusters characterized as activated pericytes, endothelia and resting pericytes. We identified differentially expressed genes (DEGs) and their enriched pathways in these clusters and detected the most transcriptional changes within activated pericytes. Using our data and a knowledge-based predictive algorithm, we discovered and prioritized molecular interactions between vascular and astrocyte clusters, the main cell types of the gliovascular unit (GVU) of the BBB. Vascular targets predicted to interact with astrocytic ligands have biological functions in signalling, angiogenesis, amyloid {beta} metabolism and cytoskeletal structure. Top astrocytic and vascular interacting molecules include both novel and known AD risk genes such as APOE, APP and ECE1. Our findings provide information on transcriptional changes in predicted vascular-astrocytic partners at the GVU, bringing insights to the molecular mechanisms of BBB breakdown in AD.

Graphical AbstractPericytes (yellow), endothelia (salmon) and astrocytes (purple) that form the gliovascular unit (GVU) at the blood brain barrier (BBB) were interrogated for their differentially expressed genes (DEG) and vascular cell (pericyte or endothelia) to astrocyte interactions using single nucleus RNA sequencing (RNAseq) transcriptome obtained from brains of Alzheimers disease (AD) patients and controls. We identified many upregulated (red) or downregulated (blue) DEGs in AD brains in these cell types. These genes have known biological functions in amyloid {beta} (A{beta}) clearance, immune modulation, astrogliosis and neuronal death. Novel predicted interactions were identified between vascular cells and astrocytic DEGs. Collectively, our findings highlight the vast transcriptome changes that occur at the GVU and provide mechanistic insights into BBB dysfunction in AD. This figure was created with Biorender.com.

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title: A Programmable Ontology Encompassing the Functional Logic of the Drosophila Brain

abstract:
The Drosophila brain has only a fraction of the number of neurons of higher organisms such as mice and humans. Yet the sheer complexity of its neural circuits recently revealed by large connectomics datasets suggests that computationally modeling the function of fruit fly brain circuits at this scale posits significant challenges.

To address these challenges, we present here a programmable ontology that expands the scope of the current Drosophila brain anatomy ontologies to encompass the functional logic of the fly brain. The programmable ontology provides a language not only for modeling circuit motifs but also for programmatically exploring their functional logic. To achieve this goal, we tightly integrated the programmable ontology with the workflow of the interactive FlyBrainLab computing platform. As part of the programmable ontology, we developed NeuroNLP++, a web application that supports free-form English queries for constructing functional brain circuits fully anchored on the available connectome/synaptome datasets, and the published worldwide literature.

In addition, we present a methodology for including a model of the space of odorants into the programmable ontology, and for modeling olfactory sensory circuits of the antenna of the fruit fly brain that detect odorant sources. Furthermore, we describe a methodology for modeling the functional logic of the antennal lobe circuit consisting of massive local feedback loops, a characteristic feature observed across Drosophila brain regions. Finally, using a circuit library, we demonstrate the power of our methodology for interactively exploring the functional logic of the massive number of feedback loops in the antennal lobe.


title: Age-associated B cells are critical for the control of latent gammaherpesvirus 68 infection

abstract: Age-associated B cells (ABCs; CD19+CD11c+T-bet+) are a unique population that are increased in an array of viral infections, though their role during latent infection is largely unexplored. Here, we use murine gammaherpesvirus 68 ({gamma}HV68) to demonstrate that ABCs remain elevated long-term during latent infection and express IFN{gamma} and TNF. Using a strain of {gamma}HV68 that is cleared following acute infection, we show that ABCs persist in the absence of latent virus, though their expression of IFN{gamma} and TNF is decreased. With a fluorescent virus we demonstrate that ABCs are infected with {gamma}HV68 at similar rates to other previously activated B cells. We find that mice without ABCs display defects in anti-viral IgG2a/c antibodies and are more susceptible to {gamma}HV68 reactivation when challenged with heterologous infection. Together, these results indicate that ABCs are a persistent effector subset during latent viral infection that restrains {gamma}HV68 reactivation.


