title: Detection of Motor-Evoked Potentials Below the Noise Floor: Rethinking the Motor Threshold

abstract:
BackgroundMotor-evoked potentials (MEP) are one of the most prominent responses to brain stimulation, such as supra-threshold transcranial magnetic stimulation (TMS) and electrical stimulation. Understanding of the neurophysiology and the determination of the lowest stimulation strength that evokes responses requires the detection of even smaller responses, e.g., from single motor units. However, available detection and quantization methods suffer from a large noise floor.

ObjectiveThis paper develops a detection method that extracts MEPs hidden below the noise floor. With this method, we aim to estimate excitatory activations of the corticospinal pathways well below the conventional detection level.

MethodsThe presented MEP detection method presents a self-learning matched-filter approach for improved robustness against noise. The filter is adaptively generated per subject through iterative learning. For responses that are reliably detected by conventional detection, the new approach is fully compatible with established peak-to-peak readings and provides the same results but extends the dynamic range below the conventional noise floor.

ResultsIn contrast to the conventional peak-to-peak measure, the proposed method increases the signal-to-noise ratio by more than a factor of 5. The first detectable responses appear to be substantially lower than the conventional threshold definition of 50 V median peak-to-peak amplitude.

ConclusionThe proposed method shows that stimuli well below the conventional 50 V threshold definition can consistently and repeatably evoke muscular responses and thus activate excitable neuron populations in the brain. As a consequence, the IO curve is extended at the lower end, and the noise cut-off is shifted. Importantly, the IO curve extends so far that the 50 V point turns out to be closer to the center of the logarithmic sigmoid curve rather than close to the first detectable responses. The underlying method is applicable to a wide range of evoked potentials and other biosignals, such as in electroencephalography.


title: Benchmarking GE-BOLD, SE-BOLD, and SS-SI-VASO sequences for depth-dependent separation of feedforward and feedback signals in high-field MRI

abstract:
Recent advances in high-field fMRI have allowed differentiating feedforward and feedback information in the grey matter of the human brain. For continued progress in this endeavor, it is critical to understand how MRI data acquisition parameters impact the read-out of information from laminar response profiles. Here, we benchmarked three different MR-sequences at 7T - gradient-echo (GE), spin-echo (SE) and vascular space occupancy imaging (VASO) - in differentiating feedforward and feedback signals in human early visual cortex (V1). The experiment (N=4) consisted of two complementary tasks: a perception task that predominantly evokes feedforward signals and a working memory task that relies on feedback signals. In the perception task, participants saw flickering oriented gratings while detecting orthogonal color-changes. In the working memory task, participants memorized the precise orientation of a grating. We used multivariate pattern analysis to read out the perceived (feedforward) and memorized (feedback) grating orientation from neural signals across cortical depth. Analyses across all the MR-sequences revealed perception signals predominantly in the middle cortical compartment of area V1 and working memory signals in the deep compartment. Despite an overall consistency across sequences, SE-EPI was the only sequence where both feedforward and feedback information were differently pronounced across cortical depth in a statistically robust way. We therefore suggest that in the context of a typical cognitive neuroscience experiment as the one benchmarked here, SE-EPI may provide a favorable trade-off between spatial specificity and signal sensitivity.

HighlightsHere, we benchmarked three sequences at high-field fMRI -GE-BOLD, SE-BOLD and VASO - in differentiating feedforward and feedback signals across grey matter depth of area V1. We show that:

O_LIAll the MR-sequences revealed the feedforward and feedback signals at the middle and deep cortical bins, respectively.
C_LIO_LISuch correspondence across the sequences indicates that widely used GE-BOLD is a suitable method for the exploration of signals in cortical depth.
C_LIO_LIOnly SE-BOLD yielded statistically reliable differences between the cortical bins carry- ing feedforward and feedback signals.
C_LI

category: neuroscience


title: Neuronal activity-driven oligodendrogenesis in selected brain regions is required for episodic memories

abstract:
The formation of long-term episodic memories requires the activation of molecular mechanisms in several regions of the medial temporal lobe, including the hippocampus and anterior cingulate cortex (ACC). The extent to which these regions engage distinct mechanisms and cell types to support memory formation is not well understood. Recent studies reported that oligodendrogenesis is essential for learning and long-term memory; however, whether these mechanisms are required only in selected brain regions is still unclear. Also still unknown are the temporal kinetics of engagement of learning-induced oligodendrogenesis and whether this oligodendrogenesis occurs in response to neuronal activity. Here we show that in rats and mice, episodic learning rapidly increases the oligodendrogenesis and myelin biogenesis transcripts olig2, myrf, mbp, and plp1, as well as oligodendrogenesis, in the ACC but not in the dorsal hippocampus (dHC). Region-specific knockdown and knockout of Myrf, a master regulator of oligodendrocyte maturation, revealed that oligodendrogenesis is required for memory formation in the ACC but not the dHC. Chemogenetic neuronal silencing in the ACC showed that neuronal activity is critical for learning-induced oligodendrogenesis. Hence, an activity-dependent increase in oligodendrogenesis in selected brain regions, specifically in the ACC but not dHC, is critical for the formation of episodic memories.

Impact statementOligodendrogenesis is required in the anterior cingulate cortex but not in the hippocampus for long-term memory consolidation.


title: Random forest modelling of neuropathological features identifies microglial activation as an accurate pathological classifier of C9orf72-related amyotrophic lateral sclerosis

abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are regarded as two ends of a pathogenetic spectrum, termed ALS-frontotemporal spectrum disorder (ALS-FTSD). However, it is currently difficult to predict where on the spectrum an individual will lie, especially for patients with C9orf72 hexanucleotide repeat expansions (HRE), a mutation associated with both ALS and FTD. It has been shown that both inflammation and protein misfolding influence aspects of ALS and ALS-FTSD disease pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. Previous studies have highlighted markers which may influence C9orf72-associated disease presentation in a targeted fashion, though there has yet to be a systematic and quantitative assessment of common immunohistochemical markers to investigate the significance of these pathways in an unbiased manner. Here we report the first extensive digital pathological assessment with random forest modelling of pathological markers often used in neuropathology practice. This study profiles glial activation and protein misfolding in a cohort of deeply clinically profiled post-mortem tissue from patients with a C9orf72 HRE, who either met the criteria for a diagnosis of ALS or ALS-FTSD. We show that microglial immunohistochemical staining features, both morphological and spatial, are the best independent classifiers of disease status and that clinicopathological associations exist between microglial activation status and cognitive dysfunction in ALS-FTSD patients with C9orf72 HRE. Furthermore, we show that spatially resolved changes in FUS staining are also an accurate predictor of disease status, implying that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9orf72 ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological associations between cohorts.


title: Robust regulatory architecture of pan-neuronal gene expression

abstract: Pan-neuronally expressed genes, such as genes involved in the synaptic vesicle release cycle or in neuropeptide maturation, are critical for proper function of all neurons, but the transcriptional control mechanisms that direct such genes to all neurons of a nervous system remain poorly understood. We show here that six members of the CUT family of homeobox genes control pan-neuronal identity specification in C. elegans. Single CUT mutants show barely any effects on pan-neuronal gene expression or global nervous system function, but such effects become apparent and progressively worsen upon removal of additional CUT family members, indicating a critical role of gene dosage. Overexpression of each individual CUT gene rescued the phenotype of compound mutants, corroborating that gene dosage, rather than the activity of specific members of the gene family, is critical for CUT gene function. Genome-wide binding profiles as well as mutation of CUT binding sites by CRISPR/Cas9 genome engineering show that CUT genes directly control the expression of pan-neuronal features. Moreover, CUT genes act in conjunction with neuron-type specific transcription factors to control pan-neuronal gene expression. Our study, therefore, provides a previously missing key insight into how neuronal gene expression programs are specified and reveals a highly buffered and robust mechanism that controls the most critical functional features of all neuronal cell types.


title: Disrupted-in-Schizophrenia-1 is required for proper pyramidal cell-interneuron communication and network dynamics in the prefrontal cortex.

abstract: We interrogated prefrontal circuit function in mice lacking Disrupted-in-schizophrenia-1 (Disc1-mutant mice), a risk factor for psychiatric disorders. Single-unit recordings in awake mice revealed reduced average firing rates of fast-spiking interneurons (INTs), including optogenetically identified parvalbumin-positive cells, and a lower proportion of INTs phase-coupled to ongoing gamma oscillations. Moreover, we observed decreased spike transmission efficacy at local pyramidal cell (PYR)-INT connections in vivo, suggesting a reduced excitatory effect of local glutamatergic inputs as a potential mechanism of lower INT rates. On the network level, impaired INT function resulted in altered activation of PYR assemblies: While assembly activations were observed equally often, the expression strength of individual assembly patterns was significantly higher in Disc1-mutant mice. Our data thus reveal a role of Disc1 in shaping the properties of prefrontal assembly patterns by setting prefrontal INT responsiveness to glutamatergic drive.


