title: Dual Effects of NV-CoV-2 Biomimetic Polymer: An Antiviral regimen against COVID-19

abstract:
Remdesivir (RDV) is the only antiviral drug so far approved for COVID-19 therapy by the FDA. However its efficacy is limited in vivo due to its low stability in presence of plasma. This paper compared the stability of RDV encapsulated with our platform technology based polymer NV-387 (NV-CoV-2), in presence of plasma in vitro and in vivo. Furthermore, a non- clinical pharmacology studies of NV-CoV-2 (Polymer) and NV-CoV-2-R (Polymer encapsulated Remdesivir) in both NL-63 infected and uninfected rats were done. In an in vitro cell culture model experiment, antiviral activity of NV-CoV-2 and NV-CoV-2-R are also compared with RDV.

The results are (i) NV-CoV-2 polymer encapsulation protects RDV from plasma- mediated catabolism in vitro and in vivo, too. (ii) Body weight measurements of the normal (uninfected) rats after administration of the test materials (NV-CoV-2, and NV-CoV-2-R) show no toxic effects on them. (iii) NL-63 infected rats body weights and their survival length were like uninfected rats after treatment with NV-CoV-2 and NV-CoV-2-R, and the efficacy as an antiviral regimen were found in the order as below: NV-CoV-2-R > NV-CoV-2 > RDV.

In brief, our platform technology based NV-387-encapsulated-RDV (NV-CoV-2-R) drug has a dual effect on coronaviruses. First, NV-CoV-2 itself as an antiviral regimen. Secondly, RDV is protected from plasma-mediated degradation in transit, rendering altogether the safest and an efficient regimen against COVID-19.

category: pharmacology and toxicology


title: Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

abstract: The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

category: genomics


title: Lipid droplets are required for lipid mediator production and cancer cell proliferation

abstract:
Lipid droplets are dynamic organelles with a central role in fatty acid metabolism. They protect cells from lipotoxicity by sequestering excess fatty acids but also provide fatty acids for metabolic reactions and signalling events. Here we show that lipid droplet turnover in cancer cells is required for production of {omega}-3 and {omega}-6 polyunsaturated fatty acid (PUFA)-derived inflammatory lipid mediators, including eicosanoids and specialised pro-resolving mediators. We show that incorporation of PUFAs into triglycerides mediated by diacylglycerol acyltransferase 1 (DGAT1), and their release by adipose triglyceride lipase (ATGL), are required for cyclooxygenase- and lipoxygenase-dependent lipid mediator production and cancer cell proliferation. The human group X secreted phospholipase A2 (hGX sPLA2) drives the delivery of membrane-derived PUFAs into lipid droplets, while ATGL promotes the incorporation of lipid droplet-derived PUFAs into phospholipids. The group IVA cytosolic PLA2 (cPLA2) acts on membrane phospholipids and complements ATGL in the regulation of PUFA trafficking between phospholipids and triglycerides. This study identifies lipid droplets as essential cellular hubs that control PUFA availability for production of lipid mediators involved in inflammation and tumorigenesis.

Synopsis

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/470010v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@13df1c6org.highwire.dtl.DTLVardef@1cc25c8org.highwire.dtl.DTLVardef@1c47cc0org.highwire.dtl.DTLVardef@da4508_HPS_FORMAT_FIGEXP  M_FIG C_FIG This study shows that lipid droplets in cancer cells control the supply of {omega}-3 and {omega}-6 polyunsaturated fatty acids (PUFAs) for the production of lipid mediators, which in turn drive cancer cell proliferation. The esterification of PUFAs into triacylglycerols (TAGs) and their release from lipid droplets are necessary for PUFA entry into lipid mediator production pathways. O_LILipid mediator production induced by the human group X secreted phospholipase A2 (hGX sPLA2), which releases PUFAs from the plasma membrane and serum lipoproteins, depends on diacylglycerol acyltransferase 1 (DGAT1)-mediated TAG synthesis.
C_LIO_LIAdipose triglyceride lipase (ATGL) liberates {omega}-3 and {omega}-6 PUFAs from TAGs and drives lipid mediator production via cyclooxygenase (COX) and lipoxygenase (LOX) pathways.
C_LIO_LIATGL promotes the incorporation of lipid droplet-derived PUFAs into phospholipids, which are targeted by the group IVA cytosolic PLA2 (cPLA2), thereby selectively supplying arachidonic acid for lipid mediator production.
C_LIO_LILipid droplets are required for cPLA2-induced lipid mediator production also in cells that do not depend on ATGL for the supply PUFAs into lipid mediator pathways.
C_LI

category: cell biology


title: Supplementary motor area contributions to rhythm perception

abstract: Timing is everything, but our understanding of the neural mechanisms of timing remains limited, particularly for timing of sequences. Temporal sequences can be represented relative to a recurrent beat (beat-based or relative timing), or as a series of absolute durations (non-beat-based or absolute timing). Neuroimaging work suggests involvement of the basal ganglia, supplementary motor area (SMA), the premotor cortices, and the cerebellum in both beat- and non-beat-based timing. Here we examined how beat-based timing and non-beat-based sequence timing were affected by modulating excitability of the supplementary motor area, the right cerebellum, and the bilateral dorsal premotor cortices, using transcranial direct current stimulation (tDCS). Participants were subjected to a sham stimulation session, followed an active stimulation session where anodal or cathodal 2mA tDCS was applied to the SMA, right premotor cortex, left premotor cortex, or the cerebellum. During both sessions, participants discriminated changes in rhythms which differentially engage beat-based or non-beat-based timing. Rhythm discrimination performance was improved by increasing SMA excitability, and impaired by decreasing SMA excitability. This polarity-dependent effect on rhythm discrimination was absent for cerebellar or premotor cortex stimulation, suggesting a crucial role of the SMA and/or its functionally connected networks in rhythmic timing mechanisms.

category: neuroscience


title: Evolution of Human-specific Alleles Protecting Cognitive Function of Grandmothers

abstract: Late-onset Alzheimers Disease (LOAD) pathology is rare in our closest living evolutionary relatives (chimpanzees), which also express much lower microglial levels of CD33(Siglec-3)-a myelomonocytic receptor inhibiting innate immune reactivity by extracellular V-set domain recognition of sialic acid(Sia)-containing "self-associated molecular patterns" (SAMPs). We earlier showed that V-set domain-deficient CD33-variant allele, protective against LOAD, is derived and specific to hominin-lineage. We now report that CD33 also harbors multiple hominin-specific V-set domain mutations and explore selection forces that may have favored such genomic changes. N-glycolylneuraminic acid (Neu5Gc), the preferred Sia-ligand of ancestral CD33 is absent in humans, due to hominin-specific, fixed loss-of-function mutation in CMAH, which generates CMP-Neu5Gc from its precursor, CMP-N-acetylneuraminic acid (Neu5Ac). Extensive mutational analysis and MD-simulations indicate that fixed change in amino acid 21 of hominin V-set domain and conformational changes related to His45 corrected for Neu5Gc-loss by switching to Neu5Ac-recognition. Considering immune-evasive "molecular mimicry" of SAMPs by pathogens, we found that human-specific pathogens Neisseria gonorrhoeae and Group B Streptococcus (affecting fertility and fetuses/neonates respectively) selectively bind huCD33 and this binding is significantly impacted by amino acid 21 modification. Alongside LOAD-protective CD33 alleles, humans harbor additional, derived, population-universal, cognition-protective variants absent in "great ape" genomes. Interestingly, 11 of 13 SNPs in these human genes (including CD33), that protect the cognitive health of elderly populations, are not shared by genomes of archaic hominins: Neanderthals and Denisovans. Finally, we present a plausible evolutionary scenario to compile, correlate and comprehend existing knowledge about huCD33 evolution and suggest that grandmothering emerged in humans.

category: evolutionary biology


title: Mars, a molecule archive suite for reproducible analysis and reporting of single molecule properties from bioimages

abstract: The rapid development of new imaging approaches is generating larger and more complex datasets revealing the time evolution of individual cells and biomolecules. Single-molecule techniques, in particular, provide access to rare intermediates in complex, multistage molecular pathways, but few standards exist for processing these information-rich datasets, posing challenges for wider dissemination. Here, we present Mars, an open-source platform for storage and processing of image-derived properties of biomolecules. Mars provides Fiji/ImageJ2 commands written in Java for common single-molecule analysis tasks using a Molecule Archive architecture that is easily adapted to complex, multistep analysis workflows. Three diverse workflows involving molecule tracking, multichannel fluorescence imaging, and force spectroscopy, demonstrate the range of analysis applications. A comprehensive graphical user interface written in JavaFX enhances biomolecule feature exploration by providing charting, tagging, region highlighting, scriptable dashboards, and interactive image views. The interoperability of ImageJ2 ensures Molecule Archives can easily be opened in multiple environments, including those written in Python using PyImageJ, for interactive scripting and visualization. Mars provides a flexible solution for reproducible analysis of image-derived properties facilitating the discovery and quantitative classification of new biological phenomena with an open data format accessible to everyone.

