title: A baton-relay mechanism for ER retrieval signal capture and proofreading by the KDEL receptor

abstract: The KDEL-retrieval pathway captures escaped ER proteins with a KDEL or variant C-terminal signal at acidic pH in the Golgi and releases them at neutral pH in the ER. To address the mechanism of signal binding and the molecular basis for differences in signal affinity, we determined the HDEL and RDEL bound structures of the KDEL-receptor. Affinity differences are explained by interactions between the variable -4 position of the signal and W120, whereas initial capture of retrieval signals by their carboxyl-terminus is mediated by a baton-relay mechanism involving a series of conserved arginine residues in the receptor. This explains how the signal is first captured and then pulled into the binding cavity. During capture, retrieval signals undergo a selective proofreading step involving two gatekeeper residues D50 and E117 in the receptor. These mechanisms operate upstream of the pH-dependent closure of the receptor and explain the selectivity of the KDEL-retrieval pathway.

category: cell biology


title: Chimpanzees communicate to coordinate a cultural practice

abstract: Human culture is considered to differ from animal culture due to its interactive nature built on shared intentionality and cognitive flexibility. Here, we investigated whether chimpanzees use communication to engage in cultural practices by analyzing grooming handclasp (GHC) interactions - a socio-cultural behavior requiring coordination. Previous accounts attributed GHC initiations to behavioral shaping whereby the initiator physically molds the partners arm into the GHC posture. Using frame-by-frame analysis and matched-control methodology, we find that chimpanzees use gestural communication to initiate GHC, which requires an active and synchronized response from the partner. This showcases a behavioral expression of joint commitment to engage in this shared cultural practice. Moreover, we show that GHC initiators used various initiation strategies, attesting to situation-contingent interactional flexibility. We conclude that chimpanzees can be jointly committed to a cultural practice, which suggests that culture predicated on shared intentionality and flexible communication may not be unique to the human species.

category: animal behavior and cognition


title: Salty surfaces deter feeding in a blood-sucking disease vector

abstract:
Salts are essential nutrients required for many physiological processes, and deficient or excessive salt results in adverse health problems. Taste is the ultimate sensory modality involved in resource quality assessment, resulting in acceptance or rejection. Here, we show that detection of high-salt substrates by a salt-sensitive antennal gustatory receptor neuron, S1-GRN, results in feeding avoidance in the hematophagous bug Rhodnius prolixus. Knock-down of two antennal-expressed amiloride-sensitive pickpocket channel receptors (PPKs; RproPPK014276 and RproPPK28) using RNA interference, prevents avoidance of bugs to high-salt substrates. Tracing antennal GRNs to the central nervous system reveals the antennal lobes as a gustatory processing center. The identification of the gustatory basis of high-salt detection in a blood feeder provides novel targets to prevent biting and feeding, as well as to promote substrate avoidance in a relevant disease vector.

Significance StatementDetection of aversive gustatory stimuli induces avoidance responses in animals. Avoidance acquires particular interest if it reduces the biting rates of blood-feeding insects of medical relevance. Here we describe the molecular and physiological basis of high-salt detection in the blood-sucking disease vector Rhodnius prolixus. We show that detection of high-salt substrates through two PPK receptors expressed in an antennal gustatory receptor neuron produces feeding avoidance. Understanding these gustatory-driven aversive responses allows the hitherto overlooked use of gustatory molecules as a complement to known olfactory repellents.

category: physiology


title: Biophysical properties governing septin assembly

abstract: Septin filaments build structures such as rings, lattices and gauzes that serve as platforms for localizing signaling and organizing cell membranes. How cells control the geometry of septin assemblies in poorly understood. We show here that septins are isodesmic polymers, in contrast to cooperative polymerization exhibited by F-actin and microtubules. We constructed a physical model to analyze and interpret how septin assemblies change in the presence of regulators in yeast extracts. Notably filaments differ in length and curvature in yeast extract compared to pure protein indicating cellular regulators modulate intrinsic biophysical features. Combining analysis of extracts from regulatory mutants with simulations, we found increased filament flexibility and reduced filament fragmentation promote assembly of septin rings, whereas reduced flexibility in crowded environments promotes local filament alignment. This work demonstrates how tuning of intrinsic features of septin filament assembly by regulatory proteins yields a diverse array of structures observed in cells.