title: Age-associated B cells are long-lasting effectors that restrain reactivation of latent γHV68


title: Biogenesis of mature piRNAs in Caenorhabditis elegans is dependent on orchestration between multiple ribonucleases and PRG-1

abstract: Piwi-interacting RNAs (piRNAs) are an animal-specific class of germline-enriched small non-coding RNAs that shape transcriptome, as well as ensure genomic integrity and fertility by regulating transposons and other selfish genetic elements. In Caenorhabditis elegans mature piRNAs are 21-nucleotides long, begin with a monophosphorylated uridine, and they associate with PRG-1 to form piRISCs that scan the transcriptome for  non-self sequences. However, these piRNAs are born as longer 5-capped transcripts, where PARN-1, a 3-5 exoribonuclease, contributes to the formation of the mature 3-end. But, till date, the 5-processing events remain elusive. We demonstrate that the recently identified endoribonuclease activity of XRN-2 is involved in the processing of the 5-end of precursor piRNAs in worms. Depletion of XRN-2 results in reduced mature piRNA levels, with concomitant increase in levels of the 5-capped precursors. We also reveal that the piRNAs born as longer precursor molecules ([≥]60 nt), prior to 5-end processing, undergo ENDU-1-mediated endoribonucleolytic processing of their 3-ends. Our in vitro RNA-protein interaction studies unravel the mechanistic interactions between XRN-2 and PRG-1 towards the formation of mature 5-ends of piRNAs. In vivo experiments employing prg-1 mutant worms indicate that XRN-2 has the potential to perform clearance of precursors that are not bound and protected by PRG-1. Finally, we also demonstrate that XRN-2 is not only important for the generation of mature piRNAs and piRNA-dependent endo-siRNAs, but through yet unknown pathways, it also affects piRNA-independent endo-siRNAs that shape transcriptome, as well as contribute to genomic integrity via regulation of transposable elements.


title: Genome-wide identification and expression analysis of the invertase gene family in common wheat

abstract:
BackgroundThe degradation of sucrose plays an important role in the process of crop biomass allocation and yield formation. Invertase (INV) irreversibly catalyzes the conversion of sucrose into glucose and fructose, which doomed its important role in plant development and stress tolerance. However, the functions of INV genes in wheat, one of the most important crops, were less studied due to the polyploidy.

ResultsHere, we systematically analyzed the INV gene family based on the latest published wheat reference genomic information. A total of 126 TaINV genes were identified and classified into three classes based on the phylogenetic relationship and their gene structure. Of which, 11 and 83 gene pairs were identified as tandem and segmental duplication genes respectively, while the Ka/Ks ratios of tandem and segmental duplication TaINV genes were less than 1. Expression profile analysis shows that 18 TaINV genes have tissue-specific expression, and 54 TaINV genes were involved in stress response. Furthermore, RNA-seq showed that 35 genes are differentially expressed in grain weight NILs N0910-81L/N0910-81S, in which 9 TaINVs were stably detected by qRT-PCR at three time-points, 4, 7 and 10 DPA. Four of them (TaCWI47, TaCWI48, TaCWI50 and TaVI27) different expressed between the NILs resided in 4 QTL segments (QTGW.nwafu-5DL.1, QTGW.nwafu-5DL.2, QTGW.nwafu-7AS.1 and QTGW.nwafu-7AS.2). These findings facilitate function investigations of the wheat INV gene family and provide new insights into the grain development mechanism in wheat.

ConclusionsOur results showed that allopolyploid events were the main reason for the expansion of the TaINV gene family in hexaploid wheat, and duplication genes might undergo purifying selection. The expression profiling of TaINV genes implied that they are likely to play an important role in wheat growth and development and adaption to stressful environments. And TaCWI47, TaCWI48, TaCWI50 and TaVI27 may have more important roles in grain developments. Our study lay a base for further dissecting the functional characterization of TaINV family members.


