title: Organ-specific microbiota enhances the terrestrial lifestyle of a brachyuran crab

abstract: Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land.


title: Conserved organ-specific microbial assemblages in different populations of a terrestrial crab

category: zoology


title: Sleep Slow Oscillations and Spindles encode ocular dominance plasticity and promote its consolidation in adult humans

abstract: Sleep and plasticity are highly interrelated, as sleep slow oscillations and sleep spindles are associated with consolidation of Hebbian-based processes. However, in adult humans, visual cortical plasticity is mainly sustained by homeostatic mechanisms, for which the role of sleep is still largely unknown. Here we demonstrate that non-REM sleep stabilizes homeostatic plasticity of ocular dominance in adult humans. We found that the effect of short-term monocular deprivation (boost of the deprived eye) was preserved at the morning awakening (>6 hours after deprivation). Subjects exhibiting stronger consolidation had increased sleep spindle density in frontopolar electrodes, suggesting distributed consolidation processes. Crucially, the individual susceptibility to visual homeostatic plasticity was encoded by changes in sleep slow oscillation rate and shape and spindle power in occipital sites, consistent with an early visual cortical site of ocular dominance homeostatic plasticity.

category: neuroscience


title: Modulation of taste sensitivity by the olfactory system in Drosophila

abstract: An animals sensory percepts are not raw representations of the outside world. Rather, they are constructs influenced by many factors including the species, past experiences, and internal states. One source of perceptual variability that has fascinated researchers for decades is the effect of losing one sensory modality on the performance of another1. Typically, dysfunction of one sense has been associated with elevated function of others, creating a type of sensory homeostasis2. For example, people with vision loss have been reported to demonstrate enhanced tactile and auditory functions, and deafness has been associated with heightened attention to visual inputs for communication3,4. By contrast, smell and taste--the two chemosensory modalities--are so intrinsically linked in their contributions to flavor that loss of smell is often anecdotally reported as leading to deficiencies in taste5-8. However, human studies specifically examining taste are mixed and generally do not support this widely-held belief, and data from animal models is largely lacking9. Here, we examine the impact of olfactory dysfunction on taste sensitivity in Drosophila melanogaster. We find that partial loss of olfactory input (hyposmia) dramatically enhances flies sensitivity to both appetitive (sugar, low salt) and aversive (bitter, high salt) tastes. This taste enhancement is starvation-independent and occurs following suppression of either first- or second-order olfactory neurons. Moreover, optogenetically increasing olfactory inputs reduces taste sensitivity. Finally, we observed that taste enhancement is not encoded in the activity of peripheral gustatory sensory neurons, but is associated with elevated sugar responses in protocerebrum anterior medial (PAM) dopaminergic neurons of the mushroom bodies. These results suggest a level of homeostatic control over chemosensation, where flies compensate for lack of olfactory input by increasing the salience of taste information.


title: Schizophrenia Risk Proteins ZNF804A and NT5C2 Interact at Synapses

abstract: The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes have been identified as robust susceptibility genes in large-scale genome-wide association studies of schizophrenia. The ZNF804A and NT5C2 proteins are highly expressed in developing and mature cortical neurons. ZNF804A has been implicated in regulating the development of neuronal morphology; it localises to synapses and is required for activity-dependent modifications of dendritic spines. NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and implicated in influencing protein synthesis in neural progenitor cells. But despite these findings, a better understanding of the role these proteins play in regulating neuronal function is needed. A recent yeast two-hybrid screen has identified ZNF804A and NT5C2 as potential interacting proteins, but whether this occurs in situ; and moreover, in cortical neurons, is unknown. Here we show that ZNF804A and Nt5C2 colocalise and interact in hEK293T cells. Furthermore, their rodent homolouges, ZFP804A and NT5C2, specifically colocalise at synapses and form a protein complex in cortical neurons. Knockdown of Zfp804A or Nt5c2 resulted in a significant decrease in synaptic expression of both proteins, suggesting that both proteins are required for the synaptic targeting of each other. Taken together, these data indicate that ZNF804A/ZFP804A and NT5C2 interact together in cortical neurons and indicate that these GWAS risk factors may function as a complex to regulate neuronal function.


title: Depleting Hypothalamic Somatostatinergic Neurons Recapitulates Diabetic Phenotypes in Brain, Bone Marrow, Adipose, and Retina

abstract: Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunologic pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation. The present data show that leptin receptor deficient (db/db) mice exhibit reduced hypothalamic somatostatinergic cells, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus (AAV) delivery of diphtheria toxin (DTA) driven by an SST-cre system to deplete these cells in SSTcre/gfp mice (SST-DTA). SST-DTA mice exhibit enhanced hypothalamic c-fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed hematopoiesis, generating excess granulocyte-monocyte precursors and increased pro-inflammatory (CCR2hi) monocytes. Visceral adipose tissue from DTA-treated animals was resistant to catecholamine induced lipolysis. Finally, SST-DTA mice exhibited a "diabetic retinopathy like" phenotype: reduced visual function by optokinetic response and electroretinogram, as well as increased percentages of retinal monocytes. Importantly, hyperglycemia was not observed in SST-DTA mice. Thus, the isolated reduction in hypothalamic somatostatinergic neurons was able to recapitulate several hallmark features of type 2 diabetes in disease relevant tissues.


title: Aging is associated with a degeneration of noradrenergic-, but not dopaminergic-neurons, in the short-lived killifish Nothobranchius furzeri.

abstract:
Parkinsons disease (PD) is characterized by phosphorylation and aggregation of the protein -Synuclein and ensuing neuronal death progressing from the noradrenergic locus coeruleus to midbrain dopaminergic neurons. In 2019, Matsui and colleagues reported in Cell Reports a spontaneous age-dependent degeneration of dopaminergic neurons and an even greater neurodegeneration of the noradrenergic neurons in the short-lived killifish Nothobranchius furzeri.

Given the great possible relevance of a spontaneous model for PD, we set to confirm their results by whole-brain clarification and 3D nuclei reconstruction to quantify total cell numbers. We observed an age dependent neurodegeneration limited to the locus coeruleus and not involving the posterior tuberculum. In addition, we observed the presence of phospho-Synuclein in the soma of locus coeruleus neurons detectable already at a young age and increasing during aging.

Our result demonstrates that N. furzeri models the early stages of PD, but not the degeneration of dopaminergic neurons.


title: Erasing motion: Scrambling direction selectivity in visual cortex during saccades

abstract: Distinguishing sensory stimuli caused by changes in the environment from those caused by an animals own actions is a hallmark of sensory processing. Saccades are rapid eye movements that shift the image on the retina. How visual systems differentiate motion of the image induced by saccades from actual motion in the environment is not fully understood. Here, we discovered that in mouse primary visual cortex (V1), the two types of motion evoke distinct activity patterns. This is because during saccades, V1 combines the visual input with a strong non-visual input arriving from the thalamic pulvinar nucleus. Thus, the pulvinar input ensures differential V1 responses to external and self-generated motion and may allow the animal to interpret sensory stimuli within the context of its actions.


title: Interpreting Null Models of Resting-State Functional MRI

abstract: Null models are necessary for assessing whether a dataset exhibits non-trivial statistical properties. These models have recently gained interest in the neuroimaging community as means to explore dynamic properties of functional Magnetic Resonance Imaging (fMRI) time series. Interpretation of null-model testing in this context may not be straightforward because (i) null hypotheses associated to different null models are sometimes unclear and (ii) fMRI metrics might be  trivial, i.e. preserved under the null hypothesis, and still be useful in neuroimaging applications. In this commentary, we review several commonly used null models of fMRI time series and discuss the interpretation of the corresponding tests. We argue that, while null-model testing allows for a better characterization of the statistical properties of fMRI time series and associated metrics, it should not be considered as a mandatory validation step to assess their relevance in neuroimaging applications.


title: Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons

abstract: Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In adult neocortex, Nav1.1 is dominant in inhibitory neurons while Nav1.2 is dominant in excitatory neurons. Although a distinct subpopulation of neocortical excitatory neurons was also reported to express Nav1.1, their nature has been uncharacterized. By using newly-generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm mutually-exclusive expressions of Nav1.1 and Nav1.2, absence of Nav1.1 in hippocampal excitatory neurons, and further show that among neocortical excitatory neurons Nav1.1 is expressed in pyramidal tract and a subpopulation of cortico-cortical while Nav1.2 in cortico-striatal, cortico-thalamic and a distinct subpopulation of cortico-cortical projection neurons. These observations now contribute to the elucidation of pathological neural circuits for epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations including sudden death in Dravet syndrome.