title: Evolution of mechanisms controlling epithelial morphogenesis across animals: new insights from dissociation - reaggregation experiments in the sponge Oscarella lobularis.

abstract:
BackgroundThe ancestral presence of epithelia in Metazoa is no longer debated. Even though Porifera seem to be the best candidates to be the sister group to all other Metazoa, hardly anything is known about the proteins involved in the composition of cell-cell junctions or about the mechanisms that regulate epithelial morphogenetic processes in this phylum.

ResultsTo get insights into the early evolution of epithelial morphogenesis, we focused on morphogenic characteristics of the homoscleromorph sponge Oscarella lobularis. Homoscleromorpha are a sponge class with a typical basement membrane and adherens-like junctions unknown in other sponge classes. We took advantage of the dynamic context provided by cell dissociation-reaggregation experiments to explore morphogenetic processes in epithelial cells in an early lineage by combining fluorescent and electronic microscopy observations and RNA sequencing approaches at key time-points of the dissociation and reaggregation processes.

ConclusionsOur results show that part of the molecular toolkit involved in the loss and restoration of epithelial features such as cell-cell and cell-matrix adhesion is conserved between Homoscleromorpha and Bilateria, suggesting their common role in the last common ancestor of animals. In addition, Sponge-specific genes are differently expressed during the dissociation and reaggregation processes, calling for future functional characterization of these genes.

category: evolutionary biology


title: A Kalirin Missense Mutation Enhances Dendritic RhoA Signaling and Leads to Regression of Cortical Dendritic Arbors Across Development

abstract:
Normally, dendritic size is established prior to adolescence then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor, Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wildtype KAL. In mice containing this missense mutation at the endogenous locus there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Tissue density of dendritic spines was also reduced. Early adult mice with these structural deficts exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.

SIGNIFICANCE STATEMENTDendrites are long branching processes on neurons that contain small processes called spines that are the site of connections with other neurons, establishing cortical circuitry. Dendrites have long been considered stable structures, with rapid growth prior to adolescence followed by maintenance of size into adulthood. However, schizophrenia is characterized by accelerated reductions of cortical gray matter volume and onset of clinical symptoms during adolescence, with reductions in dendritic length present when examined after death. We show that dendrites retain the capacity for regression, and that a mild genetic vulnerability in a regression pathway leads to onset of structural impairments in previously formed dendrites across adolescence. This suggests that targeting specific regression pathways could potentially lead to new therapeutics for schizophrenia.

category: neuroscience


title: Endosomal Acidification by NHE6-depletion Corrects ApoE4-mediated Synaptic Impairments and Reduces Amyloid Plaque Load

abstract: Apolipoprotein E4 (ApoE4) is the most important and prevalent risk factor for late-onset Alzheimers disease (AD). The isoelectric point of ApoE4 matches the pH of the early endosome (EE), causing its delayed dissociation from ApoE receptors and hence impaired endolysosomal trafficking, disruption of synaptic homeostasis and reduced amyloid clearance. We have shown that enhancing endosomal acidification by inhibiting the EE-specific sodium-hydrogen exchanger NHE6 restores vesicular trafficking and normalizes synaptic homeostasis. Remarkably and unexpectedly, loss of NHE6 effectively suppressed amyloid deposition even in the absence of ApoE4, suggesting that accelerated acidification of early endosomes caused by the absence of NHE6 occludes the effect of ApoE on amyloid plaque formation. NHE6 suppression or inhibition may thus be a universal, ApoE-independent approach to prevent amyloid buildup in the brain. These findings suggest a novel therapeutic approach for the prevention of AD by which partial NHE6 inhibition reverses the ApoE4 induced endolysosomal trafficking defect and reduces amyloid.