title: Regulation of Kidney Mitochondrial Function by Caloric Restriction

abstract: Caloric restriction (CR) prevents obesity, promotes healthy aging, and increases resilience against several pathological stimuli in laboratory rodents. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and capacities, avoiding Ca2+-induced mitochondrial permeability transition. Dietary restriction has also been shown to increase kidney resistance against damaging stimuli such as ischemia/reperfusion, but if these effects are related to similar mitochondrial adaptations had not yet been uncovered. Here, we characterized changes in mitochondrial function in response to six months of CR in rats, measuring bioenergetic parameters, redox balance and calcium homeostasis. CR promoted an increase in mitochondrial oxygen consumption rates under non-phosphorylating and uncoupled conditions. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2O2 release was enhanced, although levels of carbonylated proteins and methionine sulfoxide were unchanged. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+-induced mitochondrial permeability transition. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials or the amounts of the mitochondrial calcium uniporter (MCU). Instead, increased mitochondrial calcium uptake rates in CR kidneys correlate with a loss of MICU2, an MCU modulator. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet-sensitive regulatory role controlling mitochondrial calcium homeostasis. Together, our results highlight the organ-specific bioenergetic, redox, and ionic transport effects of CR. Specifically, we describe the regulation of the expression of MICU2 and its effects on mitochondrial calcium transport as a novel and interesting aspect of the metabolic responses to dietary interventions.


title: FastCAT accelerates absolute quantification of proteins by using multiple short non-purified chimeric standards

abstract: Absolute (molar) quantification of clinically relevant proteins determines their reference values in liquid and solid biopsies. FastCAT (for Fast-track QconCAT) method employs multiple short (<50 kDa) stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q-) peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R-) peptides that relate its abundance to a single protein standard (BSA). FastCAT not only alleviates the need in purifying CP or using SDS-PAGE, but also improves the accuracy, precision and dynamic range of the absolute quantification by grouping Q-peptides according to the expected abundance of target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantification of neurological markers in the human cerebrospinal fluid at the low ng/mL level.


title: Identifying challenges and opportunities for histone peptides in protein engineering platforms

abstract: Histone post-translational modifications are small chemical changes to histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET based systems. High throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet published studies on their use with histones have been noticeably sparse. Here we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.


title: Investigating the in vivo biodistribution of extracellular vesicles isolated from various human cell sources using positron emission tomography

abstract: Noninvasive imaging is a powerful tool for understanding the in vivo behavior of drug delivery systems and successfully translating promising platforms into the clinic. Extracellular vesicles (EVs), nano-sized vesicles with a lipid bilayer produced by nearly all cell types, are emerging platforms for drug delivery. To date, the biodistribution of EVs has been insufficiently investigated, particularly using nuclear imaging-based modalities such as positron emission tomography (PET). Herein, we developed positron-emitting radiotracers to investigate the biodistribution of EVs isolated from various human cell sources using PET imaging. Chelator conjugation did not impact EVs size and subsequent radiolabeling was found to be highly efficient and stable with Zr-89 (t1/2 = 78.4 h). In vivo tracking of EVs isolated from bone marrow-derived mesenchymal stromal cells (BMSCs EVs), primary human macrophages (M{phi} EVs), and a melanoma cell line (A375 EVs) were performed in immunocompetent ICR mice. Imaging studies revealed excellent in vivo circulation for all EVs, with a half-life of approximately 12 h. Significantly higher liver uptake was observed for M{phi} EVs, evidencing the tissue tropism of EV and highlighting the importance of carefully choosing EVs cell sources for drug delivery applications. Conversely, the liver, spleen, and lung uptake of the BMSC EVs and A375 EVs was relatively low. We also investigated the impact of immunodeficiency on the biodistribution of BMSC EVs using NSG mice. The spleen uptake drastically increased in NSG mice, which could confound results of therapeutic studies employing this mouse models. Lastly, PET imaging studies in a melanoma tumor model demonstrated efficient tumor uptake of BMSC EVs following intravenous injection. Overall, these imaging studies evidenced the potential of EVs as carriers to treat a variety of diseases, such as cancer or in regenerative medicine applications, and the necessity to understand EVs tropism to optimize their therapeutic deployment.