title: Estimating smooth and sparse neural receptive fields with a flexible spline basis

abstract: Spatio-temporal receptive field (STRF) models are frequently used to approximate the computation implemented by a sensory neuron. Typically, such STRFs are assumed to be smooth and sparse. Current state-of-the-art approaches for estimating STRFs based empirical Bayes estimation encode such prior knowledge into a prior covariance matrix, whose hyperparameters are learned from the data, and thus provide STRF estimates with the desired properties even with little or noisy data. However, empirical Bayes methods are often not computationally efficient in high-dimensional settings, as encountered in sensory neuroscience. Here we pursued an alternative approach and encode prior knowledge for estimation of STRFs by choosing a set of basis function with the desired properties: a natural cubic spline basis. Our method is computationally efficient, and can be easily applied to Linear-Gaussian and Linear-Nonlinear-Poisson models as well as more complicated Linear-Nonlinear-Linear-Nonlinear cascade model or spike-triggered clustering methods. We compared the performance of spline-based methods to no-spline ones on simulated and experimental data, showing that spline-based methods consistently outperformed the no-spline versions. We provide a Python toolbox for all suggested methods (https://github.com/berenslab/RFEst/).


title: Locus coeruleus activity improves cochlear implant performance

abstract: Cochlear implants are neuroprosthetic devices that can provide hearing to deaf patients1. Despite significant benefits offered by cochlear implants, there are highly variable outcomes in how quickly hearing is restored and perceptual accuracy after months or years of use2,3. Cochlear implant use is believed to require neuroplasticity within the central auditory system, and differential engagement of neuroplastic mechanisms might contribute to outcome variability4-7. Despite extensive studies on how cochlear implants activate the auditory system4,8-12, our understanding of cochlear implant-related neuroplasticity remains limited. One potent factor enabling plasticity is the neuromodulator norepinephrine from the brainstem locus coeruleus. Here we examined behavioral responses and neural activity in locus coeruleus and auditory cortex of deafened rats fitted with multi-channel cochlear implants. Animals were trained on a reward-based auditory task, with considerable individual differences of learning rates and maximum performance. Photometry from locus coeruleus predicted when implanted subjects would begin responding to sounds and longer-term perceptual accuracy, which were augmented by optogenetic locus coeruleus stimulation. Auditory cortical responses to cochlear implant stimulation reflected behavioral performance, with enhanced responses to rewarded stimuli and decreased distinction between unrewarded stimuli. Adequate engagement of central neuromodulatory systems is thus a potential clinically-relevant target for optimizing neuroprosthetic device use.


title: An ammonium transporter is a non-canonical olfactory receptor for ammonia

abstract: Two families of ligand-gated ion channels function as olfactory receptors in insects. Here, we show that these canonical olfactory receptors are not necessary for responses to ammonia, a key ecological odor that is attractive to many insects including disease vectors and agricultural pests. Instead, we show that a member of the ancient electrogenic ammonium transporter family, Amt, is a new type of olfactory receptor. We report two hitherto unidentified olfactory neuron populations that mediate neuronal and behavioral responses to ammonia. Their endogenous ammonia responses are Amt-dependent, and ectopic expression of either Drosophila or Anopheles Amt confers ammonia sensitivity. Amt is the first transporter known to function as an olfactory receptor in animals, and its role may be conserved across insect species.


title: NeuriteNet: A Convolutional Neural Network for determining morphological differences in neurite growth

abstract:
BackgroundDuring development or regeneration, neurons extend processes (i.e., neurites) via mechanisms that can be readily analyzed in culture. However, defining the impact of a drug or genetic manipulation on such mechanisms can be challenging due to the complex arborization and heterogeneous patterns of neurite growth in vitro.

New MethodNeuriteNet is a Convolutional Neural Network (CNN) sorting model that uses a novel adaptation of the XRAI saliency map overlay, which is a region-based attribution method. NeuriteNet compares neuronal populations based on differences in neurite growth patterns, sorts them into respective groups, and overlays a saliency map indicating which areas differentiated the image for the sorting procedure.

ResultsIn this study, we demonstrate that NeuriteNet effectively sorts images corresponding to dissociated neurons into control and treatment groups according to known morphological differences. Furthermore, the saliency map overlay highlights the distinguishing features of the neuron when sorting the images into treatment groups. NeuriteNet also identifies novel morphological differences in neurites of neurons cultured from control and genetically modified mouse strains.

Comparison with Existing MethodsUnlike other neurite analysis platforms, NeuriteNet does not require manual manipulations, such as segmentation of neurites prior to analysis, and is more accurate than experienced researchers for categorizing neurons according to their pattern of neurite growth.

ConclusionsNeuriteNet can be used to effectively screen for morphological differences in a heterogeneous group of neurons and to provide feedback on the key features distinguishing those groups via the saliency map overlay.

HighlightsO_LINeuriteNet is a machine learning model developed to identify differences in control and experimental groups of cultured neurons based on morphological criteria.
C_LIO_LINeuriteNets saliency map highlights the features of the image that associate the neuron with a particular group.
C_LIO_LINeuriteNet outperforms trained researchers in assigning neurons to control or experimental groups with a known morphological difference as well as those with no previously described difference.
C_LI


title: Perceptual and Semantic Representations at Encoding Contribute to True and False Recognition of Objects

abstract:
When encoding new episodic memories, visual and semantic processing are proposed to make distinct contributions to accurate memory and memory distortions. Here, we used functional magnetic resonance imaging (fMRI) and representational similarity analysis to uncover the representations that predict true and false recognition of unfamiliar objects. Two semantic models captured coarse-grained taxonomic categories and specific object features, respectively, while two perceptual models embodied low-level visual properties. Twenty-eight female and male participants encoded images of objects during fMRI scanning, and later had to discriminate studied objects from similar lures and novel objects in a recognition memory test. Both perceptual and semantic models predicted true memory. When studied objects were later identified correctly, neural patterns corresponded to low-level visual representations of these object images in the early visual cortex, lingual, and fusiform gyri. In a similar fashion, alignment of neural patterns with fine-grained semantic feature representations in the fusiform gyrus also predicted true recognition. However, emphasis on coarser taxonomic representations predicted forgetting more anteriorly in ventral anterior temporal lobe, left perirhinal cortex, and left inferior frontal gyrus. In contrast, false recognition of similar lure objects was associated with weaker visual analysis posteriorly in early visual and left occipitotemporal cortex. The results implicate multiple perceptual and semantic representations in successful memory encoding and suggest that fine-grained semantic as well as visual analysis contributes to accurate later recognition, while processing visual image detail is critical for avoiding false recognition errors.

Significance StatementPeople are able to store detailed memories of many similar objects. We offer new insights into the encoding of these specific memories by combining fMRI with explicit models of how image properties and object knowledge are represented in the brain. When people processed fine-grained visual properties in occipital and inferior temporal cortex, they were more likely to be recognize the objects later, and less likely to falsely recognize similar objects. In contrast, while object-specific feature representations in fusiform predicted accurate memory, coarse-grained categorical representations in frontal and temporal regions predicted forgetting. The data provide the first direct tests of theoretical assumptions about encoding true and false memories, suggesting that semantic representations contribute to specific memories as well as errors.


title: Cellular variability of nonsense-mediated mRNA decay

abstract: Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two {beta}-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.

category: cell biology


title: A DNA repair-independent role for alkyladenine DNA glycosylase in alkylation-induced unfolded protein response

abstract:
Alkylating agents damage DNA and proteins and are widely used in cancer chemotherapy. While the cellular responses to alkylation-induced DNA damage have been explored, knowledge of how alkylation damage affects global cellular stress responses is still sparse. Here, we examined the effects of the alkylating agent methylmethane sulfonate (MMS) on gene expression in mouse liver taking advantage of mice deficient in alkyladenine DNA glycosylase (Aag), the enzyme that initiates the repair of alkylated DNA bases. MMS induced a robust transcriptional response in wild-type liver that included markers of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) known to be controlled by the transcription factor XBP1, a key UPR effector. Importantly, this response is significantly reduced in the Aag knockout. To investigate a potential role for AAG in alkylation-induced UPR, the expression of UPR markers after MMS treatment was interrogated in human glioblastoma cell lines expressing different AAG levels. Alkylation induced the UPR in cells expressing AAG; conversely, AAG knock-down compromised UPR induction and led to a defect in XBP1 activation plus a decrease in the expression of the ER chaperone BiP. To verify that the DNA repair activity of AAG is required for this response, AAG knockdown cells were complemented with wild-type Aag or with a mutant version of the Aag gene producing a glycosylase-deficient AAG protein. As expected, the glycosylase-defective mutant Aag does not fully protect AAG knockdown cells against MMS-induced cytotoxicity. Remarkably, however, alkylation-induced XBP1 activation is fully complemented by the catalytically inactive AAG enzyme. This work establishes that, in addition to its enzymatic activity, AAG has non-canonical functions in alkylation-induced UPR that contribute to the overall cellular response to alkylation.