title: Behavioural consequences of Setd1a haploinsufficiency in mice: evidence for heightened emotional reactivity and impaired sensorimotor gating

abstract: A number of studies implicate the loss of function (LoF) mutations affecting the histone methyl transferase SETD1A in the aetiology of a range of neurodevelopmental disorders including schizophrenia. Here, we examined the behavioural consequences of haploinsufficiency of Setd1a in a mouse model. We find evidence for changes in a number of phenotypes of relevance to schizophrenia, including increased anxiety-related behaviour, enhanced acoustic startle response, and decreased pre-pulse inhibition of acoustic startle. The sensorimotor gating deficits in Setd1a+/- mice could not be rescued by haloperidol or risperidone, suggesting that these antipsychotics are ineffective for ameliorating schizophrenia-relevant phenotypes in Setd1a+/- mice and point to deficits in neural systems other than the monoamine system. These phenotypes are emerging as key features of a number of other mouse models of rare neurodevelopmental disorders caused by LoF mutations in genes encoding epigenome modifiers suggesting they may act in a network to modulate brain development. Taken together these data strengthen the support for the use of Setd1a haploinsufficient mice as a model for the biological basis of schizophrenia, and point towards possible underpinning neural mechanisms.


title: Active neural coordination of motor behaviors with internal states

abstract: The brain continuously coordinates skeletomuscular movements with internal physiological states like arousal, but how is this coordination achieved? One possibility is that brain simply reacts to changes in external and/or internal signals. Another possibility is that it is actively coordinating both external and internal activities. We used functional ultrasound imaging to capture a large medial section of the brain, including multiple cortical and subcortical areas, in marmoset monkeys while monitoring their spontaneous movements and cardiac activity. By analyzing the causal ordering of these different time-series, we found that information flowing from the brain to movements and heart rate fluctuations were significantly greater than in the opposite direction. The brain areas involved in this external versus internal coordination were spatially distinct but also extensively interconnected. Temporally, the brain alternated between network states for this regulation. These findings suggest that the brains dynamics actively and efficiently coordinate motor behavior with internal physiology.


title: Flexibility in the social structure of male chimpanzees (Pan troglodytes schweinfurthii) in the Budongo Forest, Uganda

abstract:
Individuals of social species face a trade-off between the competitive costs and social benefits of group living. Species show a range of social strategies to deal with this trade-off, for example atomistic fission-fusion dynamics in which temporary social groups of varying size and membership form and re-form; or molecular fission-fusion dynamics which contain stable sets of multilevel nested subgroups. Chimpanzees are considered an archetypical atomistic fission-fusion species, using dynamic changes in day-to-day association to moderate the costs of within-group competition. It has been argued that humans highly flexible social organisation allows us to live in extremely large groups. Using four years of association data from two neighbouring communities of East African chimpanzees (Pan troglodytes schweinfurthii), we describe new levels of flexibility in chimpanzee social organisation and confirm the presence of subgrouping in a second, large community of chimpanzees. We show that males from the larger Waibira community (N males 24-31) exhibited additional levels of semi-stable subgrouping, while males from the smaller Sonso community (N males 10-13) did not. Subgroup membership showed stability across some years, but flexibility across others. Our data support the hypothesis that chimpanzees can incorporate strategies other than fission-fusion to overcome costs of social living, and that their social organisation may be closer to that of modern humans than previously described.

SIGNIFICANCE STATEMENTSocial living offers benefits and costs; groups can more easily locate and defend resources, but experience increased individual competition. Many species, or individuals, flexibly adjust their social organization when faced with different competitive pressures. It is argued that humans are unique among primates in combining multigroup social organisation with fission-fusion dynamics flexibly within and across groups, and that doing so allows us to live in extremely large groups. Using four-years of association data we show new levels of flexibility in chimpanzee social organization. Males from a typically-sized community employed atomistic fission-fusion dynamics, but males in an unusually large community incorporated additional semi-stable levels of subgrouping. Our data support the hypothesis that chimpanzee males combine social organization strategies, and that doing so may allow them, like humans, to mitigate individual costs at larger community sizes.

category: animal behavior and cognition


title: The minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers

abstract: Minor splicing is a second splicing system required for the correct expression of [~]700 human minor intron-containing genes (MIGs). Many MIGs are expressed in vigorously proliferating cells and are frequently dysregulated in cancer including BRAF, ERK, JNK and p38. Minor splicing is carried out by the minor spliceosome which comprises several unique components, including a 65kDa protein encoded by RNPC3. We show that Rnpc3 heterozygosity reduces tumour burden in a broad spectrum of in vivo cancer settings, without harming normal tissues. Using the collective power of zebrafish, mouse and human cancer models, we reveal a sequence of events connecting Rnpc3 deficiency and impaired splicing of MIGs to DNA damage and activation of a Tp53-dependent transcriptional program that restricts tumour burden by inducing cell cycle arrest and apoptosis. Interrogation of human liver and lung cancer transcriptomes curated in TCGA revealed that the expression of many of the genes encoding protein components of the minor spliceosome is upregulated in these cancers. This is accompanied by upregulation of the expression of MIGs that are enriched in cell cycle and DNA damage pathways. These findings suggest that cancer cells can invoke mechanisms to increase the efficiency of minor splicing to support their high proliferation rates. Finally, Kaplan Meier survival analysis shows that highly expressed MIGs are frequently associated with poor patient survival. Taken together, these results indicate that the minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers.

category: cancer biology


title: Boron Supply Restores Aluminum-Blocked Auxin Transport by Modulation PIN2 Trafficking in the Arabidopsis Roots

abstract:
This study tested a hypothesis that boron (B) supply alleviates aluminum (Al) toxicity by modifying auxin distribution in functionally different root zones. Auxin distribution and transport at various Al and B ratios were analyzed using the range of molecular and imaging techniques. Al stress resulted in increased auxin accumulation in root apical meristem (MZ) and transition zones (TZ) while reducing its content in elongation zone (EZ). This phenomenon was explained by reduction in basipetal auxin transport caused by Al blockage of PIN2 endocytosis, regulated at posttranscriptional level. This inhibition of PIN2 endocytosis was dependent on actin filaments and microtubules. B supply facilitated the endocytosis and exocytosis of PIN2 carriers via recycling endosomes conjugated with IAA to modify Al-induced auxin depletion in the EZ. However, disruption of auxin signaling with auxinole did not alleviate Al-induced inhibition of root growth. B supply alleviates Al-induced inhibition of root growth via restoring the endocytic recycling of PIN2 proteins involved in the basipetal (shootward) auxin transport, restoring Al-induced auxin depletion in the elongation zone.

Short summaryAluminum-intensified PIN2 abundance, nontranscriptional, via repressing PIN2 endocytosis to block polar auxin transport, and this adverse effect could be alleviated by boron supply.

category: physiology


title: Dopamine D2Rs Coordinate Cue-Evoked Changes in Striatal Acetylcholine Levels

abstract: In the striatum, acetylcholine (ACh) neuron activity is modulated co-incident with dopamine (DA) release in response to unpredicted rewards and reward predicting cues and both neuromodulators are thought to regulate each other. While this co-regulation has been studied using stimulation studies, the existence of this mutual regulation in vivo during natural behavior is still largely unexplored. One long-standing controversy has been whether striatal DA is responsible for the induction of the cholinergic pause or whether D2R modulate a pause that is induced by other mechanisms. Here, we used genetically encoded sensors in combination with pharmacological and genetic inactivation of D2Rs from cholinergic interneurons (CINs) to simultaneously measure ACh and DA levels after CIN D2R inactivation. We found that CIN D2Rs are not necessary for the induction of cue induced dips in ACh levels but regulate dip lengths and rebound ACh levels. Importantly, D2R inactivation strongly decreased the temporal correlation between DA and Ach signals not only at cue presentation but also during the intertrial interval. This points to a general mechanism by which D2Rs coordinate both signals. At the behavioral level D2R antagonism increased the latency to lever press, which was not observed in CIN-selective D2R knock out mice. This latency correlated with the cue evoked dip length supporting a role of the ACh dip and its regulation by D2Rs in motivated behavior. Overall, our data indicate that striatal DA coordinate phasic ACh and DA signals via CIN D2Rs which is important for the regulation of motivated behavior.


title: Protein interaction network analysis reveals growth conditions-specific crosstalk between chromosomal DNA replication and other cellular processes in E. coli

abstract:
E. coli and many other bacterial species can alter their cell cycle according to nutrient availability. Under optimal conditions bacteria grow and divide very fast but they slow down the cell cycle when conditions deteriorate. This adaptability is underlined by mechanisms coordinating cell growth with duplication of genetic material and cell division. Several mechanisms regulating DNA replication process in E. coli have been described with biochemical details so far. Nevertheless we still dont fully understand the source of remarkable precision that allows bacterial cells to coordinate their growth and chromosome replication. To shed light on regulation of E. coli DNA replication at systemic level, we used affinity purification coupled with mass spectrometry (AP-MS) to characterize protein-protein interactions (PPIs) formed by key E. coli replication proteins, under disparate bacterial growth conditions and phases. We present the resulting dynamic replication protein interaction network (PIN) and highlight links between DNA replication and several cellular processes, like outer membrane synthesis, RNA degradation and modification or starvation response.