title: Screening of Chimeric GAA Variants in a Preclinical Study of Pompe Disease Results in Candidate Vector for Hematopoietic Stem Cell Gene Therapy

abstract:
Pompe disease is a rare genetic neuromuscular disorder caused by acid alpha-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy (ERT) is the current standard of care, which prolongs the quality of life for Pompe patients. However, ERT has limitations due to lack of enzyme penetration into the central nervous system (CNS) and skeletal muscles, immunogenicity against the recombinant enzyme, and requires life-long biweekly infusions. In a preclinical mouse model, a clinically relevant promoter to drive lentiviral vector-mediated expression of engineered GAA in autologous hematopoietic stem and progenitor cells (HSPC) was tested with nine unique human chimeric GAA coding sequences incorporating distinct peptide tags and codon-optimization iterations. Vectors including glycosylation independent lysosomal targeting (GILT) tags resulted in effective GAA enzyme delivery into key disease tissues with enhanced reduction of glycogen, myofiber and CNS vacuolation, compared to non-tagged GAA in Gaa knockout mice, a model of Pompe disease. Genetically modified microglial cells in brains were detected at low levels, but provided robust correction. Furthermore, an aminoacid substitution in the tag added to reduced capacity to induce insulin signaling and there was no evidence of off-target effects. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model providing a promising vector candidate for further investigation.

One Sentence SummaryA candidate vector for hematopoietic stem cell gene therapy of Pompe disease.


title: m7G cap-eIF4E interaction stimulates polysome formation by enhancing first-round initiation kinetics

abstract: Translation in eukaryotic cells occurs predominantly through a 7-methylguanosine (m7G) cap-dependent mechanism. m7G cap interactions with eukaryotic initiation factor 4E (eIF4E) facilitates 43S recruitment to the mRNA 5 end and enhances the translation efficiency of mRNA. However, it remains poorly understood how m7G cap-eIF4E interactions affect polysome formation kinetics. Here, we examine the role of the m7G cap in polysome formation by utilizing a single-molecule approach to track individual ribosomes during active translation. Translation was monitored in wheat germ extract with capped and uncapped synthetic mRNAs and in HeLa extract with purified human eIF4E titration. The presence of the m7G cap and the supplementation of eIF4E to eIF4E-deficient extract enhanced the kinetics of the first initiation event of polysomes. Subsequent to the first initiation event, efficient polysome-forming initiation events occurred independent of mRNA m7G capping status and eIF4E concentration. Our results indicate that m7G cap-eIF4E interactions in wheat germ and HeLa extracts promote polysome formation by enhancing first-round initiation kinetics. The dynamics of individual translation events on polysomal mRNAs suggest that first-round initiation events activate mRNAs for efficient subsequent rounds of polysome-forming initiation.


title: A spatial stochastic model reveals the role of supercoiling in transcription regulation

abstract:
In Escherichia coli, translocation of RNA polymerase (RNAP) during transcription introduces supercoiling to DNA, which influences the initiation and elongation behaviors of RNAP. To quantify the role of supercoiling in transcription regulation, we develop a spatially resolved supercoiling model of transcription, describing RNAP-supercoiling interactions, topoisomerase activities, stochastic topological domain formation, and supercoiling diffusion in all transcription stages. This model establishes that transcription-induced supercoiling mediates the cooperation of co-transcribing RNAP molecules in highly expressed genes. It reveals that supercoiling transmits RNAP-accessible information through DNA and enables different RNAP molecules to communicate within and between genes. It thus predicts that a topological domain could serve as a transcription regulator, generating substantial transcription bursting and coordinating communications between adjacent genes in the domain. The model provides a quantitative platform for further theoretical and experimental investigations of how genome organization impacts transcription.

Author SummaryDNA mechanics and transcription dynamics are intimately coupled. During transcription, the translocation of RNA polymerase overwinds the DNA ahead and underwinds the DNA behind, rendering the DNA supercoiled. The supercoiled DNA could, in return, influences the behavior of the RNA polymerase, and consequently the amount of mRNA product it makes. Furthermore, supercoils could propagate on the DNA over thousands of base pairs, impacting RNA polymerase molecules at faraway sites. These complicated interplays between supercoiling and RNA polymerase makes supercoiling an important transcription regulator. To quantitatively investigate the role of supercoiling in transcription, we build a spatially resolved model that links transcription with the generation, propagation, and dissipation of supercoiling. Our model reveals that supercoiling mediates transcription at multiple length scales. At a single-gene scale, we show that supercoiling gives rise to the collective motion of co-transcribing RNA polymerase molecules, supporting recent experimental observations. Additionally, large variations in mRNA production of a gene can arise from the constraints of supercoiling diffusion in a topological domain. At a multi-gene scale, we show that supercoiling dynamics allow two adjacent genes influence each others transcription kinetics, thus serving as a transcription regulator.