category: biophysics


title: Homologous recombination repair creates mutations in the non-coding genome that alter Topoisomerase-1 cleavage sites & orchestrates irinotecan resistance

abstract:
AbstractDevelopment of resistance to chemotherapy is a leading cause of treatment failure. Drug-resistance expression. It is commonly understood that such mutations happen randomly prior to treatment and are positively selected during the treatment. We realized that selection of drug-resistant cancer cell mutants in culture could be achieved by multiple drug exposures of cloned genetically identical cells, and thus is unlikely to result from selection of pre-existent mutations. Accordingly, adaptation must involve generation of mutations de-novo in the process of drug treatment. Here we explored both the mechanism of resistance and the origin of resistant mutations in response to a widely used Top1 inhibitor irinotecan, which triggers DNA breaks, resulting in cytotoxicity. Resistance mechanism was based on gradual accumulation of recurrent mutations in non-coding regions of DNA at sequence-specific Top1 cleavage sites. Surprisingly, cancer cells had much higher number of such sites than reference genome, which may define their increased sensitivity to irinotecan. Homologous recombination repair of DNA double strand breaks at these cleavage sites following initial drug exposures gradually reverted cleavage-sensitive cancer-related sequences back to cleavage-resistant "normal" genome sequences. These genetic changes reduced generation of double strand DNA breaks upon subsequent exposures, thus gradually increasing the drug resistance. Together, large target size for resistance mutations and their generation specifically at Top1 cleavage spots lead to gradual and rapid accumulation of such mutations, synergistically accelerating development of resistance.

Graphical abstract

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category: cancer biology


title: Allelic correlation is a marker of tradeoffs between barriers to transmission of expression variability and signal responsiveness in genetic networks

abstract: Accurately functioning genetic networks should be responsive to signals but prevent transmission of stochastic bursts of expression. Existing data in mammalian cells suggests that such transcriptional "noise" is transmitted by some genes and not others, suggesting that noise transmission is tunable, perhaps at the expense of other signal processing capabilities.However, systematic claims about noise transmission in genetic networks have been limited by the inability to directly measure noise transmission. Here we build a mathematical framework capable of modeling allelic correlation and noise transmission. We find that allelic correlation and noise transmission correspond across a broad range of model parameters and network architectures. We further find that limiting noise transmission comes with the trade-off of being unresponsive to signals, and that within the parameter regimes that are responsive to signals, there is a further trade-off between response time and basal noise transmission. Using a published allele specific single cell RNA-sequencing dataset, we found that genes with high allelic odds ratios are enriched for cell-type specific functions, and that within multiple signaling pathways, factors which are upstream in the pathway have higher allelic odds ratios than downstream factors. Overall, our findings suggest that some degree of noise transmission is required to be responsive to signals, but that minimization of noise transmission can be accomplished by trading-off for a slower response time.

category: systems biology


title: Viral dynamics and immune responses to foot-and-mouth disease virus in African buffalo (Syncerus caffer).

abstract:
Foot-and-mouth disease (FMD) is one of the most important livestock diseases restricting international trade. While it is clear that African buffalo (Syncerus caffer) act as the main wildlife reservoir, viral and immune response dynamics during FMD virus acute infection have not been described before in this species. We used experimental needle inoculation and contact infections with three Southern African Territories serotypes to assess clinical, virological and immunological dynamics for thirty days post infection. Clinical FMD in the needle inoculated buffaloes was mild and characterised by pyrexia. Despite the absence of generalised vesicles, all contact animals were readily infected with their respective serotypes within the first 2-9 days after being mixed with needle challenged buffaloes. Irrespective of the route of infection or serotype there were positive associations between the viral loads in blood and the induction of host innate pro-inflammatory cytokines and acute phase proteins. Viral loads in blood and tonsils were tightly correlated during the acute phase of the infection, however, viraemia significantly declined after a peak at 4 days post infection (dpi), which correlated with the presence of detectable neutralising antibodies. In contrast, infectious virus was isolated in the tonsils until the last sampling point (30 dpi) in most animals. The pattern of virus detection in serum and tonsil swabs was similar for all three serotypes in the direct challenged and contact challenged animals.

We have demonstrated for the first time, that African buffalo are indeed systemically affected by FMD virus and clinical FMD in buffalo is characterized by a transient pyrexia. Despite the lack of FMD lesions, infection of African buffalo was characterised by high viral loads in blood and oropharynx, rapid and strong host innate and adaptive immune responses and high transmissibility.

category: zoology


title: Reversible brain edema in experimental cerebral malaria is associated with transcellular blood-brain barrier disruption and delayed microhemorrhages

abstract:
Brain swelling occurs in cerebral malaria (CM) and may either reverse or result in fatal outcome. It is currently unknown how brain swelling in CM reverses, as investigations have been hampered by inadequate animal models. In this study, we show that reversible brain swelling in experimental murine cerebral malaria (ECM) can be induced reliably after single vaccination with radiation-attenuated sporozoites as revealed by in vivo high-field (9.4T) magnetic resonance imaging. Our results provide evidence that parenchymal fluid increase and consecutive brain swelling results from transcellular blood-brain barrier disruption (BBBD), as revealed by electron microscopy. This mechanism enables reversal of brain swelling but does not prevent persistent focal brain damage, evidenced by microhemorrhages, in areas of most severe BBBD. In a cohort of 27 pediatric and adult CM patients (n=4 fatal, n=23 non-fatal) two out of four fatal CM patients (50%) and 8 out of 23 non-fatal CM patients (35%) showed microhemorrhages on MRI at clinical field strength of 1.5T, emphasizing the translational potential of the experimental model. Our data suggest that targeting transcellular BBBD may represent a promising adjunct therapeutic approach in cerebral brain swelling to reduce edema and may ultimately lead to a reduced permanent brain damage and a better longtime neurological outcome.

Author summaryBrain swelling, which occurs in diseases such as cerebral malaria, is not necessarily fatal, and may reverse. Even upon reversal of brain swelling, neurological sequelae can still occur. The factors contributing to the reversibility of brain edema are not known, and treatment options remain therefore limited. Identifying the mechanisms leading to such reversibility could inform clinical management aimed at decreasing brain swelling and consecutive brain injury. Here we introduce a reproducible and simple animal model that allows comprehensive in vivo studies of reversible brain swelling in cerebral malaria at the peak of disease and upon recovery. We identify a specific type of blood-brain barrier disruption (BBBD) as a mechanism that occurs in brain swelling. We show that BBBD can reverse, but also highlight remaining brain damage in areas of most severe BBBD. As the ECM model introduced here bares crucial similarities to the CM in humans, our findings open strategies to study new therapeutic avenues and point to compounds that specifically target transcellular BBBD to reduce brain edema, and increase survival rates.


title: Patients with recurrent PVL+-Staphylococcus aureus infections show enhanced sensitivity to PVL-mediated formation of atypical NETs

abstract: Panton-Valentine leukocidin (PVL) is a Staphylococcus aureus (S. aureus) toxin that binds to and kills human neutrophils, resulting in the formation of neutrophil extracellular traps (NETs). Some individuals colonized with PVL-positive S. aureus (PVL-SA) suffer from recurring infections whereas others are asymptomatically colonized. We found that neutrophils from affected patients express higher levels of CD45, one of the PVL receptors, and are more susceptible to killing at a low concentration of recombinant PVL than control neutrophils. We verified that PVL induces the formation of NETs and provide genetic and pharmacological evidence that PVL-induced NET formation is independent of NADPH-oxidase and reactive oxygen species (ROS) production. Through NET proteome analysis we identified that the protein content of PVL-induced NETs is different from NETs induced by mitogen or the microbial toxin nigericin. The abundance of the proteins cathelicidin (CAMP), elastase (NE), and proteinase 3 (PRTN3) was lower on PVL-induced NETs, which were inefficient in killing S. aureus. Neutrophils from patients that suffer from recurring PVL-positive infections may be more sensitive to PVL-induced NET formation, which may impair their ability to combat the infection.

category: immunology


title: Heterocomplexes between the Atypical Chemokine MIF and the CXC-Motif Chemokine CXCL4L1 Regulate Inflammation and Thrombus Formation

abstract: To fulfil their orchestrating function in immune cell trafficking in homeostasis and disease, a network of 49 chemokines and 23 receptors capitalizes on features of specificity, redundancy, and functional selectivity such as biased agonism. The discovery of the chemokine interactome, i.e. heteromeric chemokine-chemokine interactions, even across CC- and CXC-class borders, has further expanded the complexity within the network. Moreover, some inflammatory mediators, which are not structurally linked to classical CC-, CXC-, CX3C-, or C-chemokines, can bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified the cytokine macrophage migration inhibitory factor (MIF) as an ACK that binds to the chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF may form heterocomplexes with classical chemokines. We tested this hypothesis, applying an unbiased chemokine protein binding array. The platelet chemokine CXCL4L1, but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12, was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, which also established high-affinity binding (KD{asymp}100-150 nM). The binding interface was predicted by peptide array-based mapping and molecular docking. We next determined whether heterocomplex formation modulates inflammatory and atherogenic activities of MIF. MIF-elicited T-cell chemotaxis as assessed in a 3D-matrix-based live cell-imaging set-up was abrogated, when cells were co-incubated with MIF and CXCL4L1. Heterocomplexation also blocked MIF-triggered migration of Egfp+ microglia in cortical cultures in situ. Of note, CXCL4L1 blocked the binding of Alexa-MIF to a soluble ectodomain mimic of CXCR4 and co-incubation with CXCL4L1 attenuated MIF-triggered dynamic mass redistribution in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. As MIF and CXCL4L1 are abundant platelet products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM- FRET, and proximity ligation assay visualized heterocomplexes in platelet aggregates and clinical human thrombus sections. Moreover, heterocomplex formation inhibited MIF- stimulated thrombus formation under flow and skewed the morphology of adhering platelets from a large to a small lamellipodia phenotype. Together, our study establishes a novel molecular interaction, adding to the complexity of the chemokine interactome and chemokine/receptor network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be promising target structures to modulate inflammation and thrombosis.