ImportanceDNA replication is a vital process, ensuring propagation of genetic material to progeny cells. Despite decades of studies we still dont fully understand how bacteria coordinate chromosomal DNA duplication with cell growth and cell division under optimal and stressful conditions. At molecular level, regulation of processes, including DNA replication, is often executed through direct protein-protein interactions (PPIs). In this work we present PPIs formed by the key E. coli replication proteins under three different bacterial growth conditions. We show novel PPIs with confirmed impact on chromosomal DNA replication. Our results provide also alternative explanations of genetic interactions uncovered before by others for E.coli replication machinery.

category: microbiology


title: A short prokaryotic argonaute cooperates with membrane effector to confer antiviral defense

abstract: Argonaute (Ago) proteins are widespread nucleic acid-guided enzymes that recognize targets through complementary base pairing. While in eukaryotes Agos are involved in RNA silencing, the functions of prokaryotic Agos (pAgos) remain largely unknown. In particular, a clade of truncated and catalytically inactive pAgos (short pAgos) lacks characterization. Here, we reveal that a short pAgo protein in Sulfolobus islandicus, together with its two genetically associated proteins, Aga1 and Aga2, provide robust antiviral protection via abortive infection. Aga2 is a membrane-associated toxic effector that binds anionic phospholipids via a basic pocket, which is essential for its cell killing ability. Ago and Aga1 form a stable complex that exhibits RNA-directed nucleic acid recognition ability and directly interacts with Aga2, pointing to an immune sensing mechanism. Together, our results highlight the cooperation between pAgos and their widespread associated proteins, suggesting an uncharted diversity of pAgo-derived immune systems that await to be discovered.


title: Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions

abstract: Seventy years following the discovery of peroxisomes, their proteome remains undefined. Uncovering the complete peroxisomal proteome, the peroxi-ome, is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover the peroxi-ome of the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known organellar proteome by [~]40% and paved the way for performing systematic, whole-organellar proteome assays. Coupled with targeted experiments this allowed us to discover new peroxisomal functions. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly-identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

category: cell biology


title: Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts

abstract: Osteoclasts are unique in their capacity to degrade bone tissue. To achieve this process, osteoclasts form a specific structure called the sealing zone, which creates a close contact with bone and confines the release of protons and hydrolases for bone degradation. The sealing zone is composed of actin structures called podosomes nested in a dense actin network. The organization of these actin structures inside the sealing zone at the nano scale is still unknown. Here, we combine cutting-edge microscopy methods to reveal the nanoscale architecture and dynamics of the sealing zone formed by human osteoclasts on bone surface. Random illumination microscopy allowed the identification and live imaging of densely packed actin cores within the sealing zone. A cross-correlation analysis of the fluctuations of actin content at these cores indicates that they are locally synchronized. Further examination shows that the sealing zone is composed of groups of synchronized cores linked by -actinin1 positive filaments, and encircled by adhesion complexes. Thus, we propose that the confinement of bone degradation mediators is achieved through the coordination of islets of actin cores and not by the global coordination of all podosomal subunits forming the sealing zone.


title: SMER28 attenuates PI3K/mTOR signaling by direct inhibition of PI3K p110 delta

abstract: SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and consequently growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction of phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3K{delta} and to a lesser extent p110{gamma}. The biological relevance of our observations was underscored by interference of SMER28 with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor typrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited.


title: Mechanisms underlying melanoma invasion as a consequence of MLK3 loss

abstract: Invasive melanoma is an aggressive form of skin cancer with high incidence of mortality. The process of invasion is a crucial primary step in the metastatic cascade, yet the mechanisms involved are still under investigation. Here we document a critical role for MLK3 (MAP3K11) in the regulation of melanoma cell invasion. We report that cellular loss of MLK3 in melanoma cells promotes cell invasion. Knock down of MLK3 expression results in the hyperactivation of ERK, which is linked to the formation of a BRAF/Hsp90/Cdc37 protein complex. ERK hyperactivation leads to enhanced phosphorylation and inactivation of GSK3{beta} and the stabilization of c-Jun and JNK activity. Blocking of ERK and JNK signaling as well as Hsp90 activity downstream of MLK3-silencing significantly reduces melanoma invasion. Furthermore, our studies show that ERK activation in the aforementioned context is coupled to MT1-MMP transcription as well as the TOM1L1-dependent localization of the membrane protease to invadopodia at the invasive front. These studies provide critical insight into the mechanisms that couple MLK3 loss with BRAF hyperactivation and its consequence on melanoma invasion.


title: De novo apical domain formation inside the Drosophila adult midgut epithelium

abstract: In the adult Drosophila midgut, basal intestinal stem cells give rise to enteroblasts that integrate into the epithelium as they differentiate into enterocytes. Integrating enteroblasts must generate a new apical domain and break through the septate junctions between neighbouring enterocytes, while maintaining barrier function. We observe that enteroblasts form an apical membrane initiation site when they reach the septate junction between the overlying enterocytes. A space appears above the enteroblast as Cadherin clears from its apical surface. New septate junctions then form laterally with neighbouring enterocytes and the AMIS develops into pre-apical compartment with a fully-formed brush border. Finally, the overlying septate junction dissolves and the pre-enterocyte reaches the gut lumen. Enteroblasts therefore form an apical domain before they have a free apical surface. The process of enteroblast integration resembles lumen formation in mammalian epithelial cysts, highlighting the similarities between the fly midgut and mammalian epithelia.


title: Energetic determinants of the Par-3 interaction with the Par complex

abstract: The animal cell polarity regulator Par-3 recruits the Par complex (Par-6 and atypical Protein Kinase C-aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it has been unclear how each contributes to the overall interaction. Using purified, intact Par complex and a quantitative binding assay, we found that energy for this interaction is provided by Par-3s second and third PDZ protein interaction domains. Both Par-3 PDZ domains bind to aPKCs PDZ Binding Motif (PBM) in the Par complex, with binding energy contributed from aPKCs adjacent catalytic domain. In addition to highlighting the role of Par-3 PDZ interactions with the aPKC kinase domain and PBM in stabilizing Par-3 - Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization.


title: Syntaxin 5 determines Weibel-Palade body size and Von Willebrand factor secretion by controlling Golgi architecture

abstract: Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde ER-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometrybased approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis.


title: A New Gene Set Identifies Senescent Cells and Predicts Senescence-Associated Pathways Across Tissues

abstract: Although cellular senescence is increasingly recognized as driving multiple age-related co-morbidities through the senescence-associated secretory phenotype (SASP), in vivo senescent cell identification, particularly in bulk or single cell RNA-sequencing (scRNA-seq) data remains challenging. Here, we generated a novel gene set (SenMayo) and first validated its enrichment in bone biopsies from two aged human cohorts. SenMayo also identified senescent cells in aged murine brain tissue, demonstrating applicability across tissues and species. For direct validation, we demonstrated significant reductions in SenMayo in bone following genetic clearance of senescent cells in mice, with similar findings in adipose tissue from humans in a pilot study of pharmacological senescent cell clearance. In direct comparisons, SenMayo outperformed all six existing senescence/SASP gene sets in identifying senescent cells across tissues and in demonstrating responses to senescent cell clearance. We next used SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from publicly available human and murine bone marrow/bone scRNA-seq data and identified monocytic and osteolineage cells, respectively, as showing the highest levels of senescence/SASP genes. Using pseudotime and cellular communication patterns, we found senescent hematopoietic and mesenchymal cells communicated with other cells through common pathways, including the Macrophage Migration Inhibitory Factor (MIF) pathway, which has been implicated not only in inflammation but also in immune evasion, an important property of senescent cells. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Moreover, using this senescence panel, we were able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways associated with these cells, which may be particularly useful for evolving efforts to map senescent cells (e.g., SenNet). In addition, SenMayo represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.


