title: Classification of electrically-evoked compound action potentials in the parkinsonian subthalamic nucleus of non-human primates

abstract:
BackgroundElectrically evoked compound action potentials (ECAPs) generated in the subthalamic nucleus (STN) contain features that may be useful for titrating deep brain stimulation (DBS) therapy for Parkinsons disease. While previous studies have leveraged macroscale DBS electrodes, directional DBS leads with split-band contacts are thought to enable sampling ECAPs at a higher spatial resolution both within and adjacent to the STN.

ObjectiveIn this study, we investigated the spatiotemporal features of ECAPs in and around the STN across parameter sweeps of stimulation current amplitude, pulse width, and electrode configuration.

MethodsFour non-human primates were implanted unilaterally with either a directional (n=3) or non-directional (n=1) DBS lead targeting the sensorimotor STN. Resting-state ECAPs were recorded before and after rendering the subjects parkinsonian with MPTP treatments.

ResultsECAP responses were characterized by primary features (within 1.6ms after a stimulus pulse) and secondary features (between 1.6-7.4ms after a stimulus pulse). Using these ECAP features, a linear classifier was able to differentiate electrodes within and dorsal to the STN in all four subjects.

ConclusionECAP responses varied systematically with recording and stimulating electrode location, which may provide utility in efficiently defining DBS electrode configurations to target pathways in and around the STN for treating symptoms of Parkinsons disease.


title: Polyphyly of the Niphargus stygius species group (Crustacea, Amphipoda, Niphargidae) in the Southern Limestone Alps

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe Niphargus stygius species complex is a groundwater group of large-sized, sexually dimorphic species inhabiting mainly caves and, less frequently, wells and springs. According to the taxonomists of the last century, this species complex was supposed to be present in the whole Southern Limestone Alps of Italy as well as in peninsular Italy and Slovenia. Considering the large presumed distribution area, we tested the contrasting hypotheses of monophyly versus paraphyly of this subterranean species complex, taking in account the presence of putative cryptic species. For this reason, we sampled the type localities of all the described species in the complex present in the Italian Southern Limestone Alps and neighbouring areas, and used nuclear (28S, ITS region) and mtDNA (COI) sequences to assess their phylogenetic relationships and species richness. Phylogenetic analysis confirmed that the Niphargus stygius complex in the Southern Limestone Alps is not monophyletic but comprises an eastern clade (the N. kenki clade, present in NW Italy, northern Slovenia, and southern Austria) and two western clades (the N. brixianus and N. montellianus clades), _These two clades are not closely related to the eastern one but rather form a monophyletic group together with a widely distributed Apennine clade (N. speziae clade). None of these clades is closely related to typical N. stygius. Three different molecular species delimitation methods applied to COI and ITS recognized different number of putative species, richer for the COI marker, suggesting that each clade is a species complex in itself. Bayesian time-calibrated phylogeny revealed that most clades began to split up during Miocene and Pliocene, ruling out the effect of Pleistocene glaciations in explaining their evolutionary history and justifying the presence of several putative cryptic species.

category: evolutionary biology


title: Modeling Transient Brain Coactivity Patterns in Latent Space with FMRI Data

abstract: The brain is a complex dynamic system that constantly evolves. Characterization of the spatiotemporal dynamics of brain activity is fundamental to understanding how brain works. Current studies with functional connectivity and linear models are limited by low temporal resolution and insufficient model capacity. With a generative variational auto encoder (VAE), the present study mapped the high-dimensional transient co-activity patterns (CAPs) of functional magnetic resonance imaging data to a low-dimensional latent representation that followed a multivariate gaussian distribution. We demonstrated with multiple datasets that the VAE model could effectively represent the transient CAPs in the latent space. Transient CAPs from high-intensity and low-intensity values reflected the same functional structure of brain and could be reconstructed from the same distribution in the latent space. With the reconstructed latent time courses, preceding CAPs successful predicted the following transient CAP with a long short-term memory recurrent neural network. Our methods provide a new avenue to characterize the brains transient co-activity maps and model the complex dynamics between them in a framewise manner.

category: bioengineering


title: TAMPA: interpretable analysis and visualization of metagenomics-based taxon abundance profiles

abstract: Metagenomic taxonomic profiling aims to predict the identity and relative abundance of taxa in a given whole genome sequencing metagenomic sample. A recent surge in computational methods that aim to accurately estimate taxonomic profiles, called taxonomic profilers, have motivated community driven efforts to create standardized benchmarking datasets and platforms, standardized taxonomic profile formats, as well as a benchmarking platform to assess tool performance. While this standardization is essential, there is currently a lack of tools to visualize the standardized output of the many existing taxonomic profilers, and benchmarking studies rely on a single value metrics to compare performance of tools and compare to benchmarking datasets. Here we report the development of TAMPA (Taxonomic metagenome profiling evaluation), a robust and easy-to-use method that allows scientists to easily interpret and interact with taxonomic profiles produced by the many different taxonomic profiler methods beyond the standard metrics used by the scientific community. We demonstrate the unique ability of TAMPA to provide important biological insights into the taxonomic differences between samples otherwise missed by commonly utilized metrics. Additionally, we show that TAMPA can help visualize the output of taxonomic profilers, enabling biologists to effectively choose the most appropriate profiling method to use on their metagenomics data. TAMPA is available on GitHub, Bioconda and Galaxy Toolshed at https://github.com/dkoslicki/TAMPA, and is released under the MIT license.

category: bioinformatics


title: Detergent headgroups control TolC folding in vitro

abstract: TolC is the trimeric outer membrane component of the efflux pump system in E. coli responsible for antibiotic efflux from bacterial cells. Over-expression of efflux pumps has been reported to decrease susceptibility to antibiotics in a variety of bacterial pathogens. Reliable production of membrane proteins allows for the biophysical and structural characterization needed to better understand efflux and for the development of therapeutics. Preparation of recombinant protein for biochemical/structural studies often involves the production of proteins as inclusion body aggregates from which bioactive proteins are recovered. Here we find that the in vitro folding of TolC into its functional trimeric state from inclusion bodies is dependent on the headgroup composition of detergent micelles used. Nonionic detergent favors the formation of functional trimeric TolC, whereas zwitterionic detergents induce the formation of a non-native trimeric TolC fold. We also find that nonionic detergents with shorter alkyl lengths facilitate TolC folding. It remains to be seen whether the charges in lipid headgroups have similar effects on membrane insertion and folding in biological systems.

category: biophysics


title: Senescence-Induced Immune Remodeling Facilitates Metastatic Adrenal Cancer in a Sex-Dimorphic Manner

abstract:
Aging is a carcinogen that markedly increases cancer risk, yet we have limited mechanistic understanding of cancer initiation in aged cells. Here, we demonstrate induction of the hallmark aging process cellular senescence, triggered by loss of Wnt inhibitor ZNRF3, remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer. Detailed characterization reveals a striking sexual dimorphism. Males exhibit earlier senescence activation and a greater innate immune response. This results in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and are more prone to metastatic cancer. Senescence-recruited myeloid cells become increasingly depleted with advanced tumor progression, which is recapitulated in patients where a low myeloid signature is associated with worse outcome. Collectively, our study reveals a novel role for myeloid cells in restraining adrenal cancer progression with significant prognostic value, and provides a model for interrogating pleiotropic effects of cellular senescence in cancer.

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category: cancer biology


title: Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons

abstract: CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3. Most individuals with CLN3 disease carry at least one allele with a 966 bp deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based CLN3 disease models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 {Delta} 966 bp and E295K). The isogenic deletion-corrected and unedited CLN3 patient iPSCs were used for disease modeling. iPSC-derived neurons carrying this particular CLN3 mutation (CLN3 neurons) had lower functional activity as recorded using microelectrode arrays for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis revealed hyperglycosylation of the lysosomal marker, Lysosome Associated Membrane Protein 1 (LAMP1) in CLN3 neurons at DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.

category: neuroscience


title: Immediate myeloid depot for SARS-CoV-2 in the human lung

abstract: In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2 and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.

category: immunology


title: Regenerating axolotl retinas regrow diverse cell types with modulation by Notch signaling and reconnect to the brain

abstract:
Some species successfully repair retinal injuries in contrast to non-regenerative mammalian retina. We show here that the Mexican axolotl salamander regrows its excised retina even in adulthood. During early regeneration, cell proliferation occurred in the retinal pigment epithelium (RPE). All dividing cells expressed Vimentin, and some also expressed Muller glia and neural progenitor cell marker Glast (Slc1a3), suggesting that regeneration is driven by RPE-derived retinal progenitor cells. Bulk RNA sequencing showed that genes associated with the extracellular matrix and angiogenesis were upregulated in early-to-mid retinal regeneration. The fully regenerated retina re-established nerve projections to the brain and contained all the original retinal cell types, including Muller glia. Regeneration of cellular diversity may be modulated by Notch signaling, as inhibiting Notch signaling in early regeneration promoted production of rod photoreceptors. Our study highlights the axolotl salamander as an advantageous model of adult tetrapod retinal regeneration and provides insights into its mechanisms.