title: NHE6-Depletion Corrects ApoE4-Mediated Synaptic Impairments and Reduces Amyloid Plaque Load


title: Pax6 regulates the morphological and electrophysiological development of mouse prethalamic neurons

abstract:
Pax6 is a well-known regulator of early neuroepithelial progenitor development. Its constitutive loss has a particularly strong effect on the developing prethalamus, causing it to become extremely hypoplastic. To overcome this difficulty in studying the long-term consequences of Pax6 loss for prethalamic development, we used conditional mutagenesis to delete Pax6 at the onset of neurogenesis and studied the developmental potential of the mutant prethalamic neurons in vitro. We found that Pax6 loss affected their rates of neurite elongation, the location and length of their axon initial segments and their electrophysiological properties. Our results broaden our understanding of the long-term consequences of Pax6 deletion in the developing mouse forebrain, suggesting that it can have cell autonomous effects on the structural and functional development of some neurons.

SUMMARY STATEMENTPax6 impacts neurite extension, axon initial segment properties and ability to fire normal action potentials in maturing neurons, revealing actions extending beyond those previously characterized in progenitors.

category: developmental biology


title: Pax6 loss alters the morphological and electrophysiological development of mouse prethalamic neurons


title: Entangled radicals may explain lithium effects on hyperactivity

abstract: It is known that bipolar disorder and its lithium treatment involve the modulation of oxidative stress. Moreover, it has been observed that lithiums effects are isotope-dependent. Based on these findings, here we propose that lithium exerts its effects by influencing the recombination dynamics of a naturally occurring radical pair involving oxygen. We develop a simple model inspired by the radical-pair mechanism in cryptochrome in the context of avian magnetoreception and xenon-induced anesthesia. Our model reproduces the observed isotopic dependence in the lithium treatment of hyperactivity in rats. It predicts a magnetic-field dependence of the effectiveness of lithium, which provides one potential experimental test of our hypothesis. Our findings show that Nature might harness quantum entanglement for the brains cognitive processes.


title: Optogenetic Activation of Striatal D1/D2 Medium Spiny Neurons Differentially Engages Downstream Connected Areas Beyond the Basal Ganglia

abstract:
The basal ganglia (BG) are a group of subcortical nuclei responsible for motor control, motor learning and executive function. Central to BG function are striatal medium spiny neurons (MSNs) expressing D1 and D2 dopamine receptors. D1 and D2 MSNs are typically considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. While their opposite role is well documented for certain sensorimotor loops of the BG-thalamocortical network, it is unclear whether MSNs maintain a uniform functional role across the striatum and which influence they exert on brain areas outside the BG. Here, we addressed these questions by combining optogenetic activation of D1 and D2 MSNs in the mouse ventrolateral caudoputamen (vl CPu) with whole-brain functional MRI (fMRI) recordings. Neuronal excitation of either cell population in the vl CPu evoked distinct activity patterns in key regions of the BG-thalamocortical network including the pallidum, thalamus and motor cortex. Importantly, we report that striatal D1 and D2 MSN stimulation differentially engaged cerebellar and prefrontal regions. We characterised these long-range interactions by computational modelling of effective connectivity and confirmed that changes in D1 / D2 output drive functional relationships between regions within and beyond the BG. These results suggest a more complex functional organization of MSNs across the striatum than previously anticipated and provide evidence for the existence of an interconnected fronto - BG - cerebellar network modulated by striatal D1 and D2 MSNs.