title: Candidatus Nitrosopolaris, a genus of putative ammonia-oxidizing archaea with a polar/alpine distribution

abstract: Ammonia-oxidizing archaea (AOA) are key players in the nitrogen cycle. Here, we report four novel metagenome-assembled genomes (MAGs) assigned to the genus "UBA10452", an uncultured lineage of putative AOA in the family Nitrososphaeraceae. Analysis of other eight previously reported MAGs and publicly available amplicon sequencing data revealed that the UBA10452 lineage is predominantly found in acidic polar and alpine soils. We propose a novel Candidatus genus, Ca. Nitrosopolaris, with four species representing clear biogeographical/habitat clusters. In addition to the presence of genes involved in cold adaptation, we hypothesize that the distribution of Ca. Nitrosopolaris across the cold biosphere might also be due to the combination of a polar origin and limited dispersal capabilities throughout geological time.


title: Sub-communities of the vaginal ecosystem in pregnant and non-pregnant women.

abstract: Diverse and non-Lactobacillus-dominated vaginal microbial communities are associated with adverse health outcomes such as preterm birth and acquisition of sexually transmitted infections. Despite the importance of recognizing and understanding the key risk-associated features of these communities, their heterogeneous structure and properties remain ill-defined. Clustering approaches have been commonly used to characterize vaginal communities, but they lack sensitivity and robustness in resolving community substructures and revealing transitions between potential sub-communities. We used a highly resolved approach based on mixed membership topic models with multi-domain longitudinal data from cohorts of pregnant and non-pregnant study participants to identify several non-Lactobacillus-dominated sub-communities common to both cohorts regardless of reproductive status. These sub-communities correlated with clusters in metabolite concentrations. In non-pregnant individuals, we identified non-Lactobacillus-dominated sub-communities that were more common during menses but did not predict an increased likelihood of other non-Lactobacillus-dominated communities during the rest of the menstrual cycle. The menstrual cycle was a strong driver of transitions between sub-communities and was correlated with changes in levels of cytokines and metabolites, such as elevated TNF- concentrations at the time of ovulation, and elevated kynurenine concentrations during menses, respectively. In pregnant individuals, some metabolite clusters were predictive of temporal instability in vaginal microbiota structure. Overall, our analyses based on mixed membership models revealed new substructures of the vaginal ecosystems which may have potentially important clinical or biological associations.


title: Macrophage cell lines and murine infection by Salmonella Typhi L-form bacteria

abstract: Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic-resistance genes is well-established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as the one due to treatment with bacterial cell-wall inhibiting antibiotics like ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin. This is different from in vitro drug testing where the organism is exposed to fixed drug concentrations from the beginning till the end. To mimic the mode of antibiotic exposure of microorganisms in the tissues, we cultured the wild type, ampicillin-sensitive Salmonella Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that exhibited several features of the so-called L-form of bacteria, such as the absence of cell wall, altered shape and slower growth rate compared with the parental strain. Studies on the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental strain. These results suggested that L-form generation following initiation of antibiotic treatment could lead to drug escape of S. Typhi and direct spread to new cells (macrophages), which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through faeco-oral route, highlighting the need for new approaches to decrease the reservoir of infection.


title: Timing of exposure is critical in a highly sensitive model of SARS-CoV-2 transmission

abstract: Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing. However, contrary to expectation, trends observed with sub-optimal exposure timing suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission.


title: Generation of bright autobioluminescent bacteria by chromosomal integration of the improved lux operon ilux2

abstract: The bacterial bioluminescence system enables light production in living cells without an external luciferin. Due to its relatively low levels of light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with 7-fold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.

category: biochemistry


title: Programmed knockout mutation of liver fluke granulin, Ov-grn-1, attenuates virulence and impedes malignant transformation during chronic opisthorchiasis

abstract: Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to a nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We produced programmed gene knockout flukes ({Delta}Ov-grn-1) by delivery of a CRISPR/Cas9/gRNA system by electroporation. Genome sequencing confirmed Cas9-catalyzed mutations in the targeted genes, which was accompanied by rapid depletion of transcripts and the cognate proteins. Whereas Ov-grn-1 gene-edited parasites colonized the biliary tract and developed into adult flukes, less hepatobiliary tract disease manifested during chronic infection with{Delta} Ov-grn-1 worms in comparison to hamsters infected with control parasites. Specifically, immunohistochemical analysis of thin sections of livers revealed markedly less periductal fibrosis surrounding the flukes and less liver fibrosis globally during infection with{Delta} Ov-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and markedly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade cholangiocarcinoma when infected with the{Delta} Ov-grn-1 flukes compared to controls. The clinically-relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.


title: Programmed knockout mutation of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic opisthorchiasis


title: Multiple ETS Factors Participate in the Transcriptional Control of TERT Mutant Promoter in Thyroid Cancers

abstract:
Hotspot mutations in the TERT (telomerase reverse transcriptase) gene are key determinants of thyroid cancer progression. TERT promoter mutations (TPM) create de novo consensus binding sites for the ETS ("E26 transforming sequence") family of transcription factors. In this study, we systematically knocked down each of the 20 ETS factors expressed in thyroid tumors and screened their effects on TERT expression in seven thyroid cancer cell lines with defined TPM status. We observed that, unlike in other TPM-carrying cancers such as glioblastomas, ETS factor GABPA does not unambiguously regulate transcription from the TERT mutant promoter in thyroid specimens. In fact, multiple members of the ETS family impact TERT expression, and they typically do so in a mutation-independent manner. In addition, we observe that partial inhibition of MAPK, a central pathway in thyroid cancer transformation, is more effective at suppressing TERT transcription in the absence of TPMs. Taken together, our results show a more complex scenario of TERT regulation in thyroid cancers compared to other lineages, suggest that compensatory mechanisms by ETS and other regulators likely exist and advocate for the need of a more comprehensive understanding of the mechanisms of TERT deregulation in thyroid tumors before eventually exploring TPM-specific therapeutic strategies.

Graphical Abstract

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title: Improving genomically recoded Escherichia coli for the production of proteins containing non-canonical amino acids

abstract: A genomically recoded Escherichia coli strain that lacks all amber codons and release factor 1 (C321.{Delta}A) enables efficient genetic encoding of chemically diverse, non-canonical amino acids (ncAAs) into proteins. While C321.{Delta}A has opened new opportunities in chemical and synthetic biology, this strain has not been optimized for protein production, limiting its utility in widespread industrial and academic applications. To address this limitation, we describe the construction of a series of genomically recoded organisms that are optimized for cellular protein production. We demonstrate that the functional deactivation of nucleases (e.g., rne, endA) and proteases (e.g., lon) increases production of wild-type superfolder green fluorescent protein (sfGFP) and sfGFP containing two ncAAs up to [~]5-fold. Additionally, we introduce a genomic IPTG-inducible T7 RNA polymerase (T7RNAP) cassette into these strains. Using an optimized platform, we demonstrated the ability to introduce 2 identical N6-(propargyloxycarbonyl)-L-Lysine residues site specifically into sfGFP with a 17-fold improvement in production relative to the parent. We envision that our library of organisms will provide the community with multiple options for increased expression of proteins with new and diverse chemistries.

category: synthetic biology


title: Synthetic Condensed-Phase Signaling Expands Kinase Specificity and Responds to Macromolecular Crowding

abstract: Liquid-liquid phase separation (LLPS) can concentrate biomolecules and accelerate reactions within membraneless organelles. For example, the nucleolus and PML-nuclear bodies are thought to create network hubs by bringing signaling molecules such as kinases and substrates together. However, the mechanisms and principles connecting mesoscale organization to signaling dynamics are difficult to dissect due to the pleiotropic effects associated with disrupting endogenous condensates. Here, we recruited multiple distinct kinases and substrates into synthetic LLPS systems to create new phosphorylation reactions within condensates, and generally found increased activity and broadened specificity. Dynamic phosphorylation within condensates could drive cell-cycle-dependent localization changes. Detailed comparison of phosphorylation of clients with varying recruitment valency and affinity into condensates comprised of either flexible or rigid scaffolds revealed unexpected principles. First, high client concentration within condensates is important, but is not the main factor for efficient multi-site phosphorylation. Rather, the availability of a large number of excess client binding sites, together with a flexible scaffold is crucial. Finally, phosphorylation within a suboptimal, flexible condensate was modulated by changes in macromolecular crowding. Thus, condensates readily generate new signaling connections and can create sensors that respond to perturbations to the biophysical properties of the cytoplasm.