Significance StatementStress response pathways, such as the DNA damage response (DDR) and the UPR, are critical in both the etiology and treatment of cancer and other chronic diseases. Knowledge of an interplay between ER stress and genome damage repair is emerging, but evidence linking defective DNA repair and impaired ER stress response is lacking. Here, we show that AAG is necessary for UPR activation in response to alkylating agents. AAG-deficient mice and human cancer cells are impaired in alkylation-induced UPR. Strikingly, this defect can be complemented by an AAG variant defective in glycosylase activity. Our studies suggest AAG has non-canonical functions and identify AAG as a point of convergence for stress response pathways. This knowledge could be explored to improve cancer treatment.


title: Kinetochore life histories reveal the origins of chromosome mis-segregation and correction mechanisms

abstract: Chromosome mis-segregation during mitosis leads to daughter cells with deviant karyotypes (aneuploidy) and an increased mutational burden through chromothripsis of mis-segregated chromosomes. The rate of mis-segregation and the aneuploidy state are hallmarks of cancer and linked to cancer genome evolution. Errors can manifest as "lagging chromosomes" in anaphase, although the mechanistic origins and likelihood of correction are incompletely understood. Here we combine lattice light sheet microscopy, endogenous protein labelling and computational analysis to define the life history of > 104 kinetochores throughout metaphase and anaphase from over 200 cells. By defining the "laziness" of kinetochores in anaphase, we reveal that chromosomes are at a considerable and continual risk of mis-segregation. We show that the majority of kinetochores are corrected rapidly in early anaphase through an Aurora B dependent process. Moreover, quantitative analyses of the kinetochore life histories reveal a unique dynamic signature of metaphase kinetochore oscillations that forecasts their fate in the subsequent anaphase. We propose that in diploid human cells chromosome segregation is fundamentally error prone, with a new layer of early anaphase error correction required for stable karyotype propagation.


title: Eosinophils restrict diesel exhaust particles induced cell proliferation of lung epithelial A549 cells, vial interleukin-13 mediated mechanisms: implications for tissue remodelling and fibrosis

abstract: Diesel exhaust particulates (DEPs) affect lung physiology and cause serious damage to the lungs. A number of studies have demonstrated that eosinophils play a very important role in the development of lung tissue remodelling and fibrosis. However, the exact mechanism of its pathogenesis is not known. We for the first time demonstrate that, Interleukin-13 plays a very important role in the development of tissue remodelling and fibrosis. We demonstrate that, Diesel exhaust particle significantly induce eosinophils cell proliferation and interleukin-13 release in invitro culture conditions. Supernatant collected from DEP induced eosinophils cells significantly restrict cell proliferation of epithelial cells due to exposure of diesel exhast particles. Furthermore, purified interleukin-13 decreases the proliferation of A549 cells. Notably, Etoricoxib (selective COX-2 inhibitor) did not inhibit DEP-triggered release of interleukin-13, suggesting another cell signalling pathway. In, vivo exposer of DEP to the mice lung, resulted in the high level of eosinophils degranulation as depicted by the EPX-1 immunostaining and altered level of mRNA expressions of inflammatory genes. We also found that, a-SMA, fibroblast specific protein (FSP-1) has been changed in response to DEP in the mice lungs along with the mediators of inflammation. Altogether, we elucidated the mechanistic role of eosinophils in the DEP triggered proliferation of lungs cells thus providing an inside in the pathophysiology of tissue remodelling and fibrosis of lungs.


title: High-resolution 3D reconstruction of human oocytes using FIB-SEM.

abstract: The oocyte plays a pivotal role in the reproduction of our species. Nevertheless, its biology remains poorly understood. Electron microscopy is traditionally used to inspect the ultrastructure of female gametes. However, two-dimensional micrographs contain only fragmentary information about the spatial organization of the complex oocyte cytoplasm. Here, we employed the Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to explore human oocyte intracellular morphology in three dimensions (3D). Volume reconstruction from high-resolution image stacks provided an unprecedented view of ooplasmic architecture. Organelle distribution patterns observed in 9 donor oocytes, representing 3 maturational stages, documented structural changes underlying the process by which the egg acquires developmental competence. 3D image segmentation was performed to extract information about distinct organelle populations. The quantitative analysis of the organelle abundance revealed that mitochondrion occupies ~ 4.26 % of the maturing oocyte cytoplasm. This proof-of-concept study demonstrates the potential of FIB-SEM imaging to study human oocyte morphology.


title: Differential effects of environmental and endogenous 24h rhythms within a deep-coverage spatiotemporal proteome

abstract: The cellular landscape of most eukaryotic cells changes dramatically over the course of a 24h day. Whilst the proteome responds directly to daily environmental cycles, it is also regulated by a cellular circadian clock that anticipates the differing demands of day and night. To quantify the relative contribution of diurnal versus circadian regulation, we mapped spatiotemporal proteome dynamics under 12h:12h light:dark cycles compared with constant light. Using Ostreococcus tauri, a prototypical eukaryotic cell, we achieved 85% coverage of the theoretical proteome which provided an unprecedented insight into the identity of proteins that drive and facilitate rhythmic cellular functions. Surprisingly, the overlap between diurnally- and circadian-regulated proteins was quite modest (11%). These proteins exhibited different phases of oscillation between the two conditions, consistent with an interaction between intrinsic and extrinsic regulatory factors. The relative amplitude of rhythmic protein abundance was much lower than would be expected from daily variations in transcript abundance. Transcript rhythmicity was poorly predictive of daily variation in abundance of the encoded protein. We observed coordination between the rhythmic regulation of organelle-encoded proteins with the nuclear-encoded proteins that are targeted to organelles. Rhythmic transmembrane proteins showed a remarkably different phase distribution compared with rhythmic soluble proteins, indicating the existence of a novel circadian regulatory process specific to the biogenesis and/or degradation of membrane proteins. Taken together, our observations argue that the daily spatiotemporal regulation of cellular proteome composition is not dictated solely by clock-regulated gene expression. Instead, it also involves extensive rhythmic post-transcriptional, translational, and post-translational regulation that is further modulated by environmental timing cues.


title: Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time

abstract:
Ribosomal subunit biogenesis within mammalian cells initiates in the nucleolus with the assembly of a 90S precursor particle, which is subsequently split into the pre-40S and pre-60S subunits. During further processing steps, pre-ribosomal subunits are loaded with export receptors, which enables their passage through the pore complexes (NPCs) into the cytoplasm. Here export factors are released and both subunits can form a mature ribosome. Ribosomal biogenesis has been studied in great detail by biochemical, genetic and electron microscopic approaches, however, until now live cell data on the in vivo kinetics are still missing.

We analysed export kinetics of the large ribosomal subunit ("pre-60S particle") through single NPCs in living human cells. To assess the in vivo dynamics of this process, we established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label ribosomal 60S subunits (eIF6) and NPCs (NTF2). By combining single molecule tracking and super resolution confocal microscopy in a highly customized microscopic setup, we visualized the dynamics of single pre-60S particles during the interaction with and export through single NPCs. In this way we obtained unprecedented insights into this key cellular process.

Our results revealed that for export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export process takes place with a single rate limiting step and an export dwell time of ~24 milliseconds. Only about 1/3 of attempted export events were successful. Given the molecular mass of the pre-60S particles our results show that the mass flux through a single NPC can reach up to ~125 MDa{middle dot}s-1 in vivo.


title: Dissolved organic matter from tropical peatlands impacts shelf sea light availability on coral reefs in the Singapore Strait, Southeast Asia

abstract: Shelf seas provide valuable ecosystem services, but their productivity and ecological functioning depend critically on sunlight transmitted through the water column. Anthropogenic reductions in underwater light availability are thus recognized as a serious threat to coastal habitats. The flux of strongly light-absorbing coloured dissolved organic matter (CDOM) from land to sea may have increased world-wide, but how this has altered the availability and spectral quality of light in shelf seas remains poorly known. Here, we present time-series data from the Sunda Shelf in Southeast Asia, where the monsoon-driven reversal in ocean currents supplies water enriched in CDOM from tropical peatlands for part of the year, resulting in 5-10-fold seasonal variation in light absorption by CDOM. We show that this terrigenous CDOM can dominate underwater light absorption at wavelengths up to 500 nm, and shift in the underwater irradiance spectrum towards longer wavelengths. The seasonal presence of terrigenous CDOM also causes the depth of 10% light penetration to shoal by 1-5 m, or 10-45%. We further estimate that on average 0.6 m, or 25%, of this terrigenous CDOM-mediated shoaling might be attributable to the enhanced loss of dissolved organic matter caused by peatland disturbance. We show that the seasonal change in the light environment is correlated with photo-acclimation by phytoplankton, and infer that terrigenous CDOM likely contributes to limiting the depth distribution of photosynthetic corals. Our results thus reveal an ecologically important but largely overlooked impact of human modifications to carbon fluxes that is likely becoming increasingly important in coastal seas.

category: ecology


title: The protective effect of sickle cell haemoglobin against severe malaria depends on parasite genotype

abstract: Host genetic factors can confer resistance against malaria, raising the question of whether this has led to evolutionary adaptation of parasite populations. In this study we investigated the correlation between host and parasite genetic variation in 4,171 Gambian and Kenya children ascertained with severe malaria due to Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and variation in three regions of the parasite genome, including nonsynonymous variants in the acyl-CoA synthetase family member PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The HbS-associated parasite alleles are in strong linkage disequilibrium and have frequencies which covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome, and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations.