title: Tracking bacteria at high density with FAST, the Feature-Assisted Segmenter/Tracker

abstract: Most bacteria live attached to surfaces in densely-packed communities1,2. While new experimental and imaging techniques are beginning to provide a window on the complex processes that play out in these communities, resolving the behaviour of individual cells through time and space remains a major challenge. Although a number of different software solutions have been developed to track microorganisms3-8, these approaches typically rely on a large number of user-defined parameters that must be carefully tuned to effectively track cells. Testing a given parameter combination can take hours to days depending on the size of the dataset, making iterative optimisation impractical. To overcome these limitations, we have developed FAST, the Feature-Assisted Segmenter/Tracker, which uses unsupervised machine learning to optimise tracking while maintaining ease of use. Our approach, rooted in information theory, largely eliminates the need for users to iteratively adjust parameters manually and make qualitative assessments of the resulting cell trajectories. Instead, FAST measures multiple distinguishing "features" for each cell and then autonomously quantifies the amount of unique information each feature provides. We then use these measurements to determine how data from each feature should be combined to minimize tracking errors. Comparing our algorithm with a naive approach that uses cell position alone revealed that FAST produced 4 to 10 times fewer tracking errors. The modular design of FAST combines our novel tracking method with tools for segmentation, extensive data visualisation, lineage assignment, and manual track correction. It is also highly extensible, allowing users to extract custom information from images and seamlessly integrate it into downstream analyses. FAST therefore enables high-throughput, data-rich analyses with minimal user input. It has been released for use in either Matlab or as a compiled stand-alone application, and is available at https://bit.ly/3vovDHn, along with extensive tutorials and detailed documentation.

category: microbiology


title: Noise in a metabolic pathway leads to persister formation in Mycobacterium tuberculosis

abstract: Tuberculosis is difficult to treat due to dormant cells in hypoxic granulomas, and stochastically-formed persisters tolerant of antibiotics. Bactericidal antibiotics kill by corrupting their energy-dependent targets. We reasoned that noise in the expression of an energy-generating component will produce rare persister cells. In sorted low ATP M. tuberculosis grown on acetate there is considerable cell-to-cell variation in the level of mRNA coding for AckA, the acetate kinase. Quenching the noise by overexpressing ackA sharply decreases persisters, showing that it acts as the main persister gene under these conditions. This demonstrates that a low energy mechanism is responsible for the formation of M. tuberculosis persisters and suggests that the mechanism of their antibiotic tolerance is similar to that of dormant cells in a granuloma. Entrance into a low energy state driven by stochastic variation in expression of energy-producing enzymes is likely a general mechanism by which bacteria produce persisters.


title: Comparative analysis of Mycobacterium abscessus clinical isolate virulence using an invertebrate infection model

abstract:
AimsMycobacterium abscessus causes chronic skin infections, lung diseases, and systemic or disseminated infections. We investigated the quantitative evaluation of the virulence of M. abscessus clinical isolates by calculating the LD50 using a silkworm infection model.

Methods and ResultsM. abscessus subsp. abscessus cells were injected into the hemolymph of silkworms. Silkworms died within two days post-infection with M. abscessus subsp. abscessus when reared at 37{degrees}C. Viable cell numbers of M. abscessus subsp. abscessus increased in the hemolymph of the silkworms injected with M. abscessus subsp. abscessus. Silkworms were not killed following injections with heat-killed M. abscessus subsp. abscessus cells. The administration of clarithromycin, an antibacterial drug used for the treatment of the infection, prolonged the survival time of silkworms injected with M. abscessus subsp. abscessus. The LD50 values of seven clinical isolates were determined using the silkworm infection model and differed by up to nine-fold.

ConclusionsM. abscessus proliferation is required to kill the silkworms and that the virulence of M. abscessus clinical isolates can be evaluated by the silkworm infection model.

Significance and Impact of the StudyThe silkworm infection model with M. abscessus is useful for estimating the virulence of the clinical isolates in a short period.


title: On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation

abstract: Determining the mechanisms by which genes are switched on and off during development and differentiation is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with longer periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using a newly developed on-microscope analysis, we monitored mouse -globin transcription in live cells throughout sequential stages of erythropoiesis. We find that changes in the overall levels of -globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in the patterns of transcriptional activity whereas at the peak of gene expression, in early and intermediate erythroblasts, transcription appears to be relatively stable and efficient. Later during differentiation as -globin expression declines, we again observed more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of enhancer-promoter interactions and the formation of active transcriptional compartments as gene expression is turned on and subsequently declines at sequential stages of differentiation.

category: molecular biology


title: High resolution species detection: accurate long read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing

abstract:
To monitor the effect of nature restoration projects in North Sea ecosystems, accurate and intensive biodiversity assessments are vital. DNA based techniques and especially environmental DNA (eDNA) metabarcoding from seawater is becoming a powerful monitoring tool. However, current approaches are based on genetic target regions of <500 nucleotides, which offer limited taxonomic resolution. This study aims to develop and validate a long read nanopore sequencing method for eDNA that enables improved identification of fish species.

We designed a universal primer pair targeting a 2kb region covering the 12S and 16S rRNA genes of fish mitochondria. eDNA was amplified and sequenced using the Oxford Nanopore MiniON. Sequence data was processed using the new pipeline Decona, and accurate consensus identities of above 99.9% were retrieved. The primer set efficiency was tested with eDNA from a 3.000.000 L zoo aquarium with 31 species of bony fish and elasmobranchs. Over 55% of the species present were identified on species level and over 75% on genus level. Next, our long read eDNA metabarcoding approach was applied to North Sea eDNA field samples collected at ship wreck sites, the Gemini Offshore Wind Farm, the Borkum Reef Grounds and a bare sand bottom. Here, location specific fish and vertebrate communities were obtained. Incomplete reference databases still form a major bottleneck in further developing high resolution long read metabarcoding. Yet, the method has great potential for rapid and accurate fish species monitoring in marine field studies.


title: Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase

abstract: Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors(ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Strikingly, loss of this protection results in the loss of K63-linked di-ubiquitin from the adaptors and diverts the adaptors for K48-linked poly-ubiquitination and proteasome-mediated degradation. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.


title: Meisosomes, folded membrane platforms, link the epidermis to the cuticle in C. elegans

abstract: Apical extracellular matrices (aECMs) form a physical barrier to the environment. In C. elegans, the epidermal aECM is the cuticle, composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Damage to the cuticle causes stress responses in the underlying epidermis by a process that is poorly understood. Here, we focus on structures connecting the cuticle to the epidermis, that we term "meisosomes". We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, filled with cuticle. Before moulting, meisosomes align with the underlying cytoskeleton in between furrows. In mutants lacking furrows, that exhibit a constitutive damage response in the epidermis, meisosomes are smaller and fail to align. A loss of connection between the epidermis and the cuticle is also observed in these mutants, as well as a modification of the biomechanical properties of the skin. Meisosomes are therefore an essential component of the skin, serving as attachment platforms between the cuticle and epidermis. They could also be involved in relaying tensile information from the cuticle to the underlying epidermis as part of an integrated stress response to injury and infection.


title: Effect of landscape structure on genetic structure of the Lesser horseshoe bat (Rhinolophus hipposideros) in Britanny colonies

abstract:
Some species are difficult to observe and others, need to be not disturbed because of their vulnerability. In response to the difficulty of studying the dispersal behaviors of these species, some areas of biology have been combined in order to access the information despite practical limitations. Here we present the combination of several methodologies from landscape ecology to non- invasive population genetics that allow us to obtain important information on Rinolophus hipposideros, a vulnerable European bat. We genotyped 18 georeferrenced colonies in Brittany (France) from droppings collected in their refuges. We used 6 microsatellite markers in order to obtain the genetic distances between them. On the other hand we calculated Euclidian distances between the refuges occupied by these colonies and some ecological distances with the Pathmatrix module of ArcGis 3.2. We tested hypothesis about the difficulty of dispersal of the species in areas without forest cover or with a low density of hedges. Thanks to the Monmonier algorithm we could infer possible genetic barriers between the colonies and we could compare their location to the presence of landscape barriers (areas with little tree cover). We detected a pattern of isolation by distance that reveals limited dispersal capacities in the species but no pattern linked to ecological distances. We found that some of the neighboring colonies with greater genetic distances between them were located in areas with low density of hedges which could suggest an impact of this landscape element in their movements. Finer studies should allow us to conclude on the need or not of forest cover in the dispersal of this species.