title: Constructing synthetic-protein assemblies from de novo designed 310 helices

abstract: Compared with the iconic  helix, 310 helices occur much less frequently in protein structures. The different 310-helical parameters lead to energetically less favourable internal energies, and a reduced tendency to pack into defined higher-order structures. Consequently, in natural proteins, 310 helices rarely extend past 6 residues, and do not form regular supersecondary, tertiary, or quaternary interactions. Here, we show that despite their absence in nature, synthetic protein-like assemblies can be built from 310 helices. We report the rational design, solution-phase characterisation, and an X-ray crystal structure for water-soluble bundles of 310 helices with consolidated hydrophobic cores. The design uses 6-residue repeats informed by analysing natural 310 helices, and incorporates aminoisobutyric acid residues. Design iterations reveal a tipping point between -helical and 310-helical folding, and identify features required for stabilising assemblies in this unexplored region of protein-structure space.


title: The protein organization of a red blood cell

abstract: Red blood cells (RBCs, erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles, and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we defined a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validated protein complexes through electron microscopy and chemical crosslinking, and with these data, built 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-link compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research.

category: systems biology


title: Develop an efficient and specific AAV-based labeling system for Muller glia in mice

abstract: Cell degeneration in the retina leads to several ocular diseases and vision loss. Considerable research efforts focus on reprogramming Muller glia (MG) into functional cells to rescue vision as a promising therapeutic strategy, although whether MG can convert into functional bipolar cells, retinal ganglia cells (RGCs), rods or cones in mammals remains controversial. The broad applicability of tracking MG differentiation thus presents a need for improved labeling efficiency and specificity. Here, we compared AAV-based labeling strategies with conventional lineage-tracking by crossing transgenic mouse lines. We found that reporter expression was weak and not MG-specific in mGFAP-Cre transgenic mice. Different AAV serotypes showed a range of efficiency and specificity in labeling MG, leading us to optimize a human GFAP-Cre reporter system packaged in the AAV9 serotype with the WPRE (WPRE, woodchuck hepatitis virus post-transcriptional regulatory element) removed. The hGFAP-Cre- {Delta}WPRE reporter could label 20-73.8% MGs, with non-specific RGC labeling rates ranging from 0-0.08% at doses of 1 x 108 to 1010 vector genomes (vg) per eye, an approximate 40-fold reduction compared with the AAV9-hGFAP-Cre-WPRE labeling system. Furthermore, we validated the availability of the label system to trace MG reprogramming and also detected false-positive results reported previously. The AAV9-hGFAP-Cre-{Delta}WPRE system thus represents a highly efficient and specific labeling system for MG, providing a valuable tool for tracking cell fate in vivo.


title: Transcriptomic heterogeneity of Sox2-expressing pituitary cells

abstract: The mammalian pituitary gland is a complex organ consisting of hormone-producing cells (HPC), nonhormonal folliculostellate cells (FSC) and pituicytes, vascular pericytes and endothelial cells, and putative Sox2-expressing stem cells. Here, we used scRNAseq analysis of adult female rat pituitary cells to study the heterogeneity of pituitary cells with a focus on evaluating the transcriptomic profile of the Sox2-expressing population. Samples containing whole pituitary and separated anterior and posterior lobe cells allowed the identification of all expected pituitary resident cell types and lobe-specific subpopulations of vascular cells. Sox2 was expressed uniformly in all FSC, pituicytes, and a fraction of HPC. FSC comprised two subclusters; FSC1 contained more cells but expressed less genetic diversity compared to FSC2. The latter contained proliferative cells, expressed genes consistent with stem cell niche formation, including tight junctions, and shared genes with HPC. The FSC2 transcriptome profile was also consistent with the activity of pathways regulating cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. The expression of other stem cell marker genes was common for FSC and pituicytes (Sox9, Cd9, Hes1, Vim, S100b) or cell type-specific (FSC: Prop1, Prrx1, Pitx1, Pitx2, Lhx3; pituicytes: Fgf10, Tbx3, Lhx2, Nkx2-1, Rax). FSC and pituicytes also expressed other astroglial marker genes, some common and other distinct, consistent with their identities as astroglial cells of the pituitary. These data suggest functional heterogeneity of FSC, with a larger fraction representing classical FSC, and a smaller fraction containing active stem-like cells and HPC-committed progenitors.


title: Cerebrospinal fluid formation is controlled by membrane transporters to modulate intracranial pressure

abstract: Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP. Here, we employed experimental rats to demonstrate low osmotic water permeability of the choroid plexus, lack of an osmotic gradient across this tissue, and robust CSF secretion against osmotic gradients. Together, these results illustrate that CSF secretion occurs independently of conventional osmosis, which challenges the existing assumption that CSF production is driven entirely by bulk osmotic forces across the CSF-secreting choroid plexus. Instead, we reveal that the choroidal Na+/K+/Cl- cotransporter NKCC1, Na+/HCO3- cotransporter NBCe2, and Na+/K+-ATPase are actively involved in CSF production and propose a molecular mode of water transport supporting CSF production in this secretory tissue. Further, we demonstrate that inhibition of NKCC1 directly reduces the ICP, illustrating that altered CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP.


title: Innate lymphoid cells lacking surface expression of CD90 are functional

abstract: Huge progress has been made in understanding the biology of innate lymphoid cells (ILC) by adopting several well-known concepts of T cell biology. As such flow cytometry gating strategies and markers, such as CD90, to identify ILC were discovered. Here we report that most non-NK intestinal ILC have a high expression of CD90 as expected, but surprisingly some have only a low or even no expression of this marker. CD90-negative CD127+ ILC were identified among all ILC subsets in the gut. CD90-negative cLP ILC2 were frequent at steady state. The frequency of CD90-negative CD127+ ILC was dependent on stimulatory cues in vitro and in vivo, and CD90-negative CD127+ ILC played a functional role as a source of IL-13, IFN{gamma} and IL-17A at steady state and upon dextran sulphate sodium-elicited colitis. Hence, this study highlights for the first time that CD90 is not constitutively expressed by functional ILC in the gut.

category: immunology


title: Proteome Microarray Screening Identifies Human Polyphosphate-Binding Proteins in the Phosphatidylinositol Signaling Pathway

abstract: Polyphosphates are linear chains of orthophosphate residues that are present in all living cells. Polyphosphates are released from platelet d-granules and are also produced in bacteria. Polyphosphates are procoagulant in mammalian species and in bacteria are required for energy and phosphate storage, stress resistance, chelation of metal ions and escaping host immunity. Despite these pleiotropic effects, sparse information is available on molecular binding partners of polyphosphates. Here, we used a slide-based human proteome microarray screen for the search of polyphosphate-binding proteins. This approach suggested several novel proteins with relation to the phosphatidylinositol signaling pathway. The highest signals were obtained for Disabled-1 (DAB1) and phosphatidylinositol-5-phosphate 4-kinase 2B (PIP4K2B). Isothermal titration calorimetry was used for confirmation of DAB1 interactions with long-chain polyphosphates. These results offer new rationale to further investigate the interference of polyphosphates with intracellular signaling pathways.


title: Mitochondrial fission dysfunction alleviates heterokaryon incompatibility-triggered cell death in the industrial filamentous fungus Aspergillus oryzae

abstract:
In filamentous fungi, cell-to-cell recognition is a fundamental requirement for the formation, development, and maintenance of complex hyphal networks. Basically, self/compatible individuals within the fungal species are capable of fusing together, a step important for crossbreeding, which results in the formation of viable vegetative heterokaryons. Conversely, the fusion of incompatible individuals does not result in the formation of viable hyphal networks, but it often leads to growth inhibition or cell death. Even though a number of studies have been conducted to investigate such incompatibility, the understanding of the associated molecular mechanism is still limited, and this restricts the possibility of crossbreeding incompatible individuals. Therefore, in this study, the characteristics of compatibility/incompatibility in the industrial filamentous fungus, Aspergillus oryzae, were comprehensively investigated. Protoplast fusion and co-culture assays indicated the existence of a correlation between strain phylogeny and compatibility/incompatibility features. Time-course fluorescence observations were employed to investigate the types of incompatible responses that are induced at different cellular levels upon incompatible cell fusion, which eventually lead to cell death. Propidium iodide-indicated cell death, ROS accumulation, and mitochondrial fragmentation were identified as the major responses, with mitochondrial fragmentation showing the most significant subcellular change immediately after incompatible cell fusion. Furthermore, the deletions of mitochondrial fission-related genes Aofis1 and Aodnm1 in incompatible pairing alleviated cell death, indicating that mitochondrial fission is an important mechanism by which incompatibility-triggered cell death occurs. Therefore, this study provides new insights about heterokaryon incompatibility.