category: genetics


title: Evolution of the insect PPK gene family

abstract: Insect Pickpocket (PPK) receptors mediate the detection of stimuli of diverse sensory modalities, therefore having a relevant role for environmental sounding. Notwithstanding their relevance, studies on their evolution are scarce. We have analyzed the genomes of 26 species belonging to 8 insect orders (Blattodea, Orthoptera, Hemiptera, Phthiraptera, Hymenoptera, Lepidoptera, Coleoptera, and Diptera) to identify their PPK repertoires and study the evolution of this gene family. PPKs were detected in all genomes analyzed, with a total of 578 genes identified that distributed in 7 subfamilies. Our phylogenetic analysis allowed clarifying that the ppk17 gene appears to be the most divergent family member, composing a new group designed as subfamily VII. According to our analysis, PPKs evolved under a birth-and-death model that generated lineage-specific expansions usually located in clusters and the effect of strong purifying selection was seen for several orthogroups. Subfamily V was the largest one, presenting half of all PPKs studied, including a mosquito-specific expansion that can be considered a new target for pest control. Consistently with their sensory role, PPKs present a high gene turnover that generated considerable variation in the size of insect repertoires: Musca domestica (59), Blattella germanica (41), Culex quinquefasciatus (48), and Aedes albopictus (51) presented the largest PPK repertoires, while Pediculus humanus (only ppk17), bees and ants (6-9) had the smallest ones. The expansions identified in M. domestica and Bl. germanica also show promise as specific targets for controlling these nuisance insects. Our phylogenetic analysis revealed a subset of prevalent PPKs across insect genomes, suggesting a very conserved function that resembles the case of antennal ionotropic receptors. Finally, we identified new highly conserved residues in the second transmembrane domain that may be key for receptor function. Besides, more than a hundred PPK sequences presented calmodulin binding motifs, suggesting that at least some members of this family may amplify sensory responses as previously proposed for D. melanogaster ppk25. Overall, our study is a first attempt to characterize the evolutionary history of this family of sensory receptors, revealing relevant unknown features and clade-specific expansions.

category: genomics


title: Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

abstract:
While DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterised the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinsons disease (n=27), using a combination of whole genome bisulfite sequencing (WGBS) and bisulfite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723{+/-}7,711), and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49{+/-}0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinsons disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.

Graphical abstract of the analyses and main findingsFresh-frozen brain tissue was obtained from the prefrontal cortex (Brodmann area 9) of 53 individuals, comprising 27 patients with idiopathic PD and 26 healthy controls. Tissue from the same samples was used in three different downstream analyses. WGBS was conducted on all 53 samples and the data were analysed using three different alignment strategies. Alignment against an mtDNA reference only was clearly superior as it gave the highest and most even depth of coverage. WGBS analysis revealed that mtDNA harbours very low levels of cytosine methylation, with the exception of the CpG position m.545 within the HSP1 region (lower right inset). The m.545 methylation was confirmed by bisulfite- and sequencing-independent methods, employing methylation-specific MspJI DNA digestion, followed by quantification with qPCR or fluorescent PCR and capillary electrophoresis. Finally, mtDNA transcript levels were determined by RT-qPCR and correlated to m.545 methylation levels, showing a positive association.

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title: The plastid genome characteristics of a moth orchid (Phalaenopsis wilsoniii, Orchidaceae)

abstract: Phalaenopsis wilsonii is a typical deciduous species in the horticulturally well-known genus, Phalaenopsis. Tshi species is belonging to the section Aphyllae in moth orchid, and is endemic to South China. Although the Aphyllae section display the deciduous feature that is unique in this genus, their genetic information is still insufficient and limited them as breeding parent in moth orchid. Here, we reported and characterized the complete chloroplast genome for Phalaenopsis wilsonii. We found the total size of the chloroplast genome was 145,373 bp, constituting of a large single copy (LSC) region (84,996 bp), a small single-copy region (10,668 bp) and two inverted repeats (IRs) regions (24,855 bp). Based on homologous searching on database, we annotated 76 protein-coding genes, 38 tRNA, and 8 rRNA. The phylogenetic reconstruction revealed that P. wilsonii show the closest relationship with P. lowii within subgenus Parishianae.


title: Facilitating population genomics of non-model organisms through optimized experimental design for reduced representation sequencing

abstract: Genome-wide data are invaluable to characterize differentiation and adaptation of natural populations. Reduced representation sequencing (RRS) subsamples a genome repeatedly across many individuals. However, RRS requires careful optimization and fine-tuning to deliver high marker density while being cost-efficient. The number of genomic fragments created through restriction enzyme digestion and the sequencing library setup must match to achieve sufficient sequencing coverage per locus. Here, we present a workflow based on published information and computational and experimental procedures to investigate and streamline the applicability of RRS. In an iterative process genome size estimates, restriction enzymes and size selection windows were tested and scaled in six classes of Antarctic animals (Ostracoda, Malacostraca, Bivalvia, Asteroidea, Actinopterygii, Aves). Achieving high marker density would be expensive in amphipods, the malacostracan target taxon, due to the large genome size. We propose alternative approaches such as mitogenome or target capture sequencing for this group. Pilot libraries were sequenced for all other target taxa. Ostracods, bivalves, sea stars, and fish showed overall good coverage and marker numbers for downstream population genomic analyses. In contrast, the bird test library produced low coverage and few polymorphic loci, likely due to degraded DNA. Prior testing and optimization are important to identify which groups are amenable for RRS and where alternative methods may currently offer better cost-benefit ratios. The steps outlined here are easy to follow for other non-model taxa with little genomic resources, thus stimulating efficient resource use for the many pressing research questions in molecular ecology.


title: BRED: bioluminescence energy transfer to dye for monitoring ceramide trafficking in cell

abstract: Bioluminescence resonance energy transfer (BRET) is a genetically encoded proximity-based tool to study biomolecular interactions. However, conventional BRET is usually restricted to only a few types of interactions like protein-protein or protein-ligand interactions. We here developed a spatially unbiased resonance energy transfer system, so-called BRED - bioluminescence resonance energy transfer to dye. BRED allows transferring energy from a genetically encoded bright human optimized luciferase to a fluorophore-labelled small molecule. The high efficiency of the system allows RET without specific interaction of donor and acceptor. Here, we applied BRED to monitor the trafficking of the signalling lipid ceramide, to the Golgi. This was enabled by an engineered Golgi-resident luciferase, which was used to sense the influx of BODIPY-labeled ceramide into the surrounding membrane. We demonstrated the implementation of the method via flow cytometry, thereby combining the sensitivity of bulk cell methods with the advantages of single-cell analysis. This toolbox enables simple and robust live-cell analysis of inhibitors of CERT-mediated ceramide transport. The design principle of our optogenetic tool can be applied to study intracellular trafficking of metabolites and screen for inhibitors of their key enzymes.

category: biochemistry


title: Supramolecular cylinders target bulge structures in the 5-prime UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication

abstract: The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with Molecular Dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5 UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in the stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel antiviral agents.


title: Exploring the role of glycans in the interaction of SARS-CoV-2 RBD and human receptor ACE2

abstract: COVID-19 is a highly infectious respiratory disease caused by the novel coronavirus SARS-CoV-2. It has become a global pandemic and its frequent mutations may pose new challenges for vaccine design. During viral infection, the Spike RBD of SARS-CoV-2 binds the human host cell receptor ACE2, enabling the virus to enter the host cell. Both the Spike and ACE2 are densely glycosylated, and it is unclear how distinctive glycan types may modulate the interaction of RBD and ACE2. Detailed understanding of these determinants is key for the development of novel therapeutic strategies. To this end, we perform extensive all-atom simulations of the (i) RBD-ACE2 complex without glycans, (ii) RBD-ACE2 with oligomannose MAN9 glycans in ACE2, and (iii) RBD-ACE2 with complex FA2 glycans in ACE2. These simulations identify the key residues at the RBD-ACE2 interface that form contacts with higher probabilities, thus providing a quantitative evaluation that complements recent structural studies. Notably, we find that this RBD-ACE2 contact signature is not altered by the presence of different glycoforms, suggesting that RBD-ACE2 interaction is robust. Applying our simulated results, we illustrate how the recently prevalent N501Y mutation may alter specific interactions with host ACE2 that facilitate the virus-host binding. Furthermore, our simulations reveal how the glycan on Asn90 of ACE2 can play a distinct role in the binding and unbinding of RBD. Finally, an energetics analysis shows that MAN9 glycans on ACE2 decrease RBD-ACE2 affinity, while FA2 glycans lead to enhanced binding of the complex. Together, our results provide a more comprehensive picture of the detailed interplay between virus and human receptor, which is much needed for the discovery of effective treatments that aim at modulating the physical-chemical properties of this virus.

category: biophysics


title: Fluid mechanics of mosaic ciliated tissues

abstract: In tissues as diverse as amphibian skin and the human airway, the cilia that propel fluid are grouped in sparsely distributed multiciliated cells (MCCs). We investigate fluid transport in this "mosaic" architecture, with emphasis on the trade-offs that may have been responsible for its evolutionary selection. Live imaging of MCCs in embryos of the frog Xenopus laevis shows that cilia bundles behave as active vortices that produce a flow field accurately represented by a local force applied to the fluid. A coarse-grained model that self-consistently couples bundles to the ambient flow reveals that hydrodynamic interactions between MCCs limit their rate of work so that when the system size is large compared to a single MCC, they best shear the tissue at low area coverage, a result that mirrors findings for other sparse distributions such as cell receptors and leaf stomata.