title: Clathrin-mediated Endocytosis Facilitates Internalization of Magnaporthe oryzae Effectors into Rice Cells

abstract: Many filamentous eukaryotic plant pathogens, such as fungi and oomycetes, deliver effectors into living plant cells to suppress defenses and control plant processes needed for infection. Little is known about the mechanism by which these pathogens translocate effector proteins across the plasma membrane into the plant cytoplasm. The blast fungus Magnaporthe oryzae secretes cytoplasmic effectors into a specialized biotrophic interfacial complex (BIC) before translocation. Here we show that cytoplasmic effectors within BICs are packaged into dynamic vesicles that can occasionally be found separated from BICs in the host cytoplasm. Live cell imaging with fluorescently-labelled rice lines showed that BICs are enriched in plant plasma membrane, actin, and Clathrin Light Chain-1, a marker for clathrin-mediated endocytosis (CME). We report that a novel cytoplasmic effector, Bas83, labels empty membrane vesicles surrounding BICs. Inhibition of CME using VIGS and chemical treatments results in a distinctive swollen BIC phenotype lacking effector vesicles. In contrast, fluorescent marker co-localization, VIGS and chemical inhibitor studies failed to support a major role for clathrin-independent endocytosis in effector vesicle formation. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by clathrin-mediated endocytosis in BICs and suggests a role for M. oryzae effectors in co-opting plant endocytosis.


title: Geminiviral βC1 orchestrates organellar genomic instability to augment viral infection by hijacking host RecA

abstract:
Chloroplast is the site for transforming light energy to chemical energy. It also acts as a production unit for a variety of defense-related molecules. These defense moieties are necessary to mount a successful counter defence against pathogens including viruses. Geminiviruses disrupt chloroplast homeostasis as a basic strategy for their successful infection inducing vein-clearing, mosaics and chlorosis in infected plants. Here we show that a geminiviral pathogenicity determinant protein {beta}C1 directly interferes with plastid homeostasis. {beta}C1 was capable of inducing organelle-specific nuclease to degrade plastid genome as well as diverted functions of RecA1 protein, a major player in plastid genome maintenance. {beta}C1 interacted with RecA1 in plants and its homolog in bacteria to reduce the ability of host cells to maintain genomic integrity under stresses. Further, reduction in the coding capacity of plastids severely affected retrograde signalling necessary for viral perception and activation of defense. Induction of chloroplast-specific nuclease by {beta}C1 is similar to phosphate starvation-response in which nucleotides are recycled to augment synthesis of new, potentially viral, DNA. These results indicate the presence of a novel strategy in which a viral protein alters host defence by targeting regulators of chloroplast DNA. We predict that the mechanism identified here might have similarities in other plant-pathogen interactions.

One line summary{beta}C1 alters plastid genome metabolism.


title: Subcellular dynamics and functional activity of the cleaved Na+ channel β1 subunit intracellular domain

abstract: The voltage-gated Na+ channel {beta}1 subunit, encoded by SCN1B, regulates cell surface expression and gating of  subunits, and participates in cell adhesion. {beta}1 is cleaved by /{beta} and {gamma}-secretases, releasing an extracellular domain and intracellular domain (ICD) respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on {beta}1 function in breast cancer cells. Using a series of GFP-tagged {beta}1 constructs, we show that {beta}1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal {beta}1-GFP levels occurred following {gamma}-secretase inhibition, implicating endosomes, and/or the preceding plasma membrane, as important sites for secretase processing. Using live-cell imaging, we report {beta}1-ICD-GFP accumulation in the nucleus. Furthermore, {beta}1-GFP and {beta}1ICD-GFP both increased Na+ current, whereas {beta}1STOP-GFP, which lacks the ICD, did not, thus highlighting that the {beta}1-ICD was necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is TTX-resistant (carried by Nav1.5), the Na+ current increased by {beta}1-GFP or {beta}1ICD-GFP was TTX-sensitive. Taken together, this work suggests that the {beta}1-ICD is a critical regulator of  subunit function. Our data further support the notion that {gamma}-secretase may play a key role in regulating {beta}1 function in breast cancer cells. This work thus highlights proteolytic processing of {beta}1 by secretase cleavage to be a relevant mechanism in diseases associated with abnormal {beta}1 function.