SummaryWe demonstrate that adult Mexican axolotl salamanders regenerate retinas after a retinectomy. We also show some cellular and molecular mechanisms that drive axolotl retinal regeneration.


title: Bacterial catabolic system of acetovanillone and acetosyringone useful for upgrading aromatic compounds obtained through chemical lignin depolymerization

abstract:
Acetovanillone is a major aromatic monomer produced in oxidative/base-catalyzed lignin depolymerization. However, the production of chemical products from acetovanillone has not been explored due to the lack of information on the microbial acetovanillone catabolic system. Here acvABCDEF was identified as specifically induced genes during the growth of Sphingobium sp. strain SYK-6 cells with acetovanillone and these genes were essential for SYK-6 growth on acetovanillone and acetosyringone (a syringyl-type acetophenone derivative). AcvAB and AcvF produced in Escherichia coli phosphorylated acetovanillone/acetosyringone and dephosphorylated the phosphorylated acetovanillone/acetosyringone, respectively. AcvCDE produced in Sphingobium japonicum UT26S converted the dephosphorylated phosphorylated acetovanillone/acetosyringone intermediate into vanilloyl acetic acid/3- (4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid through carboxylation. To demonstrate the feasibility of producing cis,cis-muconic acid from acetovanillone, a metabolic modification on a mutant of Pseudomonas sp. strain NGC7 that accumulates cis,cis-muconic acid from catechol was performed. The resulting strain expressing vceA and vceB required for converting vanilloyl acetic acid to vanillic acid and aroY encoding protocatechuic acid decarboxylase in addition to acvABCDEF successfully converted 1.2 mM acetovanillone to approximate equimolar cis,cis-muconic acid. Our results are expected to help improve the yield and purity of value-added chemical production from lignin through biological funneling.

IMPORTANCEIn the alkaline oxidation of lignin, aromatic aldehydes (vanillin, syringaldehyde, and p-hydroxybenzaldehyde), aromatic acids (vanillic acid, syringic acid, and p- hydroxybenzoic acid), and acetophenone-related compounds (acetovanillone, acetosyringone, and 4-hydroxyacetophenone) are produced as major aromatic monomers. Also, base-catalyzed depolymerization of guaiacyl lignin resulted in vanillin, vanillic acid, guaiacol, and acetovanillone as primary aromatic monomers. To date, microbial catabolic systems of vanillin, vanillic acid, and guaiacol have been well characterized, and the production of value-added chemicals from them has also been explored. However, due to the lack of information on the microbial acetovanillone and acetosyringone catabolic system, chemical production from acetovanillone and acetosyringone has not been achieved. This is the first study to elucidate the acetovanillone/acetosyringone catabolic system, and to demonstrate the potential of using these genes for value-added chemicals production from these compounds.

category: microbiology


title: High fat diet changes bacterial signatures in the murine pancreas

abstract: Hyperglycemia is caused by failure of pancreatic beta cells. Beta-cell inflammation contributes to beta-cell dysfunction, however (primary) immunogenic triggers are in large unknown. The gut microbiota is one potential source of pro-inflammatory molecules. A high-fat diet treatment increases pro-inflammatory bacteria in the gut microbiome, parts of which could migrate to the pancreatic beta-cell. In the present study, the bacterial DNA signature in the pancreas and intestine of C57BL6/J mice was analyzed. Mice were fed a high-fat diet (60% kcal fat) or a regular chow diet for 12 weeks. We took several precautions to avoid and map contamination. The gut microbiota was affected by high-fat diet as following: We observed several common intestinal ASVs in the pancreatic tissue. Although the pancreatic ASVs do not correlate exactly with the gut ASVs, our data implicate that the pancreas contains bacterial DNA and that this signature is altered in high-fat diet fed mice (PERMONOVA, p = 0.037; betadisper, p=0.029). Gut derived bacterial DNA might end up in the pancreas at some point in time. Hence, this work supports the concept of translocation of bacterial DNA to the pancreas, which might contribute to inflammation and dysfunction of pancreatic beta-cells.


title: Molecular Determinants of Tissue Specificity of Flavivirus Nonstructural Protein 1 Interaction with Endothelial Cells

abstract:
Members of the mosquito-borne flavivirus genus such as dengue (DENV), West Nile (WNV), and Zika (ZIKV) viruses cause distinct diseases and affect different tissues. We previously found that the secreted flaviviral nonstructural protein 1 (NS1) interacts with endothelial cells and disrupts endothelial barrier function in a tissue-specific manner consistent with the disease tropism of the respective viruses. However, the underlying molecular mechanism of this tissue-specific NS1-endothelial cell interaction is not well understood. To elucidate the distinct role(s) that the domains of NS1 ({beta}-roll, wing, and {beta}-ladder) play in NS1 interactions with endothelial cells, we constructed flavivirus NS1 chimeras that exchanged the wing and {beta}-ladder domains in a pair-wise manner between DENV, WNV, and ZIKV NS1. We found that both the NS1 wing and {beta}-ladder domains conferred NS1 tissue-specific endothelial dysfunction, with the wing conferring cell binding and the {beta}-ladder involved in inducing endothelial hyperpermeability as measured by trans-endothelial electrical resistance assay. To narrow down the amino acids dictating cell binding specificity, we utilized the DENV-WNV NS1 chimera and identified residues 91 to 93 (GDI) of DENV NS1 as a molecular motif determining binding specificity. Further, using an in vivo mouse model of localized leak, we found that the GDI motif of the wing domain was essential for triggering DENV NS1-induced vascular leak in mouse dermis. Taken together, we identify molecular determinants of flavivirus NS1 that confer NS1 binding and vascular leak and highlight the importance of the NS1 wing domain for flavivirus pathogenesis.

ImportanceFlavivirus NS1 is secreted into the bloodstream from infected cells during a viral infection. Dengue virus NS1 contributes to severe dengue pathology such as endothelial dysfunction and vascular leak independently of the virus. We have shown that multiple flavivirus NS1 proteins result in endothelial dysfunction in a tissue-specific manner consistent with their respective viral tropism. Here, we aimed to identify the molecular determinants that make some, but not other, flavivirus NS1 proteins bind to select endothelial cells in vitro and cause vascular leak in a mouse model. We identified the wing domain of NS1 as a primary determinant conferring differential endothelial dysfunction and vascular leak and narrowed the contributing amino acid residues to a three-residue motif within the wing domain. The insights from this study pave the way for future studies on the effects of flavivirus NS1 on viral dissemination and pathogenesis and offer potential new avenues for antiviral therapies.


title: 'Erythritol, a safe natural sweetener exhibits multi-stage anti-malarial activity by blocking Plasmodium falciparum membrane transporter 'aquaporin

abstract: The increased resistance of human malaria parasite Plasmodium falciparum to currently used drugs necessities the development of novel anti-malarials. In the present study, we examine the potential of erythritol, a sugar substitute, for therapeutic intervention that target a multifunctional transporter protein Plasmodium aquaglyceroporin (PfAQP) responsible for maintaining hydro-homeostasis. We show that erythritol effectively inhibited growth and progression of asexual blood stage malaria parasite by suppressing invasion and egress processes. It inhibited the liver stage (sporozoites) and transmission stage parasite (gametocytes) development that suggest its multi-stage, transmission-blocking potential. Interestingly, erythritol inhibited in vivo growth of malaria parasite in mouse experimental model. It was more effective in inhibiting parasite growth both in vivo and in vitro when tested together with a known anti-malarial  artesunate. No Evans blue staining in treated mice indicated erythritol mediated protection of blood-brain barrier integrity in mice infected with P. berghei. Additionally, erythritol showed cytokine-modulating effect which suggest its direct effect on the host immune system. Our results of cellular thermal shift assay and ammonia detection assay demonstrate that erythritol binds with PfAQP and reduce the amount of ammonia release across the parasite respectively. We performed functional complementation assays which suggest that PfAQP expression in yeast mutant restores its growth in hyperosmotic conditions but showed reduced growth in the presence of erythritol, suggesting erythritol as an inhibitor of PfAQP. Overall, our data bestow erythritol as a promising new lead compound with an attractive antimalarial profile and could possibly be combined with known drugs without losing its efficacy.

category: molecular biology


title: Cortical tracking of lexical speech units in a multi-talker background is immature in school-aged children

abstract:
Children have more difficulty perceiving speech in noise than adults. Whether these difficulties relate to immature processing of prosodic or linguistic elements of the attended speech is still unclear. To address the impact of noise on linguistic processing per se, we assessed how acoustic noise impacts the cortical tracking of intelligible speech devoid of prosody in school-aged children and adults.