title: Multiple timescales in bacterial growth homeostasis

abstract: In balanced exponential growth, bacterial cells maintain the stability of multiple properties simultaneously: cell size, growth rate, cycle time and more. These are not independent but strongly coupled variables; it is not a-priori clear which are under direct regulation and which are stabilized as a by-product of interactions. Here, we address this problem by separating different timescales in bacterial single-cell dynamics. Disentangling homeostatic set-points from fluctuations around them, we find that some properties have flexible set-points that highly sensitive to environment - defining "sloppy" variables, while other set-points are buffered and held tightly controlled - "stiff" variables. These control variables are combinations of sloppy ones that compensate one another over long times, creating a hierarchical buffering that protects them from environmental perturbations. This is manifested geometrically as a control manifold in the space of growth and division variables, whose in-plane directions span sloppy variables, while out-of-plane deviations are highly constrained. Cell size is found to be a sloppy variable, which is coupled to growth and division only on the short, single-cycle timescale. Our results show that cellular homeostasis involves multi-level regulation operating on multiple timescales. More generally, our work offers a data-driven approach for identifying control variables in a multi-dimensional system that can be applicable also in other contexts.


title: ATP allosterically stabilizes Integrin-linked kinase for efficient force generation

abstract: Focal adhesions link the actomyosin cytoskeleton to the extracellular matrix regulating cell adhesion, shape, and migration. Adhesions are dynamically assembled and disassembled in response to extrinsic and intrinsic forces, but how the essential adhesion component intergrin-linked kinase (ILK) dynamically responds to mechanical force and what role ATP bound to this pseudokinase plays remains elusive. Here, we apply force-probe molecular dynamics simulations of human ILK:-parvin coupled to traction force microscopy to explore ILK mechanotransducing functions. We identify two key saltbridge-forming arginines within the allosteric, ATP-dependent force-propagation network of ILK. Disrupting this network by mutation impedes parvin binding, focal adhesion stabilization, force generation, and thus migration. Under tension, ATP shifts the balance from rupture of the complex to protein unfolding, indicating that ATP increases the force threshold required for focal adhesion disassembly. Our study proposes a new role of ATP as an obligatory binding partner for structural and mechanical integrity of the pseudokinase ILK, ensuring efficient cellular force generation and migration.


title: Restriction of RecG translocation by DNA mispairing

abstract: The RecG DNA helicase plays a crucial role in stalled replication fork rescue as the guardian of the bacterial genome. We have recently demonstrated that single-strand DNA binding protein (SSB) promotes binding of RecG to the stalled replication fork by remodeling RecG, enabling the helicase to translocate ahead of the fork. We also hypothesized that mispairing of DNA could limit such translocation of RecG, which plays the role of roadblocks for the fork movement. Here, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing. We found that a C-C mismatch at a distance of 30 bp away from the fork position prevents translocation of RecG over this mispairing. A G-bulge placed at the same distance also has a similar roadblock efficiency. However, a C-C mismatch 10 bp away from the fork does not prevent RecG translocation, as 10 bp from fork is within the distance of footprint of RecG on fork DNA. Our findings suggest that retardation of RecG translocation ahead of the replication fork can be a mechanism for the base pairing control for DNA replication machinery.


title: Biophysical properties of a tau seed

abstract: Pathogenesis of tauopathies involves conversion of tau monomer into pathological tau conformers that serve as templates to recruit native tau into growing assemblies. Small soluble tau seeds have been proposed to drive pathological tau assembly in vitro, in cells and in vivo. We have previously described the isolation of monomeric pathogenic tau seeds derived from recombinant samples and tauopathy tissues but in-depth biophysical characterization of these species has not been done. Here we describe a chromatographic method to isolate recombinant soluble tau seeds derived from heparin treatment. We used biochemical and biophysical approaches to show that the seeds are predominantly monomeric and have the capacity to nucleate aggregation of inert forms of tau in vitro and in cells. Finally, we used crosslinking mass spectrometry to identify the topological changes in tau as it converts from an inert state to a pathogenic seed. Future studies will reveal the relationship between soluble seeds and structural polymorphs derived from tauopathies to help diagnose and develop therapeutics targeting specific tauopathies.


title: Structural insights into the activation of human calcium-sensing receptor

abstract: Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and active states. Complemented with previously reported crystal structures of CaSR extracellular domains, it suggests that there are three distinct conformations: inactive, intermediate and active state during the activation. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data shows that the agonist binding leads to the compaction of the dimer, the proximity of the cysteine-rich domains, the large-scale transitions of 7-transmembrane domains, and the inter-and intrasubunit conformational changes of 7-transmembrane domains to accommodate the downstream transducers. Our results reveal the structural basis for activation mechanisms of the CaSR.


title: Drift correction in localization microscopy using entropy minimization

abstract: Localization microscopy offers resolutions down to a single nanometer, but currently requires additional dedicated hardware or fiducial markers to reduce resolution loss from drift of the sample. Drift estimation without fiducial markers is typically implemented using redundant cross correlation (RCC). We show that RCC has sub-optimal precision and bias, which leaves room for improvement. Here, we minimize a bound on the entropy of the obtained localizations to efficiently compute a precise drift estimate. Within practical compute-time constraints, simulations show a 5x improvement in drift estimation precision over the widely used RCC algorithm. The algorithm operates directly on fluorophore localizations and is tested on simulated and experimental datasets in 2D and 3D. An open source implementation is provided, implemented in Python and C++, and can utilize a GPU if available.


title: The long non-coding RNA MaTAR20 promotes mammary tumor growth by regulating angiogenesis pathways

abstract: Long non-coding RNAs (lncRNAs) are an emerging class of regulatory molecules that have been shown to play important roles in tumorigenesis and cancer progression. Here, we studied the recently identified lncRNA Mammary Tumor Associated RNA 20 (MaTAR20) in mammary cancer progression. A CRISPR/Cas9 knockout of MaTAR20 in the metastatic 4T1 cell line led to reduced cancer cell proliferation and increased cell surface adhesion compared to control cells. Consistent with these knockout results antisense oligonucleotide (ASO) mediated knockdown of MaTAR20 resulted in reduced growth and invasion in 4T1 cells, and in primary mammary tumor organoids derived from the MMTV-PyMT mouse model of breast cancer. Injection of MaTAR20-specific ASOs subcutaneously into tumor bearing MMTV-PyMT mice resulted in smaller and highly necrotic tumors in comparison to mice injected with a scrambled control ASO. To investigate the molecular mechanism by which MaTAR20 acts to advance mammary tumor progression, we applied a combination of RNA-sequencing and RNA-pulldown coupled to DNA-sequencing. These analyses demonstrated that the nuclear retained lncRNA is associated with several essential cancer signaling pathways such as VEGF signaling. In particular, MaTAR20 directly binds to and regulates the expression of Tnfsf15. Our results indicate that MaTAR20 is an important driver of mammary tumor progression and represents a promising new therapeutic target.

category: cancer biology


title: Perivascular tenascin C triggers sequential activation of macrophages and endothelial cells to generate a pro-metastatic vascular niche in the lungs

abstract: When cancers progress to metastasis, disseminated cancer cells frequently lodge near vasculature in secondary organs. However, our understanding of the cellular crosstalk evoked at perivascular sites is still rudimentary. In this study, we identify intercellular machinery governing formation of a pro-metastatic vascular niche during breast cancer colonization in lungs. We show that four secreted factors, INHBB, SCGB3A1, OPG and LAMA1, induced in metastasis-associated endothelial cells (ECs), are essential components of the vascular niche and promote metastasis in mice by enhancing stem cell properties and survival ability of cancer cells. Notably, blocking VEGF, a key regulator of EC behavior, dramatically suppressed EC proliferation, whereas no impact was observed on the expression of the four vascular niche factors in lung ECs. However, perivascular macrophages, activated via the TNC-TLR4 axis, were shown to be crucial for EC-mediated production of niche components. Together, our findings provide mechanistic insights into the formation of vascular niches in metastasis.


title: Ovarian cancers with low CIP2A tumor expression constitute an APR-246 sensitive disease subtype

abstract: Identification of ovarian cancer (OvCa) patient subpopulations with increased sensitivity to targeted therapies could offer significant clinical benefit. We report that 22% of the high grade OvCa tumors at diagnosis express CIP2A oncoprotein at low levels. CIP2Alow OvCa tumors have significantly lower likelihood of disease relapse after standard chemotherapy, but yet a portion of relapsed tumors retain their CIP2Alow phenotype. We further discover that reactive oxygen species (ROS) inducing compound APR-246 (PRIMA-1Met/Eprenetapopt), currently in clinical development, preferentially kill CIP2Alow OvCa cells across multiple chemotherapy resistant cell lines. Consistent with CIP2Alow OvCa subtype in humans, CIP2A is dispensable for development of MISIIR-TAg-driven mouse OvCa tumors. Nevertheless, CIP2A deficient OvCa tumor cells from MISIIR-TAg mice displayed APR-246 hypersensitivity both in vitro and in vivo. Mechanistically, the lack of CIP2A expression hypersensitizes the OvCa cells to APR-246 by inhibition of NF-kB activity. Accordingly, combination of APR-246 and Nf-kB inhibitor compounds strongly synergized in killing of CIP2A positive OvCa cells. Collectively, we discover low CIP2A expression as a vulnerability for APR-246 in OvCa. The results warrant consideration of clinical testing of APR-246 for CIP2Alow OvCa tumor subtype patients, and reveal CIP2A as a candidate APR-246 combination therapy target.