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category: ecology


title: Paleozoic Protein Fossils Illuminate the Evolution of Vertebrate Genomes and Transposable Elements

abstract:
Genomes hold a treasure trove of protein fossils: fragments of formerly protein-coding DNA, which mainly come from transposable elements (TEs) or host genes. These fossils reveal ancient evolution of TEs and genomes, and many fossils have been exapted to perform diverse functions important for the hosts fitness. However, old and highly-degraded fossils are hard to identify, standard methods (e.g. BLAST) are not optimized for this task, and few Paleozoic protein fossils have been found.

Here, a recently optimized method is used to find protein fossils in vertebrate genomes. It finds Paleozoic fossils predating the amphibian/amniote divergence from most major TE categories, including virus-related Polinton and Gypsy elements. It finds 10 fossils in the human genome (8 from TEs and 2 from host genes) that predate the last common ancestor of all jawed vertebrates, probably from the Ordovician period. It also finds types of transposon and retrotransposon not found in human before. These fossils have extreme sequence conservation, indicating exaptation: some have evidence of gene-regulatory function, and they tend to lie nearest to developmental genes. Some ancient fossils suggest "genome tectonics", where two fragments of one TE have drifted apart by up to megabases, possibly explaining gene deserts and large introns.

This paints a picture of great TE diversity in our aquatic ancestors, with patchy TE inheritance by later vertebrates, producing new genes and regulatory elements on the way. Host-gene fossils too have contributed anciently-conserved DNA segments. This paves the way to further studies of ancient protein fossils.

category: bioinformatics


title: Near atomic structure of the inner ring of the Saccharomyces cerevisiae nuclear pore complex

abstract:
Nuclear pore complexes (NPCs) mediate bidirectional nucleocytoplasmic transport of substances in eukaryotic cells. However, the accurate molecular arrangement of NPCs remains enigmatic owing to their huge size and highly dynamic nature. Here we determined the structure of the asymmetric unit of the inner ring (IR monomer) at 3.73 [A] resolution by single-particle cryo-electron microscopy, and created an atomic model of the intact IR consisting of 192 copies from 8 subunits. In each IR monomer, two approximately parallel rhomboidal structures of the inner and outer layers are sandwiched with the Z-shaped Nup188-Nup192 middle layer and Nup188, Nup192 and Nic96 link all subunits to constitute a relatively stable IR monomer, while the intact IR is assembled by loose and instable interactions between IR monomer. These structures reveal various interaction modes and extensive flexible connections in the assembly, providing a structural basis for the stability and malleability of IR.

One-Sentence SummaryAccurate structure of inner ring reveals its assembly and function in dilation and constriction of nuclear pore complex.


title: Distinct and interchangeable growing patterns in colorectal cancer stem-like cells are regulated by Musashi-1

abstract: The dynamic and heterogeneous features of cancer stem-like cells (CSCs) have been widely recognized, but their nongenetic cellular plasticity mechanisms remain elusive. By using colorectal cancer organoids, we phenotypically tracked their spheroid formation and growth capacity to a single-cell resolution, and we discovered that the spheroid-forming cells exhibit a heterogeneous growth pattern, consisting of slow- and fast-growing spheroids. The isolated fast-growing spheroids seem to preserve a dual-growing pattern through multiple passages, whereas the isolated slow-growing spheroids are restricted to a slow-growing pattern. Notably, the spheroids of both patterns were tumorigenic. Moreover, the expression of CSC markers varied among the subpopulations with different growth patterns. The isolated slow-growing spheroids adopted the dual-growing pattern by various extrinsic triggers, in which Musashi-1 plays a key role. The slow-growing fraction was resistant to chemotherapy, and its successful isolation can provide an in vitro platform allowing us to elucidate their role in drug resistance.


title: Bagaza virus in wild birds, Portugal, 2021

abstract: Bagaza virus (BAGV) emerged in Spain, in 2010. Since then, BAGV was not reported in other European countries until September 2021, when BAGV was diagnosed by molecular methods in one corn bunting and several red-legged partridges, after abnormal mortality in Southern Portugal. Sequencing revealed high similarity with strains reported previously.

category: pathology


title: Spatial resolution of an integrated C4+CAM photosynthetic metabolism

abstract: C4 and CAM photosynthesis have repeatedly evolved in plants over the past 30 million years. Because both repurpose the same set of enzymes but differ in their spatial and temporal deployment, they have long been considered as distinct and incompatible adaptations. Remarkably, Portulaca contains multiple C4 species that perform CAM when droughted. Spatially explicit analyses of gene expression reveal that C4 and CAM systems are completely integrated in P. oleracea, with CAM and C4 carbon fixation occurring in the same cells and CAM-generated metabolites likely incorporated directly into the C4 cycle. Flux balance analysis corroborates the gene expression and predicts an integrated C4+CAM system under drought. This first spatially explicit description of a C4+CAM photosynthetic metabolism presents a new blueprint for crop improvement.

category: plant biology


title: Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

abstract: 1The N-terminal sequence stretch that defines subcellular targeting for most nuclear encoded chloroplast proteins is usually considered identical to the sequence that is cleaved upon import. Yet here this study shows that for nine out of ten tested Chlamydomonas chloroplast transit peptides, additional sequence past the cleavage site is required to enable chloroplast targeting. Using replacements of native post-cleavage residues with alternative sequences points to a role for unstructured sequence at mature protein N-termini.


title: Reconstruction of a global gene regulatory network for Streptomyces coelicolor: Curation, inference, and assessment

abstract: Streptomyces coelicolor A3(2) is a model microorganism for the study of Streptomycetes, antibiotic production, and secondary metabolism in general. Even though S. coelicolor has an outstanding variety of regulators among bacteria, little effort to globally study its transcription has been made. We manually curated 29 years of literature and databases to assemble a meta-curated experimentally-validated gene regulatory network (GRN) with 5386 genes and 9707 regulatory interactions ([~]41% of the total expected interactions). This provides the most extensive and up-to-date reconstruction available for the regulatory circuitry of this organism. Only [~]6% (534/9707) are supported by experiments confirming the binding of the transcription factor to the upstream region of the target gene, the so-called "strong" evidence. While for the remaining interactions there is no confirmation of direct binding. To tackle network incompleteness, we performed network inference using several methods (including two proposed here) for motif identification in DNA sequences and GRN inference from transcriptomics. Further, we contrasted the structural properties and functional architecture of the networks to assess the reliability of the predictions, finding the inference from DNA sequence data to be the most trustworthy approach. Finally, we show two applications of the inferred and the curated networks. The inference allowed us to propose novel transcription factors for the key Streptomyces antibiotic regulatory proteins (SARPs). The curated network allowed us to study the conservation of the system-level components between S. coelicolor and Corynebacterium glutamicum. There we identified the basal machinery as the common signature between the two organisms. The curated networks were deposited in Abasy Atlas (https://abasy.ccg.unam.mx/) while the inferences are available as Supplementary Material.


title: Curation, inference, and assessment of a globally reconstructed gene regulatory network for Streptomyces coelicolor


title: Input to layer 1 of somatosensory cortex: Local input outweighs long-range and can modulate the activity of layer 1 interneurons

abstract:
Neocortical layer (L) 1 is a locus for interactions between long-range inputs, L1 interneurons and apical tuft dendrites of pyramidal neurons. Even though we have a wealth of information about L1, the level and effect of local input to this layer have not been quantified. Here we characterized the input to L1 of mouse somatosensory cortex with fast blue, monosynaptic rabies and optogenetics. Our work shows that most of the input to L1 is local, and that both local and long-range inputs to this layer arise predominantly from L2/3 and L5 neurons. Subtypes of L5 and L6b neurons project to the overlying L1 with different probabilities. VIP and SST interneurons in L2/3 and L5 also innervate L1. A subset of local L5, the intratelencephalic, pyramidal neurons, drive L1 interneurons but have no effect on L5 apical tuft dendrites. Monosynaptic rabies-based retrograde labelling reveals presynaptic boutons covering the entire somato-dendritic axis of pyramidal neurons, including in L1. When fast blue application was combined with rabies virus, we found that only a fraction of local and long-range neurons was both presynaptic to L5 neurons and projected to L1. These results demonstrate that L1 receives a large proportion of its input from local neurons, and that some of these inputs specifically target interneurons. We conclude that L1 is not just a site for interaction between long-range feedback and apical tuft dendrites of pyramidal cells, it is also a site for complex modulation of pyramidal neurons and interneurons by local inputs.