Twenty adults and twenty children (7-9 years) listened to synthesized French monosyllabic words presented at 2.5 Hz, either randomly or in 4-word hierarchical structures wherein 2 words formed a phrase, and 2 phrases formed a sentence, with or without babble noise. Neuromagnetic responses to words, phrases and sentences were identified and source-localized.

Children and adults displayed significant cortical tracking of words in all conditions, and of phrases and sentences only when words formed meaningful sentences. In children compared with adults, cortical tracking of linguistic units was lower for all units in conditions without noise, and similarly impacted by the addition of babble noise for phrase and sentence units. Critically, when there was noise, adults increased the cortical tracking of monosyllabic words in the inferior frontal gyri but children did not.

This study demonstrates that the difficulties of school-aged children in understanding speech in a multi-talker background might be partly due to an immature identification of lexical but not supra-lexical linguistic units.

HighlightsO_LIChildren track the hierarchical linguistic units of clear speech devoid of prosody
C_LIO_LIThis cortical tracking is left-hemisphere dominant as the adult brain
C_LIO_LIBabble noise reduces cortical tracking of sentences in children and adults
C_LIO_LIUnlike adults, children are not able to enhance cortical tracking of words in noise
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title: HIV-1 Rev-RRE Functional Activity in Primary Isolates is Highly Dependent on Minimal Context-Dependent Changes in Rev

abstract: During HIV infection, intron-containing viral mRNAs have to be exported efficiently from the host cell nucleus to the cytoplasm in order to complete the replication cycle. To overcome cellular restrictions to export incompletely spliced transcripts, HIV encodes a protein, Rev, that is constitutively expressed from a completely spliced transcript. Rev is then imported into the nucleus where it binds to an RNA structure on intron-containing viral mRNAs called the Rev Response Element (RRE). Bound Rev multimerizes and recruits cellular factors that permit the nuclear export of the resulting ribonucleoprotein complex. Primary HIV isolates display substantial variation in the functional activity of the Rev-RRE axis, which may permit viral adaptation to differing immune environments. We describe two subtype G primary isolates with disparate Rev activity. Rev activity was correlated with in vitro fitness of replication-competent viral constructs. Amino acid differences within the oligomerziation domain, but not within the arginine-rich motif or nuclear export signal, determined the different levels of Rev activity. Two specific amino acid substitutions were demonstrated to be able to alter the low-activity Rev to a high-activity phenotype. However, introducing the original amino acids from the the low activity Rev into high activity Rev in this position did not result in significant alterations in activity, highlighting the importance of the broader sequence context for functional activity. These results demonstrate that studies of Rev and RRE activity variation, which may have broader implications for HIV transmission and pathogenesis, should include sequences from primary isolates, as findings using only laboratory-adapted strains cannot be generalized.


title: Few amino acid mutations in H6 influenza A virus from South American lineage increase pathogenicity in poultry

abstract: In chickens, infections with influenza A virus (IAV) can range from mild to severe and lethal. The study of IAV infections in poultry has been mostly limited to strains from the North American and Eurasian lineages, whereas limited information exists on similar studies with strains from the South American lineage (SAm). To better evaluate the risk of introduction of a prototypical SAm IAV strain into poultry, chickens were infected with a wild-type SAm origin strain (WT557/H6N2). The resulting virus progeny was serially passage in chickens 20 times and the immunopathological effects in chickens of the last passage virus, 20Ch557/H6N2, was compared to those of the parental strain. Comparison of complete viral genome sequences indicated that the 20Ch557/H6N2 strain contained 13 amino acid differences compared to the wild type strain. Five of these mutations are in functionally relevant regions of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, despite higher and more prolonged virus shedding in chickens inoculated with the 20Ch557/H6N2 strain compared to those that received the WT557/H6N2 strain, transmission to naive chickens was not observed for either group. Analyses by flow cytometry of mononuclear cells and lymphocyte subpopulations from the lamina propria and intraepithelial lymphocytic cells (IELs) from ileum revealed significant increase in the percentages of CD3+TCR{gamma}{delta}+ IELs in chickens inoculated with the 20Ch557/H6N2 strain compared to those inoculated with the WT557/H6N2 strain.


title: Core genome sequencing and genotyping of Leptospira interrogans in clinical samples by target capture sequencing

abstract: The life-threatening pathogen Leptospira interrogans is the most common agent of leptospirosis, an emerging zoonotic disease. However, little is known about the strains that are circulating worldwide due to the fastidious nature of the bacteria and its difficulty to be culture isolated. In addition, the paucity of bacteria in blood and other clinical samples has proven to be a considerable challenge for directly genotyping the agent of leptospirosis directly from patient material. Here, to elucidate the genomic diversity of Leptospira circulating strains, hybridization capture followed by Illumina sequencing of the core genome was performed directly from 20 biological samples that were PCR positive for pathogenic Leptospira. A set of samples subjected to capture with RNA probes covering the L. interrogans core genome resulted in 72 to 13,000-fold increase in pathogen reads when compared to standard sequencing without capture. A SNP analysis of the genomes sequenced from the biological samples using 273 Leptospira reference genome was then performed in order to determine the genotype of the infecting strain. For samples with sufficent coverage (19/20 samples with coverage >8X), we could unambigously identify L. interrogans sv Icterohaemorrhagiae (14 samples), L. kirschneri sv Grippotyphosa (4 samples) and L. interrogans sv Pyrogenes (1 sample) as the infecting strain. In conclusion, we obtained for most of our biological samples high quality genomic data at suitable coverage for confident core genome genotyping of the agent of leptospirosis. The ability to generate culture-free genomic data opens new opportunities to better understand the epidemiology and evolution of this fastidious pathogen.


title: CpG enrichment as a vaccine strategy for influenza A virus

abstract: Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. However, such recoding typically hinders virus growth. A potential solution to this is CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), leading to viral RNA degradation and/or type I interferon stimulation and consequently reduced viral yields. Therefore, in principle, removing the ZAP CpG sensor from a virus propagation system will reverse attenuation of a CpG-enriched virus. Using influenza A viruses (IAVs) engineered for increased CpG content in different genome regions, we show that ZAP mediated virus attenuation is dependent on the viral segment to which CpGs are added. In ZAP-expressing human cells, CpG introduction invariably impaired transcript and protein production, but ZAP-sensitivity did not convey type I interferon activation. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Furthermore, the ZAP-sensitive virus was fully replication competent in MDCK cells and in embryonated hens eggs, both of which are used to propagate live attenuated influenza vaccines. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform for live attenuated vaccine development.


title: Divergent transcriptional response to thermal stress among life stages could constrain coral adaptation to climate change

abstract: The ability for adaptation to keep pace with environmental change largely depends on how efficiently selection can act on heritable genetic variation. Complex life cycles may either promote or constrain adaptation depending on the integration or independence of fitness-related traits over development. Reef-building corals exhibit complex life cycles and are sensitive to increasing temperatures, highlighting the need to understand the heritable potential of the thermal stress response and how it is regulated over development. Here we used tag-based RNA-seq to profile global gene expression in inshore and offshore P. astreoides adults and their offspring recruits in response to a 16-day heat stress, and larvae from separate families in response to a 4-day heat stress, to test whether gene expression patterns differentiating adult populations, and potentially underlying differences in thermal tolerance, persist in thermally naive life stages. Host developmental stage had a major effect on both host and symbiont expression, despite symbionts being directly inherited from parent colonies, and modulated the response to thermal stress, suggesting the holobiont response to selection varies across life stages. Populations also exhibited origin-specific treatment responses, but the magnitude of the response differed among populations and life stages. Inshore parents and their juvenile offspring exhibited a more robust response to heat stress compared to offshore-origin corals, indicating expression plasticity may be heritable. However, larval populations exhibited the opposite response, possibly due to stage-specific differences or exposure duration. Overall, this study shows that putatively adaptive regulatory variation can be heritable, but the identity of thermally responsive genes are stage-specific, which will have major implications for predicting the evolutionary response of corals in a changing environment.