title: Probing the killing potency of tumor-infiltrating lymphocytes on microarrayed autologous tumoroids

abstract: Immunotherapy has shown promise as an approach to fight cancer by harnessing the immune system. However, due to the lack of biomarkers to guide treatment regimens and predict response rates, there is an unmet need for more robust ex vivo and in vitro systems that recapitulate patient-specific tumor biology and enable response prediction for immune therapies in an autologous setting. To address this issue, we developed a high-content screening-compatible assay based on microcavity arrays to study tumor-infiltrating lymphocyte (TIL) functionality on 3D tumoroid models. We validated our system using the pmel-1 activated T cell mouse model to assess both cancer immunogenicity and T cell functionality. To demonstrate the translational potential of the platform, we used it to evaluate the response of patient-derived TILs to autologous human colorectal cancer (CRC) tumoroids. Using a combination of imaging and flow cytometry, we determined several features of the antitumor activity of TILs, including the extent of tumoroid killing and secretion of cytokines. We then used the approach to identify responders to immunotherapy, such as the immune checkpoint blockade (ICB) agent Nivolumab (PD-1 inhibitor) and Ipilimumab (CTLA-4 inhibitor). Our system allows not only the identification of immunogenic tumors, but also the testing of patients for response to immunomodulators, enabling personalized immuno-oncology.


title: Caloric restriction overcomes pre-diabetic and hypertension induced by high fat diet and renal artery stenosis

abstract:
BackgroundCaloric restriction (CR) is a type of dietary intervention enjoyed as an essential tool in weight loss by modulating critical pathways of metabolic control, although it is not yet clear what repercussions this intervention model results when associated with renovascular hypertension. Here we demonstrate that CR can be beneficial in obese and hypertensive animals.

MethodsRats were divided into groups: SHAM, and two groups underwent surgery to clip the left renal artery, to induce renovascular hypertension (OH and OHR). The SHAM diet was performed: 14 weeks normolipidic diet; OH: 2 weeks normolipidic diet + 12 weeks hyperlipidic diet, both ad libitum; OHR: 2 weeks normolipidic diet + 8 weeks ad libitum high fat diet + 4 weeks restricted 40% high fat diet.

Resultsthe OHR group dissipated blood pressure, body weight and glucose homeostasis. Reductions in insulinemia, lipids, islets fibrotic areas in the OHR group were observed along with increased insulin sensitivity and normalization of the insulin-degrading enzyme. Nicotinamide phosphoribosyltransferase, insulin receptor, Sirtuin 1 and complex II protein were modulated in liver tissue in the OHR group. Strong correlations, direct or indirect, were evaluated by Spearmans model between SIRT1, AMPK, NAMPT, PGC-1 and NNMT with the reestablishment of blood pressure, weight loss, glycidic and lipid panel and mitochondrial adaptation.

ConclusionCR provided short-term beneficial effects to recover physiological parameters induced by a high-fat diet and renal artery stenosis in obese and hypertensive animals. These benefits, even in the short term, can bring physiological benefits in the long run.

category: physiology


title: A protective broadly cross-reactive human antibody defines a conserved site of vulnerability on beta-coronavirus spikes

abstract:
Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses ({beta}-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 [A] resolution and found that the peptide adopted a mainly helical structure. Conserved residues in {beta}-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on {beta}-CoV spike proteins for protective antibodies that may facilitate the development of pan-{beta}-CoV vaccines.

SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-{beta}-CoV vaccine design strategies

category: immunology


title: A human antibody reveals a conserved site on beta-coronavirus spike proteins and confers protection against SARS-CoV-2 infection


title: Immunological factors, but not clinical features, predict visceral leishmaniasis relapse in patients co-infected with HIV

abstract: Visceral leishmaniasis (VL) has emerged as a clinically important opportunistic infection in HIV patients, as VL/HIV co-infected patients suffer from frequent VL relapse. Here, we followed cohorts of VL patients with or without HIV co-infections in Ethiopia and collected detailed clinical and immunological data during 12 months of follow-up. By the end of the study 78.1% of VL/HIV patients, but none of the VL only patients, had relapsed. Despite clinically defined cure, VL/HIV patients maintained high parasite loads, low BMI, hepatosplenomegaly and pancytopenia throughout follow-up. During detailed immunological study throughout the follow-up period, we identified three markers associated with VL relapse: i) failure to restore antigen-specific production of IFN{gamma}, ii) persistently low CD4+ T cell counts, and iii) high expression of PD1 on CD4+ T cells. We show that these three markers combine well in predicting VL relapse, and that all three measurements are needed for optimal predictive power. These three immunological markers can be measured in primary hospital settings in Ethiopia and can predict VL relapse after anti-leishmanial therapy. The use of our prediction model has the potential to improve disease management and patient care.


title: Arrayed multicycle drug screens identify broadly acting chemical inhibitors for repurposing against SARS CoV 2

abstract: Coronaviruses (CoVs) circulate in humans and animals, and expand their host range by zoonotic and anthroponotic transmissions. Endemic human CoVs, such as 229E and OC43 cause limited respiratory disease, and elicit short term anti-viral immunity favoring recurrent infections. Yet, severe acute respir-atory syndrome (SARS)-CoV-2 spreads across the globe with unprecedented impact on societies and economics. The world lacks broadly effective and affordable anti-viral agents to fight the pandemic and reduce the death toll. Here, we developed an image-based multicycle replication assay for focus for-mation of -coronavirus hCoV-229E-eGFP infected cells for screening with a chemical library of 5440 compounds arrayed in 384 well format. The library contained about 39% clinically used compounds, 26% in phase I, II or III clinical trials, and 34% in preclinical development. Hits were counter-selected against toxicity, and challenged with hCoV-OC43 and SARS-CoV-2 in tissue culture and human bronchial and nasal epithelial explant cultures from healthy donors. Fifty three compounds inhibited hCoV-229E-GFP, 39 of which at 50% effective concentrations (EC50) < 2M, and were at least 2-fold separated from toxicity. Thirty nine of the 53 compounds inhibited the replication of hCoV-OC43, while SARS-CoV-2 was inhibited by 11 compounds in at least two of four tested cell lines. Six of the 11 compounds are FDA-approved, one of which is used in mouth wash formulations, and five are systemic and orally available. Here, we demonstrate that methylene blue (MB) and mycophenolic acid (MPA), two broadly available low cost compounds, strongly inhibited shedding of infectious SARS-CoV-2 at the apical side of the cultures, in either pre- or post-exposure regimens, with somewhat weaker effects on viral RNA release indicated by RT-qPCR measurements. Our study illustrates the power of full cycle screens in repurposing clinical compounds against SARS-CoV-2. Importantly, both MB and MPA reportedly act as immunosuppressants, making them interesting candidates to counteract the cytokine storms affecting COVID-19 patients.

category: microbiology


title: Spectrogram cross-correlation can be used to measure the complexity of bird vocalizations

abstract:
O_LIBirdsong is an important signal in mate attraction and territorial defense. Quantifying the complexity of these songs can shed light on individual fitness, sexual selection, and behavior. Several techniques have been used to quantify song complexity and be broadly categorized into diversity indices, measures of stationary probabilities, and measures of sequential variations. However, these methods are unable to account for important acoustic features like the frequency bandwidth and the variety in the shape of syllables which are an integral part of these vocal signals. This study proposes a new complexity measure that considers intra-song note variability and calculates a weighted index for birdsongs using spectral cross-correlation.
C_LIO_LIWe compared the previously described methods to understand the advantages and limitations based on the factors that would be affecting the complexity of songs. We developed a new method- Note Variability Index (NVI), which incorporates the spectral features of notes while quantifying complexity. This measure alleviates the need for manual annotations of notes that can be error-prone. We used Spectrogram Cross-Correlation (SPCC) to compare notes within a song and used the output values to quantify song complexity.
C_LIO_LITo check for the efficacy of the new method, we generated synthetic songs to caricature extremes in song complexity and compared selected conventional complexity measures along with the NVI. We provide case-specific limitations of these methods. Additionally, to examine the efficacy of this new method in real-world scenarios, we used natural birdsongs from multiple species across the globe with varying song structures to compare conventional methods with NVI.
C_LIO_LITo our knowledge, NVI is the only song complexity method that captures the variation of spectral features of notes in songs where the conventional methods fail to distinguish between similar song structures with different note types. As NVI does not need a manual classification of notes, it can be easily implemented for any type of birdsong with existing sound analysis softwares; it is very quick, avoids the possible biases in note classification, and can possibly be automated for large datasets in the future.
C_LI