Significance StatementHere we used traditional retrograde tracing combined with physiology and rabies virus to quantify the connectivity to L1 of somatosensory cortex. We show that local inputs far outweigh long-range inputs. We also show that subtypes of L5 neurons target the overlying L1 and depolarize L1 interneurons but have little effect on the apical tuft dendrites of L5 neurons. Local and long-range cortical inputs to L1 arise from presynaptic neurons in L2/3 and L5. The long-range input arises from a variety of cortical areas including primary motor, secondary somatosensory and motor, perirhinal and visual cortex. Thus, interneurons in L1 and the dendrites of pyramidal neurons are the site of complex interactions with local and long-range inputs.


title: Experimental Investigation Of Pulse Sterilization Of Pathogens

abstract: The results of experimental investigations of the effect of high-intensity pulsed UV radiation on reference strains of opportunistic pathogens S, aureus and E. coli are presented. The modified pulse UV sterilizer M[Pcy][Kcy]-300-3 based on an end-face plasma accelerator was used as a radiation source, which provides a power pulsed discharge in an open atmosphere. The high efficiency of inactivation of the pathogens was provided within a short period of time. The possibility of providing urgent 100% sterilization of a pathogens has been shown. The prospects for the application of pulse sterilization technology to combat pathogens are considered.


title: Staphylococcus aureus β-toxin exerts anti-angiogenic effects by inhibiting re-endothelialization and neovessel formation

abstract: Staphylococcus aureus is the causative agent of numerous severe human infections associated with significant morbidity and mortality worldwide. S. aureus often targets the vascular endothelium to interfere with proper host responses during invasive infections. In this study, we provide evidence that S. aureus {beta}-toxin inhibits wound repair mechanisms in human endothelial cells by preventing cell proliferation and migration. These findings were confirmed in a rabbit aortic explant model where {beta}-toxin impedes sprout formation. Decreased cell proliferation was accompanied by decreased production of the angiogenic proteins endothelin-1, IGFBP-3, thrombospondin-1, TIMP-1, and TIMP-4. Meanwhile, inhibited wound repair was marked by increased HGF secretion from endothelial cells, likely a marker of endothelial cell damage. Together, these findings establish a mechanistic role for {beta}-toxin where it inhibits proper tissue repair processes that likely promote S. aureus infective niche.


title: BCM3D 2.0: Accurate segmentation of single bacterial cells in dense biofilms using computationally generated intermediate image representations

abstract: Accurate detection and segmentation of single cells in three-dimensional (3D) fluorescence time-lapse images is essential for measuring individual cell behaviors in large bacterial communities called biofilms. Recent progress in machine-learning-based image analysis is providing this capability with every increasing accuracy. Leveraging the capabilities of deep convolutional neural networks (CNNs), we recently developed bacterial cell morphometry in 3D (BCM3D), an integrated image analysis pipeline that combines deep learning with conventional image analysis to detect and segment single biofilm-dwelling cells in 3D fluorescence images. While the first release of BCM3D (BCM3D 1.0) achieved state-of-the-art 3D bacterial cell segmentation accuracies, low signal-to-background ratios (SBRs) and images of very dense biofilms remained challenging. Here, we present BCM3D 2.0 to address this challenge. BCM3D 2.0 is completely complementary to the approach utilized in BCM3D 1.0. Instead of training CNNs to perform voxel classification, we trained CNNs to translate 3D fluorescence images into intermediate 3D image representations that are, when combined appropriately later, more amenable to conventional mathematical image processing than a single experimental image. Using this approach, improved segmentation results are obtained even for very low SBRs and/or high cell density biofilm images. The improved cell segmentation accuracies in turn enable improved accuracies of tracking individual cells through 3D space and time, which opens the door to investigating time-dependent phenomena in bacterial biofilms at the cellular level.


title: The role of four cholesterol-recognition motifs localized between amino acid residues 400-550 in regulating translocation and lytic activity of Adenylate Cyclase Toxin

abstract: Adenylate Cyclase Toxin (ACT or CyaA) is an important virulence factor secreted by Bordetella pertussis, the bacterium causative of whooping cough, playing an essential role in the establishment of infection in the respiratory tract. ACT is a pore-forming cytolysin belonging to the RTX (Repeats in ToXin) family of leukotoxins, capable of permeabilizing several cell types and pure lipid vesicles. Besides, the toxin delivers its N-terminal adenylate cyclase domain into the target cytosol, where catalyzes the conversion of ATP into cAMP, which affects cell signalling. In this study we have made two major observations. First, we show that ACT binds free cholesterol, and identify in its sequence 38 potential cholesterol-recognition motifs. Second, we reveal that four of those motifs are real, functional cholesterol-binding sites. Mutations of the central phenylalanine residues in said motifs have an important impact on the ACT lytic and translocation activities, suggesting their direct intervention in cholesterol recognition and toxin functionality. From our data a likely transmembrane topology can be inferred for the ACT helices constituting the translocation and the hydrophobic regions. From this topology a simple and plausible mechanism emerges by which ACT could translocate its AC domain into target cells, challenging previous views in the field. Blocking the ACT-cholesterol interactions might thus be an effective approach for inhibiting ACT toxicity on cells, and this could help in mitigating the severity of pertussis disease in humans.


title: IL-37 expression reduces acute and chronic neuroinflammation and rescues cognitive impairment in an Alzheimer s disease mouse model

abstract: The anti-inflammatory cytokine interleukin-37 (IL-37) is a member of the IL-1 family but not expressed in mice. We used a human IL-37 (hIL-37tg) expressing mouse, which has been subjected to various models of local and systemic inflammation as well as immunological challenges. Those studies demonstrate an immune-modulatory role of IL-37 which can be characterized as an important suppressor of innate immunity. We investigated the functions of IL-37 in the CNS and explored the effects of IL-37 on neuronal architecture and function, microglia phenotype, cytokine production and behavior after inflammatory challenge by intraperitoneal LPS-injection. Reduced spine density, activated microglia phenotype and impaired long-term potentiation (LTP) were observed in wild-type mice after LPS injection, whereas hIL-37tg mice showed no impairment. In addition, we crossed the hIL-37tg mouse with an animal model of Alzheimers disease (APP/PS1) to investigate the anti-inflammatory properties of IL-37 under chronic neuroinflammatory conditions. Our results show that IL-37 is able to limit inflammation in the brain after acute inflammatory events and prevent the loss of cognitive abilities in a mouse model of AD.


title: Drastic mutations can be hidden in short-read mapping: thousands of mutation clusters in human genome are explained by short-range template switching

abstract:
Variation within human genomes is distributed unevenly and variants show spatial clustering. DNA-replication related template switching is a poorly known mutational mechanism capable of causing major chromosomal rearrangements as well as creating short inverted sequence copies that appear as local mutation clusters in sequence comparisons. We reanalyzed haplotype-resolved genome assemblies representing 25 human populations and multinucleotide variants aggregated from 140,000 human sequencing experiments. We found local template switching to explain thousands of complex mutation clusters across the human genome, the loci segregating within and between populations with a small number appearing as de novo mutations. We developed computational tools for genotyping candidate template switch loci using short-read sequencing data and for identification of template switch events using both short-read data and genotype data. These tools will enable building a catalogue of affected loci and studying the cellular mechanisms behind template switching both in healthy organisms and in disease. Strikingly, we noticed that widely-used analysis pipelines for short-read sequencing data - capable of identifying single nucleotide changes - may miss TSM-origin inversions of tens of base pairs, potentially invalidating medical genetic studies searching for causative alleles behind genetic diseases.

Author summaryMutations are not randomly distributed in genomes and they often appear as clusters of nearby changes. We earlier showed that a poorly known mechanism in DNA replication can create short inverted copies of nearby sequence and that these events then show as mutation clusters in sequence comparison. Using the latest DNA sequencing and variation data we show that the human genome contains thousands of mutation clusters consistent with this mechanism and that novel mutations are created at a significant rate. Strikingly we observed that widely used methods for processing DNA sequencing data may completely miss these mutations. This has significance e.g. in medical genetic studies aiming to identify mutations causing genetic diseases.


title: Host Induced Gene Silencing of the Sclerotinia sclerotiorum ABHYDROLASE-3 gene reduces disease severity in Brassica napus

abstract: Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.


title: Inhibitory co-transmission from midbrain dopamine neurons relies on presynaptic GABA uptake

abstract: Dopamine (DA)-releasing neurons in the substantia nigra pars compacta (SNcDA) inhibit target cells in the striatum through postsynaptic activation of {gamma}-aminobutyric acid (GABA) receptors. However, the molecular mechanisms responsible for GABAergic signaling remain unclear, as SNcDA neurons lack enzymes typically required to produce GABA or package it into synaptic vesicles. Here we show that aldehyde dehydrogenase 1a1 (Aldh1a1), an enzyme proposed to function as a GABA synthetic enzyme in SNcDA neurons does not produce GABA for synaptic transmission. Instead, we demonstrate that SNcDA axons obtain GABA exclusively through presynaptic uptake using the membrane GABA transporter Gat1 (encoded by Slc6a1). GABA is then packaged for vesicular release using the vesicular monoamine transporter Vmat2. Our data therefore show that presynaptic transmitter recycling can substitute for de novo GABA synthesis and that Vmat2 contributes to vesicular GABA transport, expanding the range of molecular mechanisms available to neurons to support inhibitory synaptic communication.