title: Neryl acetate, the major component of Corsican Helichrysum Italicum essential oil, mediates its biological activities on skin barrier

abstract: Corsican Helichrysum Italicum essential oil is characterized by high concentrations of neryl acetate, and we previously demonstrated that Corsican Helichrysum Italicum essential oil increases the expression of genes that are part of the differentiation complex (involucrin, small proline rich proteins, late cornified envelope, S100 protein family). The biological activities of Helichrysum Italicum essential oil and neryl acetate were compared to identify how neryl acetate contributes to Helichrysum Italicum essential oil activity on human skin. Neryl acetate, as a part component of Helichrysum Italicum essential oil, was tested on skin explant models for 24 hours and 5 days in comparison with Helichrysum Italicum essential oil. We analyzed the biological regulations in the skin explant by transcriptomic analysis, skin barrier protein immunofluorescence, lipid staining and ceramide analysis by liquid chromatography-mass spectrometry. Transcriptomic analysis revealed that 41.5% of Helichrysum Italicum essential oil-modulated genes were also regulated by neryl acetate and a selected panel of genes were confirmed by quantitative reverse transcription PCR analysis. Those genes are involved in epidermal differentiation, skin barrier formation and ceramide synthesis. Involucrin, involved in formation of the cornified envelope, was upregulated at both gene and protein levels after 24 hours and 5 days respectively. After 5 days of treatment, total lipids and ceramides were also increased. Our results demonstrate that neryl acetate mediates a large part of Corsican Helichrysum Italicum essential oil activity on skin barrier formation.


title: Signals Generated by Neutrophil Receptors for Danger Molecules Transactivate Allosterically Modulated FFA2R: Distinct response patterns are mediated by modulators recognized by different allosteric receptor sites

abstract:
Positive allosteric modulators for free fatty acid receptor 2 (FFA2R/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFA2R was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFA2R from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFA2R in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFA2Rs, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFA2R activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFA2R activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFA2R activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFA2R modulator.

O_LIThe allosterically modulated FFA2R is transactivated by signals generated by other GPCRs.
C_LIO_LIThe PAF and ATP receptors transactivate FFA2R from the cytosolic side of the membrane.
C_LIO_LIThe mechanisms that regulates activation of FFA2R from outside and inside differ.
C_LI

category: cell biology


title: Loss of heterochromatin at endogenous retroviruses creates competition for transcription factor binding

abstract: The mammalian genome is partitioned into active and inactive regions, broadly termed euchromatin and heterochromatin, respectively. The majority of heterochromatin consists of repetitive elements, including endogenous retroviruses (ERVs). ERVs are enriched in regulatory elements containing transcription factor (TF) binding sites with individual families containing hundreds to thousands of distinct copies scattered throughout the genome. We hypothesized that epigenetic derepression of ERVs (such as that observed during early development) may alter the stoichiometry between TFs and their euchromatic target sites, with ERVs effectively competing for these factors. To test this, we modeled acute heterochromatin loss using inducible deletion of the co-repressor KAP1 in mouse embryonic stem cells (ESCs). Upon KAP1 deletion, we observe clear reductions in chromatin accessibility, histone acetylation, and TF binding at euchromatic regions. To directly test the concept of global binding site competition, we designed exogenous binding site arrays (EBSAs) to introduce upwards of 1500 copies of the OCT4 TF binding motif into ESCs. OCT4 EBSAs specifically reduce chromatin accessibility at POU family motifs and result in reduced transcription of the pluripotency machinery with subsequent differentiation. Overall, these data support a model in which heterochromatin at ERVs promotes euchromatic TF binding and transcriptional homoeostasis. We propose that regulated ERV derepression during pre-implantation may serve as a developmental siphon to weaken the robustness of ongoing transcription programs in favor of the plasticity required for cell fate specification.

category: genomics


title: Multiscale obstruction sensitivity in collective cell migration

abstract: Cellular forces and intercellular cooperation generate collective cell migration. Pathological changes in cell-level genetic and physical properties cause jamming, unjamming, and scattering in epithelial migration. Separately, changes in microenvironment stiffness and confinement can produce varying modes of cell migration. However, it remains unclear whether and how mesoscale disruptions in matrix topology alter collective cell migration. To address this question, we microfabricated matrices with stumps of defined geometry, density, and orientation, which serve as obstructions in the path of collectively migrating healthy mammary epithelial cells. Here, we show that cells lose their speed and directionality when moving through dense obstructions, compared to those sparsely spaced. On flat surfaces, leader cells are significantly stiffer than follower cells, while dense obstructions lead to the overall softening of cells. In moving through dense obstructions, epithelial cells lose the sense of leaders and followers in their physical properties, migration phenotypes, and fluidity. Although Rac inhibition reduces obstruction sensitivity, loss of cell-cell cooperation and induction of leader-like phenotype via -catenin depletion eliminates the effect of matrix obstructions on epithelial migration. Through a lattice-based model, we identify cellular protrusions, polarity, and leader-follower communication as key mechanisms for obstruction-sensitive collective cell migration. Together, microscale cytoskeletal response, mesoscale softening and disorder, and macroscale multicellular communication enable epithelial cell populations to sense topological obstructions encountered in challenging environments. These results reveal that cohesive, healthy populations are more obstruction sensitive than the dysfunctional, aggressive ones. The  obstruction-sensitivity could add to the emerging disease  mechanotypes such as cell stiffness and traction forces.


title: Model building in SHELXE

abstract:
Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method -with SAD and MAD being the most popular ones- as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well-known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson-Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, poly-alanine tracing. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, in all model building cycles sequence is docked and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests.

SynopsisSide chain tracing now completes model building in SHELXE to enhance density modification. All alternative SHELXE modes, using single or combined sources of starting phase information, are described.

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title: A quorum sensing-regulated type VI secretion system containing multiple nonredundant VgrG proteins is required for interbacterial competition in Chromobacterium violaceum

abstract:
The environmental pathogenic bacterium Chromobacterium violaceum kills Gram-positive bacteria delivering violacein packed into outer membrane vesicles, but nothing is known about its contact-dependent competition mechanisms. In this work, we demonstrate that C. violaceum utilizes a type VI secretion system (T6SS) containing multiple VgrG proteins primarily for interbacterial competition. The single T6SS of C. violaceum contains six vgrG genes, which are located in the main T6SS cluster and four vgrG islands. Using T6SS-core component null mutant strains, western blot, fluorescence microscopy, and competition assays, we show that the C. violaceum T6SS is active and required for competition against Gram-negative bacteria such as Pseudomonas aeruginosa but dispensable for C. violaceum infection in mice. Characterization of single and multiple vgrG mutants revealed that, despite having high sequence similarity, the six VgrGs show little functional redundancy, with VgrG3 showing a major role in T6SS function. Our coimmunoprecipitation data support a model of VgrG3 assembling heterotrimers with the other VgrGs. Moreover, we determined that the promoter activities of T6SS genes increased at high cell density, but the produced Hcp protein was not secreted under such condition. This T6SS growth-phase-dependent regulation was dependent on CviR but not on CviI, the components of a C. violaceum quorum sensing (QS) system. Indeed, a {Delta}cviR but not a {Delta}cviI mutant was completely defective in Hcp secretion, T6SS activity, and interbacterial competition. Overall, our data reveal that C. violaceum relies on a QS-regulated T6SS to outcompete other bacteria and expand our knowledge about the redundancy of multiple VgrGs.

IMPORTANCEThe type VI secretion system (T6SS) is a contractile nanomachine used by many Gram-negative bacteria to inject toxic effectors into adjacent cells. The delivered effectors are bound to the components of a puncturing apparatus containing the protein VgrG. The T6SS has been implicated in pathogenesis and, more commonly, in competition among bacteria. Chromobacterium violaceum is an environmental bacterium that causes deadly infections in humans. In this work, we characterized the single T6SS of C. violaceum ATCC 12472, including its six VgrG proteins, regarding its function and regulation. This previously undescribed C. violaceum T6SS is active, regulated by QS, and required for interbacterial competition instead of acute infection in mice. Among the VgrGs, VgrG3, encoded outside of the main T6SS cluster, showed a major contribution to T6SS function. These results shed light on a key contact-dependent killing mechanism used by C. violaceum to antagonize other bacteria.


title: Extensive uORF translation from HIV-1 transcripts conditions DDX3 dependency for expression of main ORFs and elicits specific T cell immune responses in infected individuals

abstract: Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus which relies on alternative splicing, translational and post-translational mechanisms to produce more than 15 functional proteins from its single [~]10kb transcriptional unit. Here, we have applied ribosome profiling and nascent protein labeling at different time points during infection of CD4+ T lymphocytes to characterize the translational landscape of cellular and viral transcripts during the course of infection. Our results indicate a strong impact of viral infection on host cellular transcript levels but a modest impact on global translation rates. Analysis of ribosome profiling reads from viral transcripts reveals extensive and productive non-AUG translation of small peptides from multiple upstream open reading-frames (uORFs) located in the 5 long terminal repeat. Remarkably, these uORFs derived peptides elicit specific T cell responses in HIV-infected individuals. uORFs are conserved among other retroviruses and, together with the TAR sequence, condition the dependency on DDX3 for efficient translation of the main viral open-reading frames.