IMPORTANCEFor a long time, it was believed that as an asexual fungus, A. oryzae does not exhibit any sexual cycle. However, the fungus has two mating types, indicating the potential for sexual reproduction besides a known parasexual cycle. Therefore, given that viable heterokaryon formation following cell fusion is an important step required for genetic crossing, we explored the mechanism of incompatibility, which restricts the possibility of cell fusion in A. oryzae. Protoplast fusion and co-culture assays led to the identification of various vegetative compatible groups. Mitochondrial fragmentation was found to be the most significant incompatible cellular response that occurred in organelles during incompatible pairing, while the deletion of mitochondrial fission-related genes was identified as a strategy used to alleviate incompatibility-triggered cell death. Thus, this study revealed a novel mechanism by which mitochondrial fission regulates incompatible responses.


title: Molecular architecture of 40S initiation complexes on the Hepatitis C virus IRES: from ribosomal attachment to eIF5B-mediated reorientation of initiator tRNA

abstract: Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Direct binding of the IRES to the 40S subunit places the initiation codon into the P site, where it base-pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Then, eIF5 and eIF5B mediate subunit joining. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. Here, we present cryo-EM structures of IRES initiation complexes at resolutions up to 3.5 [A] that cover all major stages from initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role for IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes that prepare them for subunit joining.

category: molecular biology


title: Circadian clock function does not require the histone methyltransferase MLL3

abstract:
AO_SCPLOWBSTRACTC_SCPLOWThe circadian clock controls the physiological function of tissues through the regulation of thousands of genes in a cell-type specific manner. The core cellular circadian clock is a transcription-translation negative feedback loop, which can recruit epigenetic regulators to facilitate temporal control of gene expression. Histone methyltransferase, mixed lineage leukemia gene 3 (MLL3) was reported to be required for maintenance of circadian oscillations in cultured cells. Here, we test the role of MLL3 in circadian organisation in whole animals. Using mice expressing catalytically inactive MLL3, we show that MLL3 methyltransferase activity is in fact not required for circadian oscillations in vitro in a range of tissues, nor for maintenance of circadian behavioural rhythms in vivo. In contrast to a previous report, loss of MLL3-dependent methylation did not affect global levels of H3K4 methylation in liver, indicating substantial compensation from other methyltransferases. Further, we found little evidence of genomic repositioning of H3K4me3 marks. We did, however, observe repositioning of H3K4me1 from intronic regions to intergenic regions and gene promoters, however there were no changes in H3K4me1 mark abundance around core circadian clock genes. Output functions of the circadian clock, such as control of inflammation, were largely intact in MLL3-methyltransferase deficient mice, although some gene specific changes were observed, with sexually dimorphic loss of circadian regulation of specific cytokines. Taken together, these observations call for a major reassessment of the inter-relationship between the circadian clock and MLL3-directed histone methylation, and a deeper examination of other epigenetic mechanisms which may facilitate circadian clock function.

SO_SCPLOWIGNIFICANCEC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSTATEMENTC_SCPLOWA highly cited paper published in PNAS previously reported an essential role for the histone methyltransferase MLL3 in maintaining circadian oscillations in cultured cells.

We tested the role of MLL3 in vivo and in primary tissues showing that MLL3 in fact plays no role in organising the core circadian clock, and has no functional impact on whole animal circadian behaviour. However, in further analysis, we newly discover a role for MLL3 in conferring circadian control to components of the inflammatory response, doing so in a sexually dimorphic manner. As the MLL family of histone methyltransferases are being targeted by pharmaceuticals for cancer, it is important to understand how methyltransferases may be driving circadian rhythms in gene expression.


title: Polyclonal pathogen populations accelerate the evolution of antibiotic resistance in patients

abstract:
Antibiotic resistance poses a global health threat, but the within-host drivers of resistance remain poorly understood. Pathogen populations are often assumed to be clonal within hosts, and resistance is thought to emerge due to selection for de novo variants. Here we show that pulmonary populations of the opportunistic pathogen P. aeruginosa are often polyclonal. Crucially, resistance evolves rapidly in patients colonized by polyclonal populations through selection for pre-existing resistant strains. In contrast, resistance evolves sporadically in patients colonized by monoclonal populations due to selection for novel resistance mutations. However, strong trade-offs between resistance and fitness occur in polyclonal populations that can drive the loss of resistant strains. In summary, we show that the within-host diversity of pathogen populations plays a key role in shaping the emergence of resistance in response to treatment.

One sentence summaryAntibiotic resistance evolves quickly in patients colonized by polyclonal pathogen populations.

category: evolutionary biology


title: Evolution of plasmid mobility: origin and fate of non-conjugative plasmids

abstract: Conjugation drives horizontal gene transfer of many adaptive traits across prokaryotes. Yet, only a fourth of the plasmids encode the functions necessary to conjugate autonomously, others being non-mobile or mobilizable by other elements. How these different plasmids evolve is poorly understood. Here, we studied plasmid evolution in terms of their gene repertoires and relaxases. We observed that gene content in plasmid varies rapidly in relation to the rate of evolution of relaxases, such that plasmids with 95% identical relaxases have on average fewer than 50% of homologs. The identification of 249 recent transitions in terms of mobility types revealed that they are associated with even greater changes in gene repertoires, possibly mediated by transposable elements that are more abundant in such plasmids. These changes include pseudogenization of the conjugation locus, exchange of replication initiators, and extensive gene loss. In some instances, the transition between mobility types also leads to the genesis of novel plasmid taxonomic units. Most of these transitions are short-lived, suggesting a source-sink dynamic, where conjugative plasmids constantly generate mobilizable and putatively non-mobilizable plasmids by gene deletion. Yet, in few cases such transitions resulted in the emergence of large clades of relaxases present only in mobilizable plasmids, suggesting successful specialization of these families in the hijacking of diverse conjugative systems. Our results shed further light on the huge plasticity of plasmids, suggest that many non-conjugative plasmids emerged recently from conjugative elements and allowed to quantify how changes in plasmid mobility shape the variation of their gene repertoires.


title: The 3D architecture of the pepper (Capsicum annum) genome and its relationship to function and evolution

abstract: The architecture of topologically associating domains (TADs) varies across plant genomes. Understanding the functional consequences of this diversity requires insights into the pattern, structure, and function of TADs. Here, we present a comprehensive investigation of the 3D genome organization of pepper (Capsicum annuum) and its association with gene expression and genomic variants. We report the first chromosome-scale long-read genome assembly of pepper and generate Hi-C contact maps for four tissues. The contact maps indicate that 3D structure varies somewhat across tissues, but generally the genome was segregated into subcompartments that were correlated with transcriptional state. In addition, chromosomes were almost continuously spanned by TADs, with the most prominent found in large genomic regions that were rich in retrotransposons. A substantial fraction of TAD boundaries were demarcated by chromatin loops, suggesting loop extrusion is a major mechanism for TAD formation; many of these loops were bordered by genes, especially in highly repetitive regions, resulting in gene clustering in three dimensional space. Integrated analysis of Hi-C profiles and transcriptomes showed that change in 3D chromatin structures (e.g. subcompartments, TADs, and loops) was not the primary mechanism contributing to differential gene expression between tissues, but chromatin structure does play a role in transcription stability. TAD boundaries were significantly enriched for breaks of synteny and depletion of sequence variation, suggesting that TADs constrain patterns of genome structural evolution in plants. Together, our work provides insights into principles of 3D genome folding in large plant genomes and its association with function and evolution.

category: genomics


title: Chromosome-level genome assembly of the functionally extinct northern white rhinoceros (Ceratotherium simum cottoni).

abstract: The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is functionally extinct, with only two females remaining alive. Efforts to rescue the NWR have inspired the exploration of unconventional conservation methods, including the generation of artificial gametes from induced pluripotent stem cells and somatic cell nuclear transfer. To enable the technologies required for these approaches, we used complementary sequencing and mapping methods to generate a NWR chromosome-level reference genome that meets or exceeds the metrics proposed by the Vertebrate Genome Project. It represents 40 autosomes, an X and a partially-resolved Y chromosome, and the mitochondrial genome. We compared the NWR reference genome to the southern white rhinoceros (SWR) population that has been physically separated from the NWR for tens of thousands of years. Using short-read data from the SWR and optical mapping, we found that the two populations are very similar on both the chromosome level and mitochondrial genome level. The results of this study are encouraging for the efforts underway to rescue the NWR.