title: Couching the brain's multiple-demand and language-specific systems within a macroscale gradient of cortical connectivity

abstract: The cerebrum comprises a set of specialised systems that tile across the cortical sheet, forming a tapestry-like configuration. For example, the multiple-demand and language-specific systems occupy largely separate neural estates and exhibit disparate functional profiles. Although delimiting the boundary between systems informs where cortical sheet functionally fractionates, it remains unclear why different systems topographical placements are spatially configured in typical manners and how a macroscale architecture arises from this topography. Novel approaches have tackled this challenge by condensing the topography into a principal gradient, which represents the workflow of information processing from sensory-motoric to abstract-cognitive. To understand how the multiple-demand and language-specific systems are accommodated in the gradient framework, here we used fMRI to probe cognitive operations in semantic vs. visuospatial domains and projected functional activities onto the principal gradient. We found that the two systems showed distinct trajectories of distribution along gradient tiers, suggesting different roles in the transition from sensation to cognition. Critically, when semantic processing became difficult, the brain recruited a specialised  semantic-control system that was a functional and anatomical  hybrid juxtaposed between the multi-demand and language systems. We discuss how the brains modular division can be better understood through the lens of a dimensionality-reduced gradient-like architecture.


title: Proteomic Characterization of Serum Small Extracellular Vesicles in Human Breast Cancer

abstract: There is a lack of comprehensive understanding of breast cancer (BC) specific sEVs characteristics and composition on BC unique proteomic information from human samples. Here, we interrogated the proteomic landscape of sEVs in 167 serum samples from patients with BC, benign mammary disease (BD) and from healthy donors (HD). The analysis provides a comprehensive landscape of serum sEVs with totally 9,589 proteins identified, considerably expanding the panel of sEVs markers. Of note, serum BC-sEVs protein signatures were distinct from those of BD and HD, representing stage- and molecular subtype-specific patterns. We constructed specific sEVs protein identifiers that could serve as a liquid biopsy tool for diagnosis and classification of BC from benign mammary disease, molecular subtypes, as well as assessment of lymph node metastasis. We also identified 11 potential survival biomarkers for distant metastasis. This work may provide reference value for the accurate diagnosis and monitoring of BC progression using serum sEVs.


title: An evolutionarily conserved cis-regulatory element of Nkx3.2 contributes to early jaw joint morphology in zebrafish

abstract: The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of Nkx3.2 that is deeply conserved in gnathostomes but undetectable in the jawless hagfish. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

category: developmental biology


title: PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

abstract: Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.2 [A] structure, revealing a classical {beta}-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion conducting pathway as also supported by metal binding quantification via ICP-MS and MD simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrated that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

category: biochemistry


title: Neural crest-related NXPH1/α-NRXN signaling opposes neuroblastoma malignancy by inhibiting metastasis

abstract: Neuroblastoma is a pediatric cancer that can present as low- or high-risk tumors (LR-NBs and HR-NBs), the latter group showing poor prognosis due to metastasis and strong resistance to current therapy. NBs are known to originate from alterations to cells in the sympatho-adrenal lineage derived from the neural crest, but whether LR-NBs and HR-NBs differ in the way they exploit the transcriptional program underlying their developmental origin remains unclear. Here, we compared the transcriptional landscapes of primary samples of LR-NBs, HR-NBs and human fetal adrenal gland, and thereby identified the transcriptional signature associated to NB formation that further distinguishes LR-NBs from HR-NBs. The majority of the genes comprising this signature belong to the core sympatho-adrenal developmental program, are associated with favorable patient prognosis and with diminished disease progression. The top candidate gene of this list, Neurexophilin-1 (NXPH1), encodes a ligand of the transmembrane receptors -Neurexins (-NRXNs). Our functional in vivo and in vitro assays reveal that NXPH1/-NRXN signaling has a dual impact on NB behavior: whereas NXPH1 and -NRXN1 promote NB tumor growth by stimulating cell proliferation, they conversely inhibit the ability of NB cells to form metastases. Our findings uncover a module of the neural crest-derived sympatho-adrenal developmental program that opposes neuroblastoma malignancy by impeding metastasis, and pinpoint NXPH1/-NRXN signaling as a promising target to treat HR-NBs.


title: TripletGO: Integrating Transcript Expression Profiles with Protein Homology Inferences for High-Accuracy Gene Function Annotations

abstract: Gene Ontology (GO) has been widely used to annotate functions of genes and gene products. We proposed a new method (TripletGO) to deduce GO terms of protein-coding and non-coding genes, through the integration of four complementary pipelines built on transcript expression profiling, genetic sequence alignment, protein sequence alignment and naive probability, respectively. TripletGO was tested on a large set of 5,754 genes from 8 species (human, mouse, arabidopsis, rat, fly, budding yeast, fission yeast, and nematoda) and 2,433 proteins with available expression data from the CAFA3 experiment and achieved function annotation accuracy significantly beyond the current state-of-the-art approaches. Detailed analyses show that the major advantage of TripletGO lies in the coupling of a new triplet-network based profiling method with the feature space mapping technique which can accurately recognize function patterns from transcript expressions. Meanwhile, the combination of multiple complementary models, especially those from transcript expression and protein-level alignments, improves the coverage and accuracy of the final GO annotation results. The standalone package and an online server of TripletGO are freely available at https://zhanglab.ccmb.med.umich.edu/TripletGO/.


title: A brainstem integrator for self-localization and positional homeostasis

abstract: To accurately track self-location, animals need to integrate their movements through space. In amniotes, representations of self-location have been found in regions such as the hippocampus. It is unknown whether more ancient brain regions contain such representations and by which pathways they may drive locomotion. Fish displaced by water currents must prevent uncontrolled drift to potentially dangerous areas. We found that larval zebrafish track such movements and can later swim back to their earlier location. Whole-brain functional imaging revealed the circuit enabling this process of positional homeostasis. Position-encoding brainstem neurons integrate optic flow, then bias future swimming to correct for past displacements by modulating inferior olive and cerebellar activity. Manipulation of position-encoding or olivary neurons abolished positional homeostasis or evoked behavior as if animals had experienced positional shifts. These results reveal a multiregional hindbrain circuit in vertebrates for optic flow integration, memory of self-location, and its neural pathway to behavior.


title: Neural tracking as an objective measure of auditory perception and speech intelligibility

abstract: The neural tracking framework enables the analysis of neural responses (EEG) to continuous natural speech, e.g., a story or a podcast. This allows for objective investigation of a range of auditory and linguistic processes in the brain during natural speech perception. This approach is more ecologically valid than traditional auditory evoked responses and has great potential for both research and clinical applications. In this article, we review the neural tracking framework and highlight three prominent examples of neural tracking analyses. This includes the neural tracking of the fundamental frequency of the voice (f0), the speech envelope and linguistic features. Each of these analyses provides a unique point of view into the hierarchical stages of speech processing in the human brain. f0-tracking assesses the encoding of fine temporal information in the early stages of the auditory pathway, i.e. from the auditory periphery up to early processing in the primary auditory cortex. This fundamental processing in (mostly) subcortical stages forms the foundation of speech perception in the cortex. Envelope tracking reflects bottom-up and top-down speech-related processes in the auditory cortex, and is likely necessary but not sufficient for speech intelligibility. To study neural processes more directly related to speech intelligibility, neural tracking of linguistic features can be used. This analysis focuses on the encoding of linguistic features (e.g. word or phoneme surprisal) in the brain. Together these analyses form a multi-faceted and time-effective objective assessment of the auditory and linguistic processing of an individual.


title: Layer-specific developmentally precise axon targeting of transient suppressed-by-contrast retinal ganglion cells (tSbC RGCs)

abstract:
The mouse retina encodes diverse visual features in the spike trains of more than 40 retinal ganglion cell (RGC) types. Each RGC type innervates a specific subset of the more than 50 retinorecipient brain areas. Our catalog of RGC types and feature representations is nearing completion. Yet, we know little about where specific RGC types send their information. Furthermore, the developmental strategies by which RGC axons choose their targets and pattern their terminal arbors remain obscure. Here we identify a genetic intersection (Cck-Cre and Brn3cCKOAP) that selectively labels transient Suppressed-by-Contrast (tSbC) RGCs, a member of an evolutionarily conserved functionally mysterious RGC subclass. We find that tSbC RGCs selectively innervate the dorsolateral and ventrolateral geniculate nuclei of the thalamus (dLGN and vLGN), the superior colliculus (SC), and the nucleus of the optic tract (NOT). They binocularly innervate dLGN and vLGN but project only contralaterally to SC and NOT. In each target, tSbC RGC axons occupy a specific sublayer, suggesting that they restrict their input to specific circuits. The tSbC RGC axons span the length of the optic tract by birth and remain poised there until they simultaneously innervate their four targets around postnatal day five. The tSbC RGC axons make no errors in choosing their targets and establish mature stratification patterns from the outset. This precision is maintained in the absence of Brn3c. Our results provide the first map of SbC inputs to the brain, revealing a narrow target set, unexpected laminar organization, target-specific binocularity, and developmental precision.