title: Landscape of human spinal cord cell type diversity at midgestation

abstract: Understanding spinal cord generation and assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The cellular landscape of the human spinal cord remains, however, insufficiently explored. Here, we profiled the midgestation human spinal cord with single cell-resolution and discovered, even at this fetal stage, remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped onto distinct histological domains. We discovered a surprisingly early diversification of alpha () and gamma ({gamma}) motor neurons that control and modulate contraction of muscle fibers, which was suggestive of accelerated developmental timing in human spinal cord compared to rodents. Together with mapping of disease-related genes, this transcriptional profile of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control and related disorders in humans.


title: Diversity of microglial transcriptional responses during opioid exposure and neuropathic pain

abstract: Microglia take on an altered morphology during chronic opioid treatment. This morphological change is broadly used to identify the activated microglial state associated with opioid side effects, including tolerance and opioid-induced hyperalgesia (OIH). Following chronic opioid treatment and peripheral nerve injury (PNI) microglia in the spinal cord display similar morphological responses. Consistent with this observation, functional studies have suggested that microglia activated by PNI or opioids engage common molecular mechanisms to induce hypersensitivity. Here we conducted deep RNA sequencing of acutely isolated spinal cord microglia from male mice to comprehensively interrogate transcriptional states and mechanistic commonality between multiple OIH and PNI models. Following PNI, we identify a common early proliferative transcriptional event across models that precedes the upregulation of histological markers of activation, followed by a delayed and injury-specific transcriptional response. Strikingly, we found no such transcriptional responses associated with opioid-induced microglial activation, consistent with histological data indicating that microglia number remain stable during morphine treatment. Collectively, these results reveal the diversity of pain-associated microglial transcriptomes and point towards the targeting of distinct insult-specific microglial responses to treat OIH, PNI, or other CNS pathologies.


title: Biological Aging of CNS-Resident Cells Alters the Clinical Course and Immunopathology of Autoimmune Demyelinating Disease

abstract: Biological aging is the strongest factor associated with the clinical phenotype of multiple sclerosis (MS). Relapsing remitting MS (RRMS) typically presents in the third or fourth decade, while the mean age of presentation of progressive MS (pMS) is 45 years old. Here we show that experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of encephalitogenic CD4+ Th17 cells, is more severe, and less like to remit, in middle-aged compared with young adult mice. Donor T cells and neutrophils are more abundant, while B cells are relatively sparse, in central nervous system (CNS) infiltrates of the older mice. Experiments with reciprocal bone marrow chimeras demonstrate that radio-resistant, non-hematopoietic cells play a dominant role in shaping age-dependent features of the neuroinflammatory response, as well as the clinical course, during EAE. Reminiscent of pMS, EAE in middle-aged adoptive transfer recipients is characterized by widespread microglial activation. Microglia from older mice express a distinctive transcriptomic profile, suggestive of enhanced chemokine synthesis and antigen presentation. Collectively, our findings suggest that drugs that suppress microglial activation, and acquisition or expression of aging-associated properties, may be beneficial in the treatment of progressive forms of inflammatory demyelinating disease.


title: Species-specific mitochondria dynamics and metabolism regulate the timing of neuronal development

abstract: The evolution of species involves changes in the timeline of key developmental programs. Among these, neuronal development is considerably prolonged in the human cerebral cortex compared with other mammals, leading to brain neoteny. Here we explore whether mitochondria influence the species-specific properties of cortical neuron maturation. By comparing human and mouse cortical neuronal maturation at high temporal and cell resolution, we found a slower pattern of mitochondria development in human cortical neurons compared with the mouse, together with lower mitochondria metabolic activity, particularly oxidative phosphorylation. Stimulation of mitochondria metabolism in human neurons resulted in accelerated maturation, leading to excitable and complex cells weeks ahead of time. Our data identify mitochondria as important regulators of the pace of neuronal development underlying human-specific features of brain evolution.