title: Geometrical frustration of phase-separated domains in Coscinodiscus diatom frustules

abstract: Diatoms are single-celled organisms with a cell wall made of silica, called the frustule. Even though their elaborate patterns have fascinated scientists for years, little is known about the biological and physical mechanisms underlying their organization. In this work, we take a top-down approach and examine the micron-scale organization of diatoms from the Coscinodiscus family. We find two competing tendencies of organization, which appear to be controlled by distinct biological pathways. On one hand, micron-scale pores organize locally on a triangular lattice. On the other, lattice vectors tend to point globally toward a center of symmetry. This competition results in a frustrated triangular lattice, populated with geometrically necessary defects whose density increases near the center.

category: biophysics


title: Single Molecule Analysis of differential functional mechanisms of MtRecG while being exposed to variants of stalled replication-fork

abstract: Helicases are motor proteins involved in multiple activities to carry out manipulation of the nucleic acids for efficient gene regulation. In case of roadblocks that can lead the replication machinery to get halted, a complex molecular surveillance system utilizing helicases as its key player ensures the halted fork to resume its duplication process. RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. Here, through adoption of single-molecule techniques we have attempted to probe the DNA unwinding features by RecG and tried to capture several stages of genetic rearrangement. An elevated processivity of RecG has been observed for the kinds of stalled fork where progression of lagging daughter strand is ahead than that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. In summary, we have featured that RecG adopts asymmetric mode of locomotion to unwind the lagging daughter strand to facilitate Holliday junction creation which acts as a suitable intermediate for recombinational repair pathway.


title: Genetic Subtypes of Smoldering Multiple Myeloma are associated with Distinct Pathogenic Phenotypes and Clinical Outcomes

abstract: Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with significant heterogeneity in disease progression. Existing clinical models of progression risk do not fully capture this heterogeneity. Here we integrated 42 genetic alterations from 214 SMM patients using unsupervised binary matrix factorization (BMF) clustering and identified six distinct genetic subtypes. These subtypes were differentially associated with established MM-related RNA signatures, oncogenic and immune transcriptional profiles, and evolving clinical biomarkers. Three subtypes were associated with increased risk of progression to active MM in both the primary and validation cohorts, indicating they can be used to better predict high and low-risk patients within the currently used clinical risk stratification model.


title: 3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib

abstract: Osimertinib sensitive and resistant NSCLC NCI-H1975 clones were used to model osimertinib acquired resistance in humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation were found in resistant clones. Resistant tumors in humanized mice were initially partially responsive to osimertinib, then aggressive tumor regrowth occurred accompanied by an immunosuppressive tumor microenvironment. 3-phosphoinositide-dependent kinase 1 (PDK1) was identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitized resistant clones and a patient derived xenograft (PDX) with acquired resistance to osimertinib. PDK1 knock-out dysregulated PI3K/Akt/mTOR signaling, promoted cell cycle arrest at the G1 phase, and inhibited nuclear translocation of yes-associated protein (YAP). Higher expression of PDK1 was found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP.


title: A mechanistic investigation of enhanced nitrogen use efficiency in wheat seedlings after treatment with a precision engineered Ascophyllum nodosum biostimulant

abstract: Reduction in the emissions of the greenhouse gas nitrous oxide and nitrogen (N) pollution of ground water by improving nitrogen use efficiency (NUE) in crops is urgently required in pursuit of a sustainable agricultural future. Utilising an engineered biostimulant (PSI-362) derived from the brown seaweed Ascophyllum nodosum, we examined its effect on wheat seedling growth dynamics and mechanistic spatiotemporal changes at transcriptional and biochemical levels in relation to N uptake, assimilation and NUE. PSI-362-mediated biomass increase was associated with increased nitrate uptake and N assimilation in the form of glutamate, glutamine, free amino acids, soluble proteins and total chlorophyll. Phenotypical and biochemical analysis were supported by evaluation of differential expression of genetic markers involved in nitrate perception and transport (TaNRT1.1/NPF6.3), and assimilation (TaNR1 and TaNiR1, TaGDH2, TaGoGAT, TaGS1). Finally, a comparative analysis of the PSI-362 and two generic Ascophyllum nodosum extracts (ANEs) demonstrated that the NUE effect greatly differs depending on the ANE biostimulant used. In the current context of climate warming the transition of agriculture to a more sustainable model is urgently required. Application and adoption of precision biostimulants creates an opportunity for sustainable crop management, reduced production cost and environmental pollution, while maintaining yields.

category: plant biology


title: Leptin-mediated suppression of food intake by conserved Glp1r-expressing neurons prevents obesity

abstract: The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A previously unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-sequencing of enriched mouse hypothalamic LepRb cells, as well as with total hypothalamic cells from multiple mammalian species. In addition to identifying known LepRb neuron types, this analysis identified several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations display strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons that express more Lepr and respond more robustly to exogenous leptin than other LepRb populations. Ablating LepRb from these cells provoked hyperphagic obesity without impairing energy expenditure. Conversely, reactivating LepRb in Glp1r-expressing cells decreased food intake and body weight in otherwise LepRb-null mice. Furthermore, LepRb reactivation in GABA neurons improved energy balance in LepRb-null mice, and this effect required the expression of LepRb in GABAergic Glp1r-expressing neurons. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the control of food intake and body weight by leptin.


title: A new animal model signifying a decisive bridge between innate immunity and the pathognomonic morphological characteristics of Type 1 Diabetes

abstract:
Available animal models for Type 1 Diabetes (T1D) show limited similarities with the human disease and have no predictive value in screening for effective intervention therapies. Heat-inactivated bacteria instilled in the ductal compartment of the pancreas in healthy rats rapidly cause periductal inflammation and accumulation of mainly granulocytes and monocytes in the exocrine pancreas and in the peri-islet area mimicking the acute pancreatic inflammation in subjects with recent onset T1D. After three weeks, the triggered exocrine inflammation had vanished and pancreases showed normal morphology. However, a distinct accumulation of both CD4+ and CD8+ T cells within and adjacent to affected islets was found in one third of the rats, mimicking the pathognomonic insulitis in T1D. As in human T1D, the insulitis affected a fraction of all islets and was observed only in certain lobes of the pancreases. The presented animal model for T1D will allow detailed mechanistic studies to unravel a previously unknown interplay between bacteria-activated innate immunity and an acquired cellular immunity forming the immunopathological events described in humans at different stages of T1D.

Summary StatementThe results presented signify a previously unknown decisive bridge between innate immunity and formation of the pathognomonic immunopathological events described in subjects with recent onset T1D.


title: Myosin phosphatase target subunit 1 governs integrity of the embryonic gut epithelium to circumvent atresia development in medaka, Oryzias latipes

abstract:
BackgroundIntestinal atresia (IA) is a congenital gut obstruction caused by the absence of gut opening. Genetic factors are assumed to be critical for the development of IA, in addition to accidental vascular insufficiency or mechanical strangulation. However, the molecular mechanism underlying IA remains poorly understood.

ResultsIn this study, to better understand such a mechanism, we isolated a mutant of Oryzias latipes (the Japanese rice fish known as medaka) generated by N-ethyl-N-nitrosourea mutagenesis, in which IA develops during embryogenesis. Positional cloning identified a nonsense mutation in the myosin phosphatase target subunit 1 (mypt1) gene. Consistent with known Mypt1 function, the active form of myosin regulatory light chain (MRLC), which is essential for actomyosin contraction, and F-actin were ectopically accumulated in the intestinal epithelium of mutant embryos, whereas cell motility, proliferation and cell death were not substantially affected. Corresponding to the accumulation site of F-actin/active MRLC, the intestinal epithelium architecture was disordered. Importantly, blebbistatin, a non-muscle myosin inhibitor, attenuated the development of IA in the mutant.

ConclusionsCytoskeletal contraction governed by mypt1 regulates the integrity of the embryonic intestinal epithelium. This study provides new insight into our understanding of the mechanism of IA development in humans.