title: You will know by its tail: a method for quantification of heterogeneity of bacterial populations using single cell MIC profiling

abstract: Severe non-healing infections are often caused by multiple pathogens or by genetic variants of the same pathogen exhibiting different levels of antibiotic resistance. For example, polymicrobial diabetic foot infections double the risk of amputation compared to monomicrobial infections. Although these infections lead to increased morbidity and mortality, standard antimicrobial susceptibility methods are designed for homogenous samples and are impaired in quantifying heteroresistance. Here, we propose a droplet-based label-free method for quantifying the antibiotic response of the entire population at the single-cell level. We used Pseudomonas aeruginosa and Staphylococcus aureus samples to confirm that the shape of the profile informs about the coexistence of diverse bacterial subpopulations, their sizes, and antibiotic heteroresistance. These profiles could therefore indicate the outcome of antibiotic treatment in terms of the size of remaining subpopulations. Moreover, we studied phenotypic variants of a S. aureus strain to confirm that the profile can be used to identify tolerant subpopulations, such as small colony variants, associated with increased risks for the development of persisting infections. Therefore, the profile is a versatile instrument for quantifying the size of each bacterial subpopulation within a specimen as well as their individual and joined heteroresistance.


title: In situ structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen

abstract: Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of cryo-electron tomography (cryoET) and single particle cryoEM. We show that microsporidian spores contain ribosomes primed for host cell invasion and thus shed new light on the infection mechanism of this important pathogen. Prior to host infection, virtually all ribosomes are locked in the 100 S dimeric state, which appears to be formed by a unique dimerization mechanism that is distinct from its bacterial counterparts. Within the dimer, the hibernation factor MDF1 is bound within the E site, locking the L1 stalk in a closed conformation, and thus preventing the translation of mRNAs to polypeptides.


title: Novel CRISPR-based detection of Leishmania species

abstract: Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods lead to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. CRISPR-Cas systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with L. (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 x 10-2 (kDNA) or 5 x 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.


title: Actin cytoskeleton signaling via MRTF/SRF entrains circadian clock

abstract:
The circadian clock is entrained to daily environmental cues. Integrin-linked intracellular signaling via actin cytoskeleton dynamics transduces extracellular matrix interactions to Myocardin-related Transcription Factor (MRTF)/Serum Response Factor (SRF)-mediated transcription. Actin cytoskeleton organization in liver displays diurnal oscillations and SRF-MRTF activity exert transcriptional control to entrain clock. By interrogating disparate upstream events involved in actin cytoskeleton-MRTF-A/SRF signaling cascade, here we show that this signaling cascade transduce cellular niche cues to modulate circadian clock function. Pharmacological inhibitions of MRTF-A/SRF, by disrupting actin polymerization or blocking ROCK kinase, induced period lengthening with augmented clock amplitude, and genetic loss-of-functions of Srf or Mrtf-a mimic that of actin-depolymerizing agents. In contrast, actin-polymerization induced by Jasplakinolide shortened period with attenuated amplitude. In addition, interfering with cell-matrix interaction through blockade of integrin, inhibition of focal adhesion kinase or attenuating matrix rigidity led to reduced period length while enhancing amplitude. Mechanistically, we identify that core clock repressors, Per2, Nr1d1, and Nfil3, are direct transcriptional targets of MRTF-A/SRF in mediating actin dynamic-induced clock response. Collectively, our findings defined an integrin-actin cytoskeleton-MRTF/SRF pathway in linking clock entrainment with extracellular microenvironment that may facilitate cellular adaptation to its physical niche.

Summary statementOur study revealed the role of actin cytoskeleton-MRTF/SRF signaling in entraining circadian clock to its extracellular physical niche environment.


title: Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration

abstract: In aging, skeletal muscle strength and regenerative capacity declines due, in part, to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here we capitalize on mass-cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blocking of CD47 alternative polyadenylation or antibody blockade of CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.


title: Off-target integron activity leads to rapid plasmid compensatory evolution in response to antibiotic selection pressure

abstract: Integrons are mobile genetic elements that have played an important role in the dissemination of antibiotic resistance. Under stress, the integron can generate combinatorial variation in resistance cassette expression by cassette re-shuffling, accelerating the evolution of resistance. However, the flexibility of the integron integrase site recognition motif hints at potential off-target effects of the integrase on the rest of the genome that may have important evolutionary consequences. Here we test this hypothesis by selecting for increased piperacillin resistance populations of P.aeruginosa with a mobile integron containing a hard-to-mobilise beta-lactamase cassette to minimize the potential for adaptive cassette re-shuffling. We found that integron activity can both decrease overall survival rate but also improve the fitness of the surviving populations. Off-target inversions mediated by the integron accelerated plasmid adaptation by disrupting costly conjugative genes otherwise mutated in control populations lacking a functional integrase. Plasmids containing integron-mediated inversions were associated with lower plasmid costs and higher stability than plasmids carrying mutations, albeit at a cost of reduced conjugative ability. These findings highlight the potential for integrons to create structural variation that can drive bacterial evolution, and they provide an interesting example showing how antibiotic pressure can drive the loss of conjugative genes.


title: New insights from short and long reads sequencing to explore cytochrome b variants of Plasmopara viticola populations collected in vineyard and related to resistance to complex III inhibitors

abstract: Downy mildew is caused by Plasmopara viticola, an obligate oomycete plant pathogen, a devasting disease for grapevine. To preserve plants from the disease, complex III inhibitors are among the widely used fungicides that specifically target the mitochondrial cytochrome b (cytb) of the pathogen to block cellular respiration mechanisms. In French vineyard, P. viticola developed resistance against a first category of these fungicides, the Quinone outside inhibitors, by exhibiting a single amino acid substitution G143A in its cytb mitochondrial sequence. Their usage was restricted and another kind of fungicides, Quinone inside inhibitors, targeting the same gene and highly effective against oomycetes, were used instead. Recently however, less sensitive P. viticola populations were detected after treatments with some inhibitors, in particular ametoctradin and cyazofamid. By isolating resistant single-sporangia strains of P. viticola to these fungicides, we characterized new variants in cytb sequences associated with cyazofamid resistance: a point mutation (L201S) and more strikingly, two insertions (E203-DE-V204, E203-VE-V204). In parallel with classical tools, pyrosequencing and RT-PCR, we then benchmarked both short and long-reads NGS technologies (Ion Torrent, Illumina, Oxford Nanopore Technologies) to sequence the complete cytb with the prospect to detect and assess the proportion of resistant variants of P. viticola at a natural population scale. Eighteen populations collected from French vineyard fields in 2020 were analysed: 12 show a variable proportion of G143A, 11 of E203-DE-V204 and 7 populations of the S34L variant that confers resistance to ametoctradin. Interestingly, long reads were able to identify variants, including SNPs, with confidence and detect a small proportion of P. viticola showing several variants along the same cytb sequence. Altogether, NGS appear promising methods to evaluate pathogen resistance towards fungicides related to cytb modifications at a population scale in the field. This approach could be rapidly a robust decision-support management tool for vineyard in future.


title: Selenocyanate Derived Se-Incorporation into the Nitrogenase Fe Protein Cluster

abstract: The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein independent substrate (CO2) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein Fe4S4 cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the Fe4S4 cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source. The observed chalcogenide exchange illustrates that this Fe4S4 cluster is capable of core substitution reactions under certain conditions, adding to the Fe proteins repertoire of unique properties.

category: biochemistry


title: Nanobodies identify an activated state of the TRIB2 pseudokinase

abstract: Tribbles proteins (TRIB1-3) are a pseudokinase-only branch of the human kinome, which recruit substrates to the COP1 ubiquitin-ligase for ubiquitination. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukaemia oncogene, by way of recruiting the C/EBPa transcription factor for degradation by COP1. Here we report selection and characterisation of nanobodies against the TRIB2 pseudokinase domain from a synthetic yeast surface-display library. We identified nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of Nb4.103 in complex with TRIB2 identified a mode of binding to the N-terminal lobe of the pseudokinase, in a manner that enables specific recognition of TRIB2 over TRIB1 and TRIB3. In the nanobody-stabilised state, TRIB2 adopts an activated conformation that is remarkably similar to the C/EBPa-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilise a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors, and nanobody tools for future investigation of TRIB2 function.


title: Cells function as a ternary logic gate to decide migration direction under integrated chemical and fluidic cues

abstract: Cells sense various environmental cues and process intracellular signals to decide their migration direction in many physiological and pathological processes. Although several signaling molecules have been identified in these directed migrations, it still remains elusive how cells decipher multiple cues, specifically chemical and fluidic cues. Here, we investigated the cellular signal processing machinery by reverse-engineering directed cell migration under integrated chemical and fluidic cues. We exposed controlled chemical and fluidic cues to cells using a microfluidic platform and analyzed the extracellular coupling of the cues with respect to the cellular detection limit. Then, the cells migratory behavior was reverse-engineered to build the cells intrinsic signal processing system as a logic gate. Our primary finding is that the cellular signal processing machinery functions as a ternary logic gate to decipher integrated chemical and fluidic cues. The proposed framework of the ternary logic gate suggests a systematic approach to understand how cells decode multiple cues to make decisions in migration.