Significance statementIn recent years, we have learned a lot about the visual features encoded by retinal ganglion cells (RGCs), the eyes output neurons. In contrast, we know little about where RGCs send their information and how RGC axons, which carry this information, target specific brain areas during development. Here, we develop an intersectional strategy to label a unique RGC type, the tSbC RGC, and map its projections. We find that tSbC RGC axons are highly selective. They innervate few retinal targets and restrict their arbors to specific sublayers within these targets. The selective tSbC RGC projection patterns develop synchronously and without trial and error, suggesting molecular determinism and coordination.


title: Long-term consequences of alcohol use in early adolescent mice: focus on neuroadaptations in GR, CRF and BDNF

abstract: Our aim was to assess the cognitive and emotional state, as well as related-changes in the glucocorticoid receptor (GR), the corticotropin-releasing factor (CRF) and the brain-derived neurotrophic factor (BDNF) expression of adolescent C57BL/6J male mice after a five-week two-bottle choice protocol (postnatal day (pd) 21 to pd52). Additionally, we wanted to analyse whether the behavioural and neurobiological effects observed in late adolescence (pd62) lasted until adulthood (pd84). Behavioural testing revealed that alcohol during early adolescence increased anxiety-like and compulsive-related behaviours, which was maintained in adulthood. Concerning cognition, working memory was only altered in late adolescent mice, whereas object location test performance was impaired in both ages. In contrast, novel object recognition remained unaltered. Immunohistochemical analysis showed that alcohol during adolescence diminished BDNF+ cells in the cingulate cortex, the hippocampal CA1 layer and the central amygdala. Regarding hypothalamic-pituitary-adrenal axis (HPA) functioning, alcohol abuse increased the GR and CRF expression in the hypothalamic paraventricular nucleus and the central amygdala. Besides this, GR density was also higher in the prelimbic cortex and the basolateral amygdala, regardless of the animals age. Our findings suggest that adolescent alcohol exposure led to long-term behavioural alterations, along with changes in BDNF, GR and CRF expression in limbic brain areas involved in stress response, emotional regulation, and cognition.


title: Simultaneous pathway engagement of translation in astrocytes, circadian rhythm in GABAergic neurons and cytoskeleton in glutamatergic neurons precede electroencephalographic changes in neurodegenerative prion disease

abstract: Selective vulnerability is an enigmatic feature of neurodegenerative diseases (NDs), whereby a widely expressed protein causes lesions in specific brain regions and cell types. This selectivity may arise from cells possessing varying capacities to regain proteostasis when stressed by cytotoxic protein conformers. Using the RiboTag method in mice, translational responses of five neural subtypes to acquired prion disease (PrD) were measured. Pre-onset and disease onset timepoints were chosen based on longitudinal electroencephalography (EEG) that revealed a gradual increase in theta power between 10- and 18-weeks after prion injection, resembling a clinical feature of human PrD. At disease onset, marked by significantly increased theta power and histopathological lesions, mice had pronounced translatome changes in all five cell types despite having a normal outward appearance. Remarkably, at a pre-onset stage, prior to EEG and neuropathological changes, we found that 1) translatomes of astrocytes indicated a sharply reduced synthesis of ribosomal and mitochondrial components, 2) excitatory neurons showed increased expression of cytoskeletal genes, and 3) inhibitory neurons revealed reduced expression of circadian rhythm network genes. Further assessment for the role of circadian rhythms using a jet lag paradigm modestly exacerbated disease. These data demonstrate that early translatome responses to neurodegeneration emerge prior to other signs of disease and are unique to different cell types. Therapeutic strategies may need to target multiple pathways, each in specific populations of cells, early in the disease process.


title: Increasing the Spatial Extent of Attention Strengthens Surround Suppression

abstract: Here we investigate how the extent of spatial attention affects center-surround interaction in visual motion processing. To do so, we measured motion direction discrimination thresholds in humans using drifting gratings and two attention conditions. Under the narrow attention condition, attention was limited to the central part of the visual stimulus, whereas under the wide attention condition, it was directed to both the center and surround of the stimulus. We found stronger surround suppression under the wide attention condition. The magnitude of the attention effect increased with the size of the surround when the stimulus had low contrast, but did not change when it had high contrast. Results also showed that attention had a weaker effect when the center and surround gratings drifted in opposite directions. Next, to establish a link between the behavioral results and the neuronal response characteristics, we performed computer simulations using the divisive normalization model. Our simulations showed that the model can successfully predict the observed behavioral results using parameters derived from the medial temporal (MT) area of the cortex. These findings reveal the critical role of spatial attention on surround suppression and establish a link between neuronal activity and behavior. Further, these results also suggest that the reduced surround suppression found in certain clinical disorders (e.g., schizophrenia and autism spectrum disorder) may be caused by abnormal attention mechanisms.


title: Nociceptive stimuli activate the hypothalamus-habenula circuit to inhibit the mesolimbic reward system

abstract: Nociceptive signals interact with various regions of the brain, including those involved in physical sensation, reward, cognition, and emotion. Emerging evidence points to a role of nociception in the modulation of the mesolimbic reward system. The mechanism by which nociception affects dopamine (DA) signaling and reward is unclear. The lateral hypothalamus (LH) and the lateral habenula (LHb) receive somatosensory inputs and are structurally connected with the mesolimbic DA system. Here we show that the LH-LHb pathway is necessary for nociceptive modulation of this system. Our extracellular single-unit recordings and head-mounted microendoscopic calcium imaging revealed that nociceptive stimulation by tail-pinch excited LHb and LH neurons, which was inhibited by chemical lesion of the LH. Tail-pinch decreased extracellular DA release in the nucleus accumbens ventrolateral shell, which was blocked by disruption of the LH. Furthermore, tail-pinch attenuated cocaine-induced locomotor activity, 50-kHz ultrasonic vocalizations and reinstatement of cocaine-seeking behavior, which was inhibited by chemogenetic silencing of the LH-LHb pathway. Our findings suggest that nociceptive stimulation recruits the LH-LHb pathway to inhibit mesolimbic DA system and drug reinstatement.


title: Evidence for alternate stable states in an Ecuadorian Andean Cloud Forest

abstract: We analyzed a set of historical data from rapid vegetation inventories in a tropical montane cloud forest in northern Andean Ecuador. Trees in plots from several types of forest were counted and measured, including: (1) primary forest, including mature and recently closed-canopy sites and naturally formed gaps, (2) abandoned pasture, and (3) intensively-farmed sites. The goal of the study was to understand in a specific period of time the similarities and differences among natural and anthropogenic disturbances and their potential long term effect on the forest plant community. We found that mature and intermediate close canopy sites are similar. Also old forest is quite resilient to gap-forming disturbance. Natural gaps are quickly colonized by old-forest-associated plant species, and return to an old-forest-type community of trees in a short time. In contrast, forests regenerating from anthropogenic disturbance appear to have multiple possible states: some regenerating forest sites where the anthropogenic disturbance were low are coming to closely resemble old-forest-type communities, but some where the anthropogenic disturbance was intense appear to be changing in a very different direction, which does not resemble any other vegetation community type currently in the forest. A major predictor of present ecological state is the type of land use before reforestation: pastures can occasionally transition back to the pre-disturbance state of forest. More intensively used sites, many of which are abandoned sugar cane plantations, do not return to a pre-disturbance ecological state, instead forming a new and different kind of forest, dominated by a different community of trees. We examined tree-seedling communities to understand the trajectory of the forest into the future, and find that new forest types may be forming that do not resemble any existing associationsintensive agricultural sites. We also found that Los Cedros is extremely diverse in tree species. We estimate approximately 500 species of tree in only the small southeastern area of the reserve that has been explored scientifically. Additionally, the forest tree community shows extremely rapid distance decay (beta-diversity), approaching near complete turn-over in the limited spatial extent of the study. This suggests that hundreds of other tree species remain to be observed in the reserve, in addition to the 350+ that are directly observed in the present study, including new observations of species with IUCN threatened-endangered status. We also highlight the conservation value of Reserva Los Cedros, which has managed to reverse deforestation within its boundaries despite a general trend of extensive deforestation in the surrounding region, and to protect large, contiguous areas of highly-endangered Andean primary cloud forest habitat.


title: Effect of species, size, and chimerism on the susceptibility of Caribbean brain coral recruits to stony coral tissue loss disease (SCTLD)

abstract: Stony coral tissue loss disease (SCTLD) has devastated coral populations along Floridas Coral Reef and beyond. Although widespread infection and mortality of adult colonies have been documented, no studies have yet investigated the susceptibility of recruits to this disease. Here, we exposed eight-month-old Diploria labyrinthiformis recruits and four-month-old Colpophyllia natans recruits to two sequential doses of SCTLD in the laboratory to track infection and assess potential resilience. Both species began to develop lesions as early as 48 h after exposure began. During the first dose, 59.0% of C. natans recruits lost all tissue (died) within two to eight days of developing lesions, whereas D. labyrinthiformis recruits experienced significantly slower rates of tissue loss and minimal eventual mortality. In C. natans, larger recruits and those fused into groups of multiple genets (chimeras) exhibited the highest survivorship. In contrast, smaller and/or single (ungrouped) recruits had the lowest survivorship (9.9 - 26.5%). After 20 days, a second SCTLD dose was delivered to further test resistance in remaining recruits, and all recruits of both species succumbed within 6 days. Although no recruits showed absolute resistance to SCTLD following repeated exposures, our results provide evidence that interactions between species, size, and chimerism can impact relative resistance. This study represents the first report of SCTLD in Caribbean coral recruits and carries implications for natural species recovery and reef restoration efforts. Additional research on the susceptibility of coral juveniles to SCTLD is urgently needed, to include different species, locations, parents, and algal symbionts, with the goal of assessing relative susceptibility and identifying potential sources of resilience for this critical life history stage.