title: Piecemeal regulation of convergent neuronal lineages by bHLH transcription factors in C. elegans

abstract: Classic cell lineage studies in the nematode Caenorhabditis elegans as well as recent lineage tracing in vertebrates have shown that cells of the same type can be generated by distinct cellular lineages that originate in different parts of the developing embryo ("lineage convergence"). Several C. elegans neuron classes composed of left/right or radially symmetric class members display such lineage convergence, in that individual neurons of the same class derive from distinct, non-bilaterally symmetric lineages. We show here that the C. elegans Atonal homolog lin-32/Ato, a bHLH transcription factor, is differentially expressed in neuronal lineages that give rise to left/right or radially symmetric class members. Loss of lin-32/Ato results in the selective loss of the expression of panneuronal markers and terminal selector-type transcription factors that confer neuron class-specific features. We discovered that another bHLH transcription factor, the Achaete Scute-homolog hlh-14 is expressed in mirror image pattern to lin-32/Ato in a subset of the left/right symmetric neuron pairs and is required to induce neuronal identity and terminal selector expression on the contralateral side of the animal. These findings demonstrate that distinct lineage histories converge via distinct bHLH factors on the level of induction of terminal selector identity determinants, which thus serve as integrators of distinct lineage histories. We also describe neuron-to-neuron identity transformations in lin-32/Ato mutants, which we propose to also be the result of misregulation of terminal selector gene expression.

category: developmental biology


title: Insight into Canary Island pine physiology provided by stable isotope patterns of water and plant tissues along an altitudinal gradient

abstract: The Canary Islands, an archipelago east of Moroccos Atlantic coast, present steep altitudinal gradients covering various climatic zones from hot deserts to subalpine Mediterranean, passing through fog-influenced cloud forests. Unlike the majority of the Canarian flora, Pinus canariensis grow along most of these gradients, allowing the study of plant functioning in contrasting ecosystems. Here we assess the water sources (precipitation, fog) of P. canariensis and its physiological behavior in its different natural environments. We analyzed carbon and oxygen isotope ratios of water and organics from atmosphere, soil and different plant organs and tissues (including 10-year annual time series of tree-ring cellulose) of six sites from 480 to 1990 m asl on the Canary island La Palma. We found a decreasing {delta}18O trend in source water that was overridden by an increasing {delta}18O trend in needle water, leaf assimilates and tree-ring cellulose with increasing altitude, suggesting site-specific tree physiological responses to relative humidity. Fog-influenced and fog-free sites showed similar {delta}13C values, suggesting photosynthetic activity to be limited by stomatal closure and irradiance at certain periods. Besides, we observed an 18O-depletion (fog-free and timberline sites) and 13C-depletion (fog-influenced and fog-free sites) in latewood compared to earlywood caused by seasonal differences in: (i) water uptake (i.e. deeper ground water during summer drought, fog water frequency and interception) and (ii) meteorological conditions (stem radial growth and latewood {delta}18O correlated with winter precipitation). In addition, we found evidence for foliar water uptake and strong isotopic gradients along the pine needle axis in water and assimilates. These gradients are likely the reason for an unexpected underestimation of pine needle water {delta}18O when applying standard leaf water {delta}18O models. Our results indicate that soil water availability and air humidity conditions are the main drivers of the physiological behavior of pine along the Canary Islands altitudinal gradients.


title: Genome- and Transcriptome-wide Splicing Associations with Problematic Alcohol Use and Alcohol Use Disorder

abstract: Genetic mechanisms of alternative mRNA splicing have been shown in the brain for a variety of neuropsychiatric traits, but not substance use disorders. Our study used RNA-sequencing data on alcohol use disorder (AUD) in the brains reward circuitry (n=56; ages 40-73; 100%  Caucasian; four brain regions) and genome-wide association data on problematic alcohol use (n=435,563, ages 22-90; 100% European-American) to investigate potential genetic links with alcohol-related alternative mRNA splicing. Polygenic scores of problematic alcohol use predicted alternative mRNA brain splicing associated with AUD, which depended on brain region. Across brain regions, we found 714 differentially spliced genes in various putative addiction genes and other novel gene targets. We found 6,463 splicing quantitative trait loci (sQTLs) that were associated with the AUD differentially spliced genes. sQTLs were enriched in loose chromatin genomic regions and downstream gene targets. Additionally, the heritability of problematic alcohol use was significantly enriched for DNA variants in and around differentially spliced genes associated with AUD. Our study also performed splicing transcriptome-wide association studies (TWASs) of problematic alcohol use and other drug use traits that unveiled individual genes for follow-up and robust splicing correlations across SUDs. Finally, we show that differentially spliced genes associated showed significant overlap in primate models of chronic alcohol consumption at the gene-level in similar brain regions. Altogether, our study illuminates substantial genetic contributions of alternative mRNA splicing in relation to problematic alcohol use and AUD.


title: Automatic Multi-functional Integration Program (AMFIP) towards All-optical Mechanobiology Interrogation

abstract: Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for biomedical studies of active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. Manager is a widely used open-source software platform for controlling many optoelectronic instruments. Due to limited development since its introduction, Manager lacks compatibility with some of the latest microscopy equipment. To address this unmet need, we have developed a novel software-based automation program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent plugin for Manager. Without extra hardware, AMFIP enables the functional synchronization of Manager, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for many hardware and software. Users can customize AMFIP according to their own specific experimental requirements and hardware environments. To verify AMFIPs performance, we applied it to elucidate the relationship between cell spreading and spatial-temporal cellular expression of Yes-associated protein (YAP), a mechanosensitive protein that shuttles between cytoplasm and nucleus upon mechanical stimulation, in an epithelial cell line. We found that the ratio of YAP expression in nucleus and cytoplasm decreases as the spreading area of cells increases, suggesting that the accumulation of YAP in the nucleus decreases throughout the cell spreading processes. In summary, AMFIP provides a new open-source and charge-free solution to integrate multiple hardware and software to satisfy the need of automatic imaging operations in the scientific community.

category: bioengineering


title: Influence of substrate stiffness on barrier function in an iPSC-derived in vitro blood-brain barrier model

abstract:
IntroductionVascular endothelial cells respond to a variety of biophysical cues such as shear stress and substrate stiffness. In peripheral vasculature, extracellular matrix (ECM) stiffening alters barrier function, leading to increased vascular permeability in atherosclerosis and pulmonary edema. The effect of ECM stiffness on blood-brain barrier (BBB) endothelial cells, however, has not been explored. To investigate this topic, we incorporated hydrogel substrates into an in vitro model of the human BBB.

MethodsInduced pluripotent stem cells were differentiated to brain microvascular endothelial-like (BMEC-like) cells and cultured on hydrogel substrates of varying stiffness. Cellular changes were measured by imaging, functional assays such as transendothelial electrical resistance (TEER) and p-glycoprotein efflux activity, and bulk transcriptome readouts.

ResultsThe magnitude and longevity of TEER in iPSC-derived BMEC-like cells is enhanced on compliant substrates. Quantitative imaging shows that BMEC-like cells form fewer intracellular actin stress fibers on substrates of intermediate stiffness (20 kPa relative to 1 kPa and 150 kPa). Chemical induction of actin polymerization leads to a rapid decline in TEER, agreeing with imaging readouts. P-glycoprotein activity is unaffected by substrate stiffness. Modest differences in RNA expression corresponding to specific signaling pathways were observed as a function of substrate stiffness.

ConclusionsiPSC-derived BMEC-like cells exhibit differences in passive but not active barrier function in response to substrate stiffness. These findings may provide insight into BBB dysfunction during neurodegeneration, as well as aid in the optimization of more complex threedimensional neurovascular models utilizing compliant hydrogels.


title: ADAPTATION OF DOLPHINS (TURSIOPS TRUNCATUS) LOCATION SIGNALS WHEN SEARCHING AND RECOGNIZING OBJECTS HIDDEN BY SEA SEDIMENTS

abstract:
An experimental study was conducted to research dolphins sonar adaptation capabilities for location of objects obscured by marine sediments. It was shown that dolphins are able to alter spectral and temporal characteristics of their location signals. Adaptive change of length, spectral width, and amplitude of impulses provides optimal tools to fight interference and allows dolphins to effectively identify objects.

SUMMARYThis work describes dolphins search, location and recognition objects hidden by sea sediments and changes in its location signal structure.


title: Furanoditerpenoid biosynthesis in the bioenergy crop switchgrass is catalyzed by an alternate metabolic pathway

abstract:
Specialized diterpenoid metabolites are important mediators of stress resilience in monocot crops. A deeper understanding of how species-specific diterpenoid-metabolic pathways and functions contribute to plant chemical defenses can enable crop improvement strategies. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop switchgrass (Panicum virgatum). Combinatorial pathway reconstruction showed that CYP71Z25-29 catalyze furan ring addition to diterpene alcohol intermediates derived from distinct class II diterpene synthases, thus bypassing the canonical role of class I diterpene synthases in plant diterpenoid metabolism. Transcriptional co-expression patterns and presence of select diterpenoids in droughted switchgrass roots support possible roles of CYP71Z25-29 in abiotic stress responses. Integrating molecular dynamics, structural analysis, and targeted mutagenesis, identified active site determinants controlling distinct CYP71Z25-29 catalytic specificities and, combined with broad substrate promiscuity for native and non-native diterpenoids, highlights the potential of these P450s for natural product engineering.