title: Robustness of the autophagy pathway to somatic copy number losses

abstract: Autophagy allows cells to temporarily tolerate energy stress by replenishing critical metabolites through self-digestion, thereby attenuating the cytotoxic effects of anticancer drugs that target tumor metabolism. Autophagy defects could therefore mark a metabolically vulnerable cancer state and open a therapeutic window. While mutations of autophagy genes (ATGs) are notably rare in cancer, haploinsufficiency network analyses across many cancers have shown that the autophagy pathway is frequently hit by somatic copy number losses of ATGs like MAP1LC3B/ATG8F (LC3), BECN1/ATG6 (Beclin-1), and ATG10. Here, we used CRISPR/Cas9 technology to delete increasing numbers of copies of one or more of these ATGs in non-small cell lung cancer cells and examined the effects on sensitivity to compounds targeting aerobic glycolysis, a hallmark of cancer metabolism. Whereas complete knock-out of one ATG blocked autophagy and led to profound metabolic vulnerability, this was not the case for combinations of different non-homozygous deletions. In cancer patients, the effect of ATG copy number loss was blunted at the protein level and did not lead to accumulation of p62 as a sign of reduced autophagic flux. Thus, the autophagy pathway is shown to be markedly robust and resilient, even with concomitant copy number loss of key autophagy genes.


title: The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases

abstract:
BackgroundHumans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions.

MethodsWe used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimers disease, Parkinsons disease and amyotrophic lateral sclerosis.

ResultsWe found a significant depletion of positively-selected SNPs in the heritability of Parkinsons disease, raising the possibility that these variants may modulate disease risk, in addition to conferring an evolutionary advantage. For Alzheimers disease and amyotrophic lateral sclerosis, common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk.

ConclusionsThese findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.


title: Nipsnap1- A Regulatory Factor Required for Long-Term Maintenance of Non-Shivering Thermogenesis

abstract: The molecular activation of non-shivering thermogenesis (NST) has strong potential to combat obesity and metabolic disease. However, the mechanisms surrounding the maintenance of NST once it is fully activated, remain unexplored. Here, we present 4-Nitrophenylphosphatase Domain and Non-Neuronal SNAP25-Like 1 (Nipsnap1) as a critical regulator of long-term thermogenic maintenance in brown adipose tissue (BAT). Nipsnap1 localizes to the mitochondrial matrix and increases its transcript and protein levels in response to both chronic cold and {beta}3 adrenergic signaling. Through the generation of BAT-specific Nipsnap1 knockout mice (N1-KO), we show that these mice are unable to sustain activated energy expenditure and fail to protect their body temperature in the face of an extended cold challenge. Mechanistically, we demonstrate that Nipsnap1 integrates with lipid metabolism and BAT-specific ablation of Nipsnap1 leads to severe defects in {beta}-oxidation capacity. Our findings identify Nipsnap1 as a potent regulator of long-term NST maintenance.


title: Histone deacetylase 6 inhibition promotes microtubule acetylation and facilitates autophagosome-lysosome fusion in dystrophin-deficient mdx mice

abstract: Duchenne Muscular Dystrophy (DMD) is a severe X-linked genetic disorder. Defective autophagy and disorganized microtubule network contributes to DMD pathogenesis, yet the mechanisms by which microtubule alterations regulate autophagy remain elusive. We show decreased acetylated -tubulin and enhanced histone deacetylase (HDAC6) expression in mdx mice. Pharmacological inhibition of HDAC6 increases tubulin acetylation and enhances Q-SNARE complex formation, leading to improved autophagosome-lysosome fusion. HDAC6 inhibition reduces apoptosis, inflammation, muscle damage and prevents contraction induced force loss. HDAC6 inhibition restores peroxiredoxin (PrxII) by increasing its acetylation and protecting it from hyper-oxidation, hence modulating intracellular redox status in mdx mice. Genetic inhibition of Nox2 activity in mdx mice promotes autophagosome maturation. Our data highlight that autophagy is differentially regulated by redox and acetylation in mdx mice. By restoring tubulin acetylation HDAC6 inhibition enhances autophagy, ameliorates the dystrophic phenotype and improves muscle function, suggesting a potential therapeutic target for treating DMD.


title: Aβ42 and Aβ40 oligomers form transient and persistent pores with different time evolutions and toxicities

abstract: In Alzheimers disease (AD), formation of harmful self-gating pores formed by the insertion of amyloid beta oligomers (A{beta}Os) into the plasma membrane have been shown to cause disruption of Ca2+ homeostasis, leading to neuronal malfunctioning and degeneration. Among different isoforms, the most studied A{beta}40 and A{beta}42 are also believed to be the most toxic ones. Using single channel imaging, we show that both isoforms can form functionally distinct populations of Ca2+ permeable pores, we named transient and persistent pores. The transient pores could be seen only for a few tens of milliseconds, while persistent pores can be observed for more than an hour. However, while the Ca2+-toxicity of pores formed by A{beta}42Os tend to increase over time by displaying higher open probability and larger Ca2+ permeability, pores formed by A{beta}40Os show opposite time dependent behavior. We conclude that although both isoforms can form Ca2+ permeable pores in the cells plasma membrane, pores due to A{beta}42Os display worsening Ca2+ toxicity over time.


title: Transcription factor binding sites are frequently under accelerated evolution in primates

abstract: Recent comparative genomic studies have identified many human accelerated elements (HARs) with elevated substitution rates in the human lineage. However, it remains unknown to what extent transcription factor binding sites (TFBSs) are under accelerated evolution in humans and other primates. Here, we introduce two pooling-based phylogenetic methods with dramatically enhanced sensitivity to examine accelerated evolution in TFBSs. Using these new methods, we show that more than 6,000 TFBSs annotated in the human genome have experienced accelerated evolution in Hominini, apes, and Old World monkeys. Although these TFBSs individually show relatively weak signals of accelerated evolution, they collectively are more abundant than HARs. Also, we show that accelerated evolution in Pol III binding sites may be driven by lineage-specific positive selection, whereas accelerated evolution in other TFBSs might be driven by nonadaptive evolutionary forces. Finally, the accelerated TFBSs are enriched around neurodevelopmental and pluripotency genes, suggesting that accelerated evolution in TFBSs may drive the divergence of neurodevelopmental processes between primates.


title: Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction

abstract:
2.Complete, accurate, cost-effective, and high-throughput reconstruction of bacterial genomes for large-scale genomic epidemiological studies is currently only possible with hybrid assembly, combining long- (typically using nanopore sequencing) and short-read (Illumina) datasets. Being able to utilise nanopore-only data would be a significant advance. Oxford Nanopore Technologies (ONT) have recently released a new flowcell (R10.4) and chemistry (Kit12), which reportedly generate per-read accuracies rivalling those of Illumina data. To evaluate this, we sequenced DNA extracts from four commonly studied bacterial pathogens, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, using Illumina and ONTs R9.4.1/Kit10, R10.3/Kit12, R10.4/Kit12 flowcells/chemistries. We compared raw read accuracy and assembly accuracy for each modality, considering the impact of different nanopore basecalling models, commonly used assemblers, sequencing depth, and the use of duplex versus simplex reads. Duplex nanopore reads combined with "super accuracy" (sup) basecalling have high per-read accuracies and could be used to robustly reconstruct bacterial genomes without the use of Illumina data. However, the per-run yield of duplex reads generated in our hands with standard sequencing protocols was low (typically <10%), with substantial implications for cost and throughput if relying on nanopore data only to enable bacterial genome reconstruction. In addition, recovery of small plasmids with the best-performing long-read assembler (Flye) was inconsistent. R10.4/Kit12 combined with sup basecalling holds promise as a singular sequencing technology in the reconstruction of commonly studied bacterial genomes, but hybrid assembly (Illumina+R9.4.1 hac) currently remains the highest throughput, most robust, and cost-effective approach.

3. Impact statementOur understanding of microbes has been greatly enhanced by the capacity to evaluate their genetic make-up using a technology known as whole genome sequencing. Sequencers represent microbial genomes as stretches of shorter sequence known as  reads, which are then assembled using computational algorithms. Different types of sequencing approach have advantages and disadvantages with respect to the accuracy and length of the reads they generate; this in turn affects how reliably genomes can be assembled.