Bullet PointsO_LIMedaka mypt1 mutants display intestinal atresia.
C_LIO_LIThe level of phosphorylated myosin regulatory light chain was higher in mypt1 mutant embryos than in wild-type embryos.
C_LIO_LIThe levels of F-actin appeared elevated in the intestinal epithelium of mypt1 mutants.
C_LIO_LIBlebbistatin, an inhibitor of non-muscle myosin II, rescued intestinal atresia in mypt1 mutant embryos.
C_LI

category: developmental biology


title: The population genetics of adaptation through copy-number variation in a fungal plant pathogen

abstract: Microbial pathogens can rapidly adapt to changing environments such as the application of pesticides or host resistance. Copy number variations (CNV) are a major source of adaptive genetic variation for recent adaptation. Here, we analyze how a major fungal pathogen of barley, Rhynchosporium commune, has adapted to host environment, fungicide and temperature challenges. We screen the genomes of 126 isolates sampled across a worldwide set of populations and identify a total of 7879 gene duplications and 116 gene deletions. Most gene duplications result from segmental chromosomal duplications. We find that genes showing recent gains or losses are enriched in functions related to host exploitation (i.e. effectors and cell wall degrading enzymes). We perform a phylogeny-informed genome-wide association study (GWAS) and identify 191 copy-number variants associated with different pathogenesis and temperature related traits, including a large segmental duplication of CYP51A that has contributed to the emergence of azole resistance. Additionally, we use a genome-wide SNP dataset to replicate the GWAS and contrast it with the CNV-focused analysis. We find that frequencies of adaptive CNV alleles show high variation among populations for traits under strong selection such as fungicide resistance. In contrast, adaptive CNV alleles underpinning temperature adaptation tend to be near fixation. Finally, we show that transposable elements are important drivers of recent gene copy-number variation. Loci showing signatures of recent positive selection are enriched in miniature inverted repeat transposons. Our findings show how extensive segmental duplications create the raw material for recent adaptation in global populations of a fungal pathogen.


title: Human pathogenic RNA viruses establish non-competing lineages by occupying independent niches

abstract:
Many pathogenic viruses are endemic among human populations and can cause a broad variety of diseases, some potentially leading to devastating pandemics. How virus populations maintain diversity and what selective pressures drive population turnover, is not thoroughly understood. We conducted a large-scale phylodynamic analysis of 27 human pathogenic RNA viruses spanning diverse life history traits in search of unifying trends that shape virus evolution. For most virus species, we identify multiple, co-circulating lineages with low turnover rates. These lineages appear to be largely noncompeting and likely occupy semi-independent epidemiological niches that are not regionally or seasonally defined. Typically, intra-lineage mutational signatures are similar to inter-lineage signatures. The principal exception are members of the family Picornaviridae, for which mutations in capsid protein genes are primarily lineage-defining. The persistence of virus lineages appears to stem from limited outbreaks within small communities so that only a minor fraction of the global susceptible population is infected at any time. As disparate communities become increasingly connected through globalization, interaction and competition between lineages might increase as well, which could result in changing selective pressures and increased diversification and/or pathogenicity. Thus, in addition to zoonotic events, ongoing surveillance of familiar, endemic viruses appears to merit global attention with respect to the prevention or mitigation of future pandemics.

SignificanceNumerous pathogenic viruses are endemic in humans and cause a broad variety of diseases, but what is their potential of causing new pandemics? We show that most human pathogenic RNA viruses form multiple, co-circulating lineages with low turnover rates. These lineages appear to be largely noncompeting and occupy distinct epidemiological niches that are not regionally or seasonally defined, and their persistence appears to stem from limited outbreaks in small communities so that a minor fraction of the global susceptible population is infected at any time. However, due to globalization, interaction and competition between lineages might increase, potentially leading to increased diversification and pathogenicity. Thus, endemic viruses appear to merit global attention with respect to the prevention of future pandemics.


title: Extend Mixed Models to Multi-layer Neural Networks for Genomic Prediction Including Intermediate Omics Data

abstract: With the growing amount and diversity of intermediate omics data complementary to genomics (e.g., DNA methylation, gene expression, and protein abundance), there is a need to develop methods to incorporate intermediate omics data into conventional genomic evaluation. The omics data helps decode the multiple layers of regulation from genotypes to phenotypes, thus forms a connected multi-layer network naturally. We developed a new method named NN-LMM to model the multiple layers of regulation from genotypes to intermediate omics features, then to phenotypes, by extending conventional linear mixed models ("LMM") to multi-layer artificial neural networks ("NN"). NN-LMM incorporates intermediate omics features by adding middle layers between genotypes and phenotypes. Linear mixed models (e.g., pedigree-based BLUP, GBLUP, Bayesian Alphabet, single-step GBLUP, or single-step Bayesian Alphabet) can be used to sample marker effects or genetic values on intermediate omics features, and activation functions in neural networks are used to capture the nonlinear relationships between intermediate omics features and phenotypes. NN-LMM had significantly better prediction performance than the recently proposed single-step approach for genomic prediction with intermediate omics data. Compared to the single-step approach, NN-LMM can handle various patterns of missing omics measures, and allows nonlinear relationships between intermediate omics features and phenotypes. NN-LMM has been implemented in an open-source package called "JWAS".

category: genetics


title: Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology

abstract: NADK2 encodes the mitochondrial isoform of NAD Kinase, which phosphorylates nicotinamide adenine dinucleotide (NAD). Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here we describe two chemically-induced mouse mutations in Nadk2, S326L and S330P, which cause a severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5 weeks of age and muscle atrophy by 11 weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on brain, muscle, liver, and spinal cord at the same ages. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency, and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNAseq and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, such as translation, and those processes that are gene-specific such as glycolysis and acetylcholine binding. These findings improve our understanding of the pathophysiology of both Nadk2 and Pla2g6 mutations, as well as pathways common to neuromuscular/neurodegenerative diseases.


title: Phylogenomic analyses of Snodgrassella isolates from honeybees and bumblebees reveals taxonomic and functional diversity

abstract:
Snodgrassella is a Betaproteobacteria genus found in the gut of honeybees (Apis spp.) and bumblebees (Bombus spp). It is part of a conserved microbiome that is composed of few core phylotypes and is essential for bee health and metabolism. Phylogenomic analyses using whole genome sequences of 75 Snodgrassella strains from 4 species of honey bees and 14 species of bumblebees showed that these strains formed a monophyletic lineage within the Neisseriaceae family, that Snodgrassella isolates from Asian honeybees diverged early on from the other species in their evolution, that isolates from honeybees and bumblebees were well separated and that this genus consists of at least seven species. We propose to formally name two new Snodgrassella species that were isolated from bumblebees, i.e. Snodgrassella gandavensis sp. nov. and Snodgrassella communis sp. nov. Possible evolutionary scenarios for 107 species or group specific genes revealed very limited evidence for horizontal gene transfer. Functional analyses revealed the importance of small proteins, defense mechanisms, amino acid transport and metabolism, inorganic ion transport and metabolism and carbohydrate transport and metabolism among these 107 specific genes.

ImportanceThe microbiome of honeybees (Apis spp.) and bumblebees (Bombus spp.) is highly conserved and represented by few phylotypes. This simplicity in taxon composition makes the bees microbiome an emergent model organism for the study of gut microbial communities. Since the description of the Snodgrassella genus, which was isolated from the gut of honeybees and bumblebees in 2013, a single species, i.e. Snodgrassella alvi, has been named. Here we demonstrate that this genus is actually composed of at least seven species, two of them (Snodgrassella gandavensis sp. nov. and Snodgrassella communis sp. nov.) being formally described in the present publication. We also report the presence of 107 genes specific to Snodgrassella species, showing notably the importance of small proteins and defense mechanisms in this genus.

Data summaryO_LICornet L and Vandamme P, European Nucleotide Archive (ENA), Project accession: PRJEB47378
C_LIO_LICornet L and Vandamme P, European Nucleotide Archive (ENA), Reads accessions: SAMEA9570070 - SAMEA9570078
C_LIO_LICornet L and Vandamme P, European Nucleotide Archive (ENA), Genome accessions: GCA_914768015, GCA_914768025, GCA_914768035, GCA_914768045, GCA_914768055, GCA_914768065, GCA_914768075, GCA_914768085, GCA_914768095.
C_LI


title: Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

abstract: ATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.


title: Manipulating Histone Acetylation Leads to Adverse Effects in Hemangiosarcoma Cells

abstract: Canine hemangiosarcoma (HSA) is a malignant tumour derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. SAHA and VPA treatment in HSA cell lines upregulated inflammatory-related genes, thereby attracting macrophages. This implies that SAHA and VPA can induce anti-tumour immunity. JQ1 stimulated autophagy and inhibited the cell cycle. Finally, JQ1 suppressed HSA tumour cell proliferation in vivo. These results suggest that HDACi and BETi can be alternative drugs for HSA treatment. Although further research is required, this study provides useful insights for developing new treatments for HSA.