title: Recurrent evolution of cross-resistance in response to selection for improved post-infection survival in Drosophila melanogaster

abstract: The host susceptibility to one pathogen can decrease, increase, or remain unaffected by virtue of the host evolving resistance towards a second pathogen. Negative correlations between a host susceptibility to different pathogens is an often-cited explanation for maintenance of genetic variation in immune function determining traits in a host population. In this study, we investigated the change in susceptibility of Drosophila melanogaster flies to various novel bacterial pathogens after being experimentally selected for increased resistance to one particular bacterial pathogen. We independently selected flies to become more resistant towards Enterococcus faecalis and Pseudomonas entomophila, and baring a few exceptions the evolved populations exhibited cross-resistance against the range of pathogens tested in the study. Neither the identity of the native pathogen nor the host sex was major determining factors in predicting the pattern of cross-resistance exhibited by the selected populations. We therefore report that a generalized cross-resistance to novel pathogens can repeatedly evolve in response to selection for resistance against a single pathogen.


title: Fitness costs associated with spinetoram resistance in Spodoptera frugiperda is driven by host plants

abstract:
Insecticide resistance is usually associated with fitness costs. The magnitude of fitness costs is affected by environmental and ecological factors. Here, we explored how host plants could affect fitness costs associated with insecticide resistance. Initially, spinetoram-resistant (RR) and susceptible (SS) strains of Spodoptera frugiperda were selected using F2 screen from a population collected in Sao Desiderio, Bahia State, Brazil in 2018. Besides de RR and SS strains, fitness costs were also assessed for a heterozygous strain (RS). Life-history traits were evaluated to estimate population growth parameters of neonate larvae of each strain fed on corn, soybean and cotton plants. Compared to the SS strain, the relative fitness of the RR strain, based on intrinsic rate of population increase, was 1.06, 0.84 and 0.67 on plants of corn, soybean and cotton respectively. The relative fitness of the RS strain was similar to the SS strain regardless the host plant, suggesting a recessive fitness cost. No differences were found between the strains fed on corn plants. The larval development time was greater for RR strain fed on soybean and cotton plants compared to RS and SS strain. Low survival rate and fecundity of the RR strain were found when larvae fed on plants of soybean and cotton. The results of this study demonstrated that fitness costs of spinetoram resistance in S. frugiperda depend strongly on the host plants that S. frugiperda larvae fed on. Such information can be used to design resistance management strategies considering the host plants of the agricultural landscape.

Key messagesO_LIThe presence of fitness costs associated with resistance can be exploited in resistance management strategies.
C_LIO_LIHost plant influences the fitness costs associated with spinetoram resistance in S. frugiperda.
C_LIO_LIInformation considering the host plants in an agricultural landscape is essential to design effective resistance management programs.
C_LI


title: Genetic and thermal variation influences adaptation to fluctuating temperature in the seed beetle, Callosobruchus maculatus

abstract: The impacts of climate change on biological systems are notoriously difficult to measure, and laboratory studies often do not realistically represent natural fluctuations in environmental conditions. To date, most experimental studies of thermal adaptation test populations at constant temperatures, or they make incremental changes to an otherwise constant mean background state. To address this, we examined the long-term effects of stressful fluctuating daily temperature on several key life history traits in two laboratory populations of the seed beetle, Callosobruchus maculatus. These populations were kept for 19 generations at either a constant control temperature, T=29{degrees}C, or at a fluctuating daily cycle with Tmean = 33{degrees}C, Tmax=40, and Tmin=26. The latter being a simple representation of daily temperatures in southern central India in May, where this species originally evolved. We found that beetles that had evolved in stressful environments were smaller in body size when switched to a constant 29{degrees}C and had far greater reproductive fitness in comparison to beetles from both the constant control and continuously stressful 33{degrees}C environments. This suggests that beetles raised in environments with stressful fluctuating temperatures were more phenotypically plastic and had greater genetic variability than control treatment beetles and indicates that populations that experience fluctuations in temperature may be better able to respond to short-term changes in environmental conditions.


title: The structure of the Physcomitrium Patens Photosystem I Reveals a Unique Lhca2 Paralogue replacing Lhca4

abstract: The moss Physcomitrium patens diverged from green algae shortly after the colonization of land by ancient plants. This colonization posed new environmental challenges which drove evolutionary processes. The photosynthetic machinery of modern flowering plants is adapted to the high light conditions on land. Red shifted Lhca4 antennae are present in the photosystem I light harvesting complex of many green lineage plants but absent from P. patens. The Cryo-EM structure of the P. patens photosystem I light harvesting complex I supercomplex (PSI-LHCI) at 2.8 [A] reveals that Lhca4 is replaced by a unique Lhca2 paralogue in moss. This PSI-LHCI supercomplex also retains the PsaM subunit, present in cyanobacteria and several algal species but lost in higher plants, and the PsaO subunit responsible for binding light harvesting complex II. The blue shifted Lhca2 paralogue and chlorophyll b enrichment relative to higher plants make the P. patens PSI-LHCI spectroscopically unique among other green lineage supercomplexes. Overall, the structure represents an evolutionary intermediate PSI with the crescent shaped LHCI common in higher plants and contains a unique Lhca2 paralogue which facilitates the mosses adaptation to low light niches.


title: Multiparametric quantitative phase imaging for real-time, single cell, drug screening in breast cancer

abstract: Quantitative phase imaging (QPI) measures the growth rate of individual cells by quantifying changes in mass versus time. Here, we use the breast cancer cell lines MCF-7, BT-474, and MDA-MB-231 to validate QPI as a multiparametric approach for determining response to single-agent therapies. Our method allows for rapid determination of drug sensitivity, cytotoxicity, heterogeneity, and time of response for up to 100,000 individual cells or small clusters in a single experiment. We find that QPI EC50 values are concordant with CellTiter-Glo (CTG), a gold standard metabolic endpoint assay. In addition, we apply multiparametric QPI to characterize cytostatic/cytotoxic and rapid/slow responses and track the emergence of resistant subpopulations. Thus, QPI reveals dynamic changes in response heterogeneity in addition to average population responses, a key advantage over endpoint viability or metabolic assays. Overall, multiparametric QPI reveals a rich picture of cell growth by capturing the dynamics of single-cell responses to candidate therapies.

category: bioengineering


title: Experimental analysis and biaxial biomechanical behaviour of ex-vivo sheep trachea

abstract:
PurposeThe purpose of this study is to investigate the mechanical behaviour of the tracheal tissue under biaxial tensile loading. Furthermore, the study examines the material properties of the tissue through a study of the model parameters for six constitutive models.

Materials and methodsThe fourteen (n = 13) trachea sheep (Vleis Merino) pieces of tissues measured to be ~ 30 x 20 mm where only the effective area subjected to engineering strain was ~ 25 x 16 mm. In this study, we assume that the tracheal tissue is anisotropic and incompressible, therefore we apply and study the material parameters from six models namely the Fung, Choi-Vito, Holzapfel (2000), Holzapfel (2005), Polynomial (Anisotropic) and Four-Fiber Family models.

ResultsThe results show that the trachea tissue is twice as stiff along the circumferential direction as it is along the longitudinal direction. It is also observed that the material properties are different (non-homogeneous) along the trachea.

ConclusionsThe findings of this study will benefit computational models for the study of tracheal diseases or injuries. Furthermore, these findings will assist in the development of regenerative medicine for different tracheal pathologies and in the bioengineering of replacement tissue in cases of damage.


title: In Vitro Proliferation and Long-Term Preservation of Functional Primary Rat Hepatocytes in Cell Fibers

abstract: Primary hepatocytes are essential cellular resource for drug screening and medical transplantation. Since culture systems for them have already succeeded in reconstituting the biomimetic microenvironment, acquiring additional capabilities both to expand primary hepatocytes and to handle them easily would be expected as progress to the next stage. This paper describes a culture system for primary rat hepatocytes that is equipped with scalability and handleability relying on cell fiber technology. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium through the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated they increase in number while keeping their viability and their hepatic specific functions for up to thirty days of subsequent culture. Then, we demonstrated the potency of the primary rat hepatocytes that proliferate in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Therefore, our culture system could serve for innovating strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.


title: MECP2pedia: A Comprehensive Transcriptome Portal for MECP2 Disease Research

abstract: Mutations in MeCP2 result in a crippling neurological disease, but we lack a lucid picture of MeCP2s molecular role. Focusing on individual transcriptomic studies yields inconsistent differentially expressed genes. We have aggregated and homogeneously processed modern public MeCP2 transcriptome data, which we present in a web portal. With this big data, we discovered a commonly perturbed core set of genes that transcends the limitations of any individual study. We then found distinct consistently up and downregulated subsets within these genes. We observe enrichment for this mouse core in other species MeCP2 models and see overlap between this core and ASD models. Analysis of signal to noise finds that many studies lack enough biological replicates. By integrating and examining transcriptomic data at scale, we have generated a valuable resource and insight on MeCP2 function.