Currently, to completely reconstruct bacterial genomes in a high-throughput and cost-effective manner, researchers tend to use two different types of sequencing data, namely Illumina (short-read) and nanopore (long-read) data. Illumina data are highly accurate; nanopore data are much longer, and this combination facilitates accurate and complete bacterial genomes in a so-called "hybrid assembly". However, new developments in nanopore sequencing have reportedly greatly improved the accuracy of nanopore data, hinting at the possibility of requiring only a single sequencing approach for bacterial genomics.

Here we evaluate these improvements in nanopore sequencing in the reconstruction of four bacterial reference strains, where the true sequence is already known. We show that although these improvements are extremely promising, for high-throughput, low-cost reconstruction of bacterial genomes hybrid assembly currently remains the optimal approach.

4. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article, through supplementary data files, or in publicly accessible repositories.

Nanopore fast5 and fastq data are available in the ENA under project accession: PRJEB51164.

Assemblies have been made available at: https://figshare.com/articles/online_resource/q20_comparison_genome_assemblies/19683867.

Code and analysis outputs are available at: https://gitlab.com/ModernisingMedicalMicrobiology/assembly_comparison_analysis/-/tree/main (tagged version v0.5.5).


title: H4K16ac activates transcription of transposable elements and contribute to their cis regulatory function

abstract: Mammalian genomes harbor a large number of transposable elements (TEs) and their remnants. Most TEs are incapable of retrotransposition. Although most TEs are epigenetically repressed, transcriptional silencing is partially released to permit developmental or tissue-specific expression of TEs. Some TEs have also evolved as cis-regulatory elements (CREs), enabling them to recruit host-encoded transcription factors. Understanding the contribution of TEs in the regulation of the mammalian genome is an active area of research. Previously, the noncoding long terminal repeat (LTR) part of the endogenous retrovirus (ERV) families has been shown to function as enhancers. We show that new LTR families and the promoter region of LINE1 (L1) are enriched with H4K16ac and H3K122ac and the chromatin features associated with active enhancers. Depletion of MSL complex and H4K16ac levels leads to a significant reduction in the expression of L1 and ERV/LTRs. We demonstrate that H4K16ac regulates TE transcription by maintaining a permissive chromatin structure. Furthermore, CRISPR-based perturbation of candidate TEs led to the downregulation of genes in cis. We conclude that H4K16ac and H3K122ac regulate a significant portion of the mammalian genome by opening local chromatin structure and transcriptional activity at TEs.


title: DeepSlice: rapid fully automatic registration of mouse brain imaging to a volumetric atlas

abstract: Registration of data to a common frame of reference is an essential step in the analysis and integration of diverse neuroscientific data modalities. To this end, volumetric brain atlases enable histological datasets to be spatially registered and analysed, yet accurate registration remains expertise-dependent and slow. We have trained a neural network, DeepSlice, to register mouse brain histology to the Allen Brain Atlas, retaining accuracy while improving speed by >1000 fold.


title: The Features Underlying the Memorability of Objects

abstract:
Despite decades of study of memory, it remains unclear what makes an image memorable. There is considerable debate surrounding the underlying determinants of memory, including the roles of semantic (e.g., animacy, utility) and visual features (e.g., brightness) as well as whether the most prototypical or most atypical items are best remembered. Prior studies have relied on constrained stimulus sets, limiting any generalized view of the features that may contribute to memory. Here, we collected over one million memory ratings (N=13,946) for THINGS (Hebart et al., 2019), a naturalistic dataset of 26,107 object images designed to comprehensively sample concrete objects. First, we establish a model of object features that is predictive of image memorability, capturing over half of the explainable variance. For this model, we find that semantic features have a stronger influence than visual features on what people will remember. Second, we examined whether memorability could be accounted for by the typicality of the objects, by comparing human behavioral data, object feature dimensions, and deep neural network features. While prototypical objects tend to be the most memorable, the relationship between memorability and typicality is more complex than a simple positive or negative association and typicality alone cannot account for memorability.

SIGNIFICANCE STATEMENTWhy is it that we seem to remember and forget the same things? Our lived experiences differ, but there is remarkable consistency in what is remembered across people. Here, we collected memory performance scores for a comprehensive and diverse collection of natural object images to identify which properties determine our ability to remember. We created a model for predicting memory from object features showing that semantic properties more than visual properties contribute primarily to memorability. Further, we find that it is neither the most prototypical or atypical images that are best remembered, which suggests that typicality alone cannot account for memorability. Our findings challenge decades of prior research that suggest that the most atypical items are most memorable, informing our understanding of the features and organizational principles of memory.


title: Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities

abstract: Palladin (PALLD) belongs to the PALLD/myopalladin (MYPN)/myotilin family of actin-associated immunoglobulin-containing proteins in the sarcomeric Z-line. PALLD is ubiquitously expressed in several isoforms and its longest 200 kDa isoform, predominantly expressed in striated muscle, shows high structural homology to MYPN. MYPN gene mutations are associated with human cardiomyopathies, whereas the role of PALLD in the heart has remained unknown, partly due to embryonic lethality of PALLD knockout mice. In a yeast two-hybrid screening, CARP/Ankrd1 and FHOD1 were identified as novel interaction partners of PALLDs N-terminal region. To study the role of PALLD in the heart, we generated conditional (cPKO) and inducible (cPKOi) cardiomyocyte-specific PALLD knockout mice. While cPKO mice exhibited no pathological phenotype, ablation of PALLD in adult cPKOi mice caused progressive cardiac dilation and systolic dysfunction, associated with reduced cardiomyocyte contractility, intercalated disc abnormalities, and fibrosis, demonstrating that PALLD is essential for normal cardiac function. Double cPKO and MYPN knockout mice exhibited a similar phenotype as MKO mice, suggesting that MYPN does not compensate for the loss of PALLD in cPKO mice. Transcript levels of MYPN and the PALLD long isoform were significantly increased in myocardial tissue from human dilated cardiomyopathy patients, suggesting a role of PALLD in cardiac disease.


title: Intracellular injection of brain extracts from Alzheimer's disease patients trigger unregulated Ca2+ release from intracellular stores that hinders cellular bioenergetics

abstract: Strong evidence indicates that amyloid beta (A{beta}) inflicts its toxicity in Alzheimers disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic A{beta}42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous A{beta}s oligomers. To investigate this possibility, we use human postmortem brain extracts from control and AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of samples from AD patients revealed elevated content of soluble A{beta} oligomers, detected by the conformation-dependent OC-antibody, whereas no immunoreactivity was detected in the normal samples. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients rising within few seconds after injection and persisting for several seconds. Pre-incubation of brain extracts with the conformation specific OC antibody completely suppressed brain extract ability to trigger cytosolic Ca2+ events. Comparison of the elementary events triggered by brain extracts and synthetic A{beta}42 oligomer showed comparable temporal evolution and amplitudes to events triggered by direct injection of IP3. Moreover, bath application of caffeine reversibly inhibited local and global Ca2+ signals in all the samples confirming the involvement of Ca2+ release from the ER. Analysis of the recorded Ca2+ fluorescence signals by computational modeling allowed quantification of the IP3 and Ca2+ generated by each sample. The model further shows that the abnormal increase of Ca2+ and IP3 may affect mitochondrial bioenergetics. These results, supports the hypothesis that endogenous amyloid oligomer contained in neurons of AD-affected brains may represent the toxic agents responsible for neurons malfunctioning and death, associated with the disruption of neuronal Ca2+ homeostasis.


title: An improved pipeline for reprogramming human induced pluripotent stem cells with TET1

abstract: Despite offering insights into regenerative medicine, induced pluripotent stem cells (iPSCs) exhibit inconsistent differentiation potential, which may negatively impact their downstream applications. Here, we potentiated the reprogramming process of iPSCs by adding ten-eleven translocation 1 (TET1), a DNA demethylase, to a conventional reprogramming cocktail to produce TET1-iPSCs (T-iPSCs). We selected differentiation-elite iPSC clones through testing using a default differentiation procedure that challenged the evolutionarily conserved differentiation propensity toward neuroectoderm formation. By comparing the differentiation efficiencies of 46 in-house- generated iPSC clones, we identified extraembryonic markers that may indicate differentiation defects. Elite T-iPSCs showed an epithelialized morphology and differentiated into midbrain dopaminergic neurons with unprecedented fidelity and efficiency. We propose the T-iPSC production pipeline described here as a de facto standard for obtaining human pluripotent stem cells with enhanced differentiation potential.


title: SPTLC1 variants associated with childhood onset amyotrophic lateral sclerosis produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

abstract: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Mutations in the SPTLC1 subunit of serine-palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased sphingolipid synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause the peripheral sensory neuropathy HSAN1 due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.


title: Hyperimmune bovine colostrum containing lipopolysaccharide antibodies (Imm124-E) has a non-detrimental effect on gut microbial communities in unchallenged mice.