Significance StatementDiterpenoids play important roles in stress resilience and chemically mediated interactions in many plant species, including major food and bioenergy crops. Enzymes of the cytochrome P450 monooxygenase family catalyze the various functional decorations of core diterpene scaffolds that determine the large diversity of biologically active diterpenoids. This study describes the identification and mechanistic analysis of an unusual group of cytochrome P450 monooxygenases, CYP71Z25-29, from the bioenergy crop switchgrass (Panicum virgatum). These enzymes catalyze the furan ring addition directly to class II diterpene synthase products, thus bypassing the conserved pairwise reaction of class II and class I diterpene synthases in labdane diterpenoid metabolism. Insight into the distinct substrate-specificity of CYP71Z25-29 offers opportunity for engineering of furanoditerpenoid bioproducts.

category: plant biology


title: Reducing publication delay to improve the efficiency and impact of conservation science

abstract: Evidence-based decision making is most effective with comprehensive access to scientific studies. If studies face delays or barriers to being published, the useful information they contain may not reach decision-makers in a timely manner. This represents a potential problem for mission-oriented disciplines where access to the latest data is paramount to ensure effective actions are deployed. We sought to analyse the severity of publication delay in conservation science -- a field that requires urgent action to prevent the loss of biodiversity. We used the Conservation Evidence database to assess the length of publication delay (time from finishing data collection to publication) in the literature that tests the effectiveness of conservation interventions. From 7,415 peer-reviewed and non-peer-reviewed studies of conservation interventions published over eleven decades, we find that the mean publication delay (time from completing data collection to publication) was 3.6 years and varied by conservation subject -- a smaller delay was observed for studies focussed on the management of captive animals. Publication delay was significantly smaller for studies in the non-journal literature (typically non-peer-reviewed) compared to studies published in scientific journals. Although we found publication delay has marginally increased over time (1912-2020), this change was weak post-1980s. Publication delay also varied inconsistently between studies on species with different IUCN Red List statuses and there was little evidence that studies on more threatened species were subject to a smaller delay. We discuss the possible drivers of publication delay and present suggestions for scientists, funders, publishers, and practitioners to reduce the time taken to publish studies. Although our recommendations are aimed at conservation science, they are highly relevant to other mission-driven disciplines where the rapid dissemination of scientific findings is important.

category: scientific communication and education


title: Impact of fenbendazole resistance in Ascaridia dissimilis on the economics of production in turkeys

abstract: Feed conversion efficiency is among the most important factors affecting profitable production of poultry. Infections with parasitic nematodes can decrease efficiency of production, making parasite control through the use of anthelmintics an important component of health management. In ruminants and horses, anthelmintic resistance is highly prevalent in many of the most important nematode species, which greatly impacts their control. Recently, we identified resistance to fenbendazole in an isolate of Ascaridia dissimilis, the most common intestinal helminth of turkeys. Using this drug-resistant isolate, we investigated the impact that failure to control infections has on weight gain and feed conversion in growing turkeys. Birds were infected on Day 0 with either a fenbendazole-susceptible or -resistant isolate, and then half were treated with fenbendazole (SafeGuard(R) Aquasol) at 4- and 8-weeks post infection. Feed intake and bird weight were measured for each pen weekly throughout the study, and feed conversion rate was calculated. Necropsy was performed on birds from each treatment group to assess worm burdens at weeks 7 and 9 post infection. In the birds infected with the susceptible isolate, fenbendazole-treated groups had significantly better feed conversion as compared to untreated groups. In contrast, there were no significant differences in feed conversion between the fenbendazole-treated and untreated groups in the birds infected with the resistant isolate. At both weeks 7 and 9, worm burdens were significantly different between the treated and untreated birds infected with the drug-susceptible isolate, but not in the birds infected with the drug-resistant isolate. These significant effects on feed conversion were seen despite having a rather low worm establishment in the birds. Overall, these data indicate that A. dissimilis can produce significant reductions in feed conversion, and that failure of treatment due to the presence of fenbendazole-resistant worms can have a significant economic impact on turkey production. Furthermore, given the low worm burdens and an abbreviated grow out period of this study, the levels of production loss we measured may be an underestimate of the true impact that fenbendazole-resistant worms may have on a commercial operation.


title: Fenbendazole resistance in Heterakis gallinarum, the vector of Histomonas meleagridis, the causative agent of Blackhead Disease in poultry.

abstract: Due to their ubiquity, management of parasites is a common and important factor for profitable production of poultry. Heterakis gallinarum, the cecal nematode, is the most common nematode parasite of poultry. While typically causing no pathology on its own, H. gallinarum is the vector of Histomonas meleagridis, a protozoan parasite that causes blackhead disease. Histomonas meleagridis is highly pathogenic in turkeys, potentially causing high mortality. In contrast, disease caused by H. meleagridis is much less severe in chickens, where it primarily reduces productivity without manifestations of clinical disease. There are no approved treatments for H. meleagridis, making control reliant on control of the helminth vector through the use of fenbendazole (FBZ) the only drug labeled for treatment of H. gallinarum in the United States We were contacted by an industry veterinarian regarding health-related concerns in a broiler-breeder house due to histomoniasis, despite frequent anthelmintic treatments. Since we had recently diagnosed resistance to FBZ in Ascaridia dissimilis, a closely related nematode of turkeys, we were interested to determine if H. gallinarum had also evolved resistance to FBZ. Heterakis gallinarum eggs were isolated from litter collected from the house and used to infect 108 chickens. Treatment groups included a non-treated control, a label-dose and a 2X-label dose of FBZ, with 36 birds per group divided into two replicate pens of 18 birds each. Birds were placed at 1-day post hatch, and at 3 weeks of age were infected with 150 embryonated eggs via oral gavage. Two weeks post infection, treated birds were administered a minimum of either a label- or 2X label-dose of FBZ in water for 5 days (SafeGuard(R) Aquasol, 1mg/kg BW). To ensure that all birds consumed the full intended dose at a minimum, the dosage was calculated using 1.25 times the average body weight. One-week post treatment, birds were euthanized, ceca removed, and parasites enumerated. Efficacy was calculated by comparing the total numbers of worms recovered from each treatment group to the numbers recovered in the non-treated control group. There were no significant differences in worm numbers recovered from any of the three groups (p-value=0.81). There also was no efficacy benefit to treatment with a 2X dose; H. gallinarum worm counts were reduced by 42.7% and 41.4%, for the label and 2X dosages, respectively. These data provide strong evidence that H. gallinarum has developed resistance to FBZ. Consequently, in houses infected with FBZ-resistant H. gallinarum, H. meleagridis will be able to cycle through the birds in an unrestricted manner. Further investigation is needed to determine the prevalence of resistance in H. gallinarum on chicken farms, but it is clear this has the potential to have a large-scale economic impact on the poultry industry. These data when viewed together with our recent findings of FBZ resistance in A. dissimilis, suggest that drug resistance in ascarid nematodes may be an important emerging problem on poultry operations.


title: The effect of double biofeedback on functional hemispheric asymmetry and activity: a pilot study

abstract: In the current pilot study, we attempt to find out how double neurofeedback influences functional hemispheric asymmetry and activity. We examined 30 healthy participants (8 males; 22 females, mean age = 29; SD= 8). To measure functional hemispheric asymmetry and activity, we used computer laterometry in the  two-source lead-lag dichotic paradigm. Double biofeedback included 8 minutes of EEG oscillation recording with five minutes of basic mode. During the basic mode, the current amplitude of the EEG oscillator gets transformed into feedback sounds while the current amplitude of alpha EEG oscillator is used to modulate the intensity of light signals. Double neurofeedback did not directly influence the asymmetry itself but accelerated individual sound perception characteristics during dichotic listening in the preceding effect paradigm. Further research is needed to investigate the effect of double neurofeedback training on functional brain activity and asymmetry taking into account participants age, gender, and motivation.


title: Attention improves information flow between neuronal populations without changing the communication subspace

abstract: Visual attention allows observers to flexibly use or ignore visual information, suggesting that information can be flexibly routed between visual cortex and neurons involved in decision-making. We investigated the neural substrate of flexible information routing by analyzing the activity of populations of visual neurons in the medial temporal area (MT) and oculomotor neurons in the superior colliculus (SC) while rhesus monkeys switched spatial attention. We demonstrated that attention increases the efficacy of visuomotor communication: trial-to-trial variability of the population of SC neurons was better predicted by the activity of MT neurons (and vice versa) when attention was directed toward their joint receptive fields. Surprisingly, this improvement in prediction was not explained or accompanied by changes in the dimensionality of the shared subspace or in local or shared pairwise noise correlations. These results suggest a mechanism by which visual attention can affect perceptual decision-making without altering local neuronal representations.