abstract: 1Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea in travelers, military personnel and children in developing countries. Infection has the potential to cause long-term gastrointestinal dysfunction. Preventative treatments for ETEC-induced diarrhea exist, yet the effects of these treatments on gastrointestinal commensals in healthy individuals is unclear. Whether administration of a prophylactic preventative treatment for ETEC-induced diarrhea causes specific shifts in gut microbial populations in controlled environments is also unknown. Here we studied the effects of a hyperimmune bovine colostrum (IMM-124E) used in the manufacture of Travelan(R) (AUST L 106709) on gastrointestinal bacteria in healthy C57BL/6 mice. Using next generation sequencing, we aimed to test the onset and magnitude of potential changes to the mouse gut microbiome in response to the anti-diarrheagenic hyperimmune bovine colostrum product, rich in immunoglobulins against select ETEC strains (Travelan(R), Immuron Ltd). We engineered changes in mouse fecal and cecal bacterial communities by delivering lipopolysaccharide (LPS) antibodies derived from bovine colostrum via dietary supplementation. Holstein Friesian and Jersey cows between 28- and 35-weeks gestation stimulated by subcutaneous delivery of three important pathogenic and antigenic determinants; LPS, flagella, and colonization factor antigen (CFA), produced a hyperimmune colostrum (IMM-124E) with demonstrated beneficial effects on health via modulation of metabolic pathways and immune function. We show that in mice administered colostrum containing LPS antibodies there was an increased abundance of potentially gut-beneficial bacteria, such as Akkermansia and Desulfovibrio, without disrupting the underlying ecology of the gastrointestinal tract. Compared to controls, there was no difference in overall weight gain, body or cecal weights or small intestine length following LPS antibody colostrum supplementation. Overall, dietary supplementation with colostrum containing LPS antibodies produced subtle alterations in gut bacterial composition of mice. Primarily, Travelan(R) LPS antibody treatment decreased the ratio of Firmicutes/Bacteroidetes in gut microbial populations in unchallenged healthy mice. Further studies are required to examine the effect of Travelan(R) LPS antibody treatment to engineer the microbiome in a diseased state and during recovery.


title: MEIG1/PACRG associated and non-associated functions of axonemal dynein light intermediate polypeptide 1 (DNALI1) in mammalian spermatogenesis

abstract:
Axonemal dynein light intermediate polypeptide 1 (DNALI1) was originally cloned from Chlamydomonas reinhardtii in an effort to find motor proteins essential for flagellar motility. Here we report that DNALI1 is a binding partner of parkin co-regulated gene 1 (PACRG), which forms a complex with meiosis expressed gene 1 (MEIG1) in the manchette, a transient and unique structure only present in the elongating spermatids and required for normal spermiogenesis of the male germ cell differentiation process. DNALI1 recruits the PACRG protein in transfected CHO cells, and also stabilizes PACRG in bacteria and transfected mammalian cells. The untagged DNALI1 could also be co-purified with His-tagged PACRG in the gel filtration assay. Immunofluorescence staining on isolated male germ cells revealed that DNALI1 was present in the manchette of elongating spermatids, and colocalized with PACRG in this structure. In Pacrg mutant mice, localization of DNALI1 in the manchette was not changed, suggesting that DNALI1 and PACRG form a complex in the manchette, with DNALI1 being an upstream molecule. Mice deficiency in DNALI1 specifically in male germ cells showed dramatically reduced sperm numbers and were infertile. In addition, majority of the sperm exhibited abnormal morphology including misshapen heads, bent tails and enlarged midpiece, discontinuous accessory structure, and loss of sperm individualization, emphasizing the importance of DNALI1 in sperm development. Examination of testis histology revealed impaired spermiogenesis in the conditional Dnali1 knockout mice. Electron microscopy revealed disrupted ultrastructure in sperm of the Dnali1 mutant mice. Testicular levels of MEIG1, PACRG and SPAG16L proteins were not changed in the Dnali1 mutant mice. However, MEIG1 and SPAG16L were no longer present in the manchette in the absence of DNALI1. These findings demonstrate that DNALI1 is involved in the connection of the MEIG1/PACRG complex to carry cargo proteins along the manchette microtubules for sperm flagella formation. Given that Dnali1 mutant mice showed impaired sperm individualization that was not observed in the MEIG1 nor PACRG-deficient mice, DNALI1 might fulfill other functions beyond its role associated with the MEIG1/PACRG complex. Thus, DNALI1 plays multiple roles in sperm cell differentiation and function.

Summary statementAxonemal dynein light intermediate polypeptide 1 (DNALI1) is required for sperm formation and male fertility. It associates with the MEIG1/PACRG complex in the manchette and is involved in a cargo transport system. In addition, it might be related to IFT and sperm individualization.


title: Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells

abstract: The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DC) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.


title: Thyroid hormone and ALK5 inhibitor improve maturation of human pluripotent stem cell derived hepatocytes

abstract: Hepatocytes derived from human pluripotent stem cells (PSCs) hold great promise for modeling human liver disease, in vitro hepatotoxicity testing, and future cellular therapy. However, current protocols generate hepatocyte-like cells (HLCs) that resemble fetal hepatocytes, and thus do not accurately recapitulate the molecular identity and functions of the adult liver. To address this, we compared the transcriptomes of human fetal and adult liver to PSC-derived HLCs during progressive stages of in vitro differentiation. This revealed that during the final stages of in vitro differentiation the hepatic transcription factors HNF4A and CEBPA were sub-optimally expressed. Computational analyses predicted that ALK5i II (TGF-{beta} receptor inhibitor) and thyroid hormone (T3) would be able to rectify this and improve HLC maturation. We next show that application of these molecules during hepatocyte differentiation indeed increases CEBPA and HNF4A mRNA and protein expression, and that these HLCs show enhanced albumin secretion, a 25-fold increase in CYP3A4 activity, and 10 to 100-fold increased expression of mature hepatic markers. We demonstrate that this improved maturation is effective across different cell lines and HLC differentiation protocols, and exemplifies that our approach provides a tractable template for identifying and targeting additional factors that that will fully mature human liver cells from human pluripotent stem cells.

category: developmental biology


title: Divergence of trafficking and polarity mechanisms for PIN auxin transporters during land plant evolution

abstract: The phytohormone auxin and its directional transport through tissues play a fundamental role in plant development. This polar auxin transport relies on the PIN auxin exporters, whose polarization at the plasma membrane determines the overall direction of auxin flow. Hence, PIN polarization is crucial for development, but its evolution during the rise of developmental complexity in land plants remains unclear. Here, we investigated the evolution of PIN trafficking and polarization by comparing two model bryophytes, Physcomitrella patens and Marchantia polymorpha, with the angiosperm model Arabidopsis thaliana. All examined PINs show a conserved auxin export function, which is reflected by comparable bryophytic growth defects in overexpression lines. In bryophytes, bryophytic PINs polarize to filamentous apices, while Arabidopsis PINs distribute symmetrically on the membrane with additional intracellular puncta. In the Arabidopsis root epidermis, bryophytic PINs show no defined polarity. Pharmacological interference revealed a strong cytoskeleton dependence of bryophytic but not Arabidopsis PIN polarization, which, in contrast, strongly relies on Brefeldin A-sensitive trafficking. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues in individual species. These results collectively reveal a divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and suggest co-evolution of PIN sequence-based and cell-based polarity mechanisms.


title: Machine learning-based detection of label-free cancer stem-like cell fate

abstract: Most imaging methods rely on labelling biological samples in order to provide specific and easily detectable features. However, label-free imaging is a non-invasive and non-toxic alternative that requires accurate image analysis algorithm based on cell morphology. Such analysis has to deal with a high image variability while fewer features are extractable, so far, fast analysis of label-free brightfield microscopy (LFBM) images remains a challenging task. With the development of microfabricated devices during the last decades, high throughput image generation makes it possible to use machine learning-based algorithms in order to analyse LFBM images. Fast algorithms are also crucially needed to analyze high throughput experiments. In this paper, we provide a data-driven study in order to assess the complexity of LFBM time-lapses monitoring isolated cancer stem-like cells (CSCs) fate in non-adherent conditions. We combined for the first time individual cell fate and cell state temporality analysis in a unique algorithm. Several image analysis algorithms of increasing processing capacities were tested: a classical computer vision algorithm (CCVA), a shallow learning-based algorithm (SLBA) and a deep learning-based algorithm (DLBA). We show that our optimized DLBA has by far the best accuracy compared to CCVA and SLBA, is at least as accurate as other state-of-the-art DLBAs while being faster. With this study, we demonstrate that optimizing our DLBA accordingly to the image analysis problem can overall provide better results than pretrained models. Such a fast and accurate DLBA is therefore compatible with the generation of high throughput data and opens the route for on-the-fly analysis of CSC fate from LFBM time-lapses.