Graphical Abstract

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title: Recognition of familiar objects in tortoise hatchlings (Testudo spp.)

abstract: Tortoises do not show parental care and live solitary except for the context of reproduction. Despite their limited need to interact with conspecifics, we previously observed that young tortoises, at their first experiences with conspecifics, can discriminate between familiar and unfamiliar conspecifics after just one encounter with another tortoise. Tortoise hatchlings ignored familiar conspecifics, while they first explored and then actively avoided unfamiliar conspecifics. It remains to be established whether the different reactions to unfamiliar and familiar individuals in tortoise hatchlings are reactions to novelty, or whether they are specific to the interactions with living animals. To test this, we familiarized one-month-old tortoise hatchlings with an object (a brown cone vs. a blue sphere) and then tested them in a novel arena once with the familiar object and once with an unfamiliar one. To measure the reactions toward familiar and unfamiliar objects, we measured the distance between the tortoise and the object throughout the test. Differently from what happened with unfamiliar and familiar conspecifics, we found no difference in behavior toward familiar and unfamiliar objects. This shows that the different reactions toward familiar and unfamiliar conspecifics previously observed are specific for social interactions and are not a mere reaction to the novelty effect. The behavioral responses displayed by young tortoises for unfamiliar conspecifics, but not for unfamiliar objects, show the relevance of social behavior from the beginning of life, even for solitary species.


title: Label-free screening of brain tissue myelin content using phase imaging with computational specificity (PICS)

abstract: Inadequate myelination in the central nervous system is associated with neurodevelopmental complications. Thus, quantitative, high spatial resolution measurements of myelin levels are highly desirable. We used spatial light interference microcopy (SLIM), a highly sensitive quantitative phase imaging (QPI) technique, to correlate the dry mass content of myelin in piglet brain tissue with dietary changes and gestational size. We combined SLIM micrographs with an AI classifying model that allows us to discern subtle disparities in myelin distributions with high accuracy. This concept of combining QPI label-free data with AI for the purpose of extracting molecular specificity has recently been introduced by our laboratory as phase imaging with computational specificity (PICS). Training on nine thousand SLIM images of piglet brain tissue with the 71-layer transfer learning model Xception, we created a two-parameter classification to differentiate gestational size and diet type with an accuracy of 82% and 80%, respectively. To our knowledge, this type of evaluation is impossible to perform by an expert pathologist or other techniques.

category: bioinformatics


title: Analysis of immune subtypes across the epithelial-mesenchymal plasticity spectrum

abstract: Epithelial-mesenchymal plasticity plays a critical role in many solid tumor types as a mediator of metastatic dissemination and treatment resistance. In addition, there is also a growing appreciation that the epithelial/mesenchymal status of a tumor plays a role in immune evasion and immune suppression. A deeper understanding of the immunological features of different tumor types has been facilitated by the availability of large gene expression datasets and the development of methods to deconvolute bulk RNA-Seq data. These resources have generated powerful new ways of characterizing tumors, including classification of immune subtypes based on differential expression of immunological genes. In the present work, we combine scoring algorithms to quantify epithelial-mesenchymal plasticity with immune subtype analysis to understand the relationship between epithelial plasticity and immune subtype across cancers. We find heterogeneity of epithelial-mesenchymal transition (EMT) status both within and between cancer types, with greater heterogeneity in the expression of EMT-related factors than of MET-related factors. We also find that specific immune subtypes have associated EMT scores and differential expression of immune checkpoint markers.

category: cancer biology


title: Cancer models and statistical analysis of age distribution of cancers

abstract:
In this paper, mathematical mutational models of the age distribution of cancers are obtained. These are two models - a simple model and a complex model, which takes into account the growth of the cell population and the transmission of mutations to daughter cells. Using the resulting formulas, we approximated real age-specific cancer incidence datasets in women (colon, lung, mammary, stomach) and men (colon, lung, prostate, stomach). We estimated parameters such as the average number of mutations (per cell per unit of time) and number of mutations required for cancer to occur.

The number of mutations averaged (over four types of cancer) required for cancer to occur is 5.5 (mutations per cell for women) and 6.25 (mutations per cell for men) for the complex mutational model.

As an alternative to mutational models, we also consider the model of delayed carcinogenic event.


title: Antidepressant and antipsychotic drugs reduce viral infection by SARS-CoV-2 and fluoxetine show antiviral activity against the novel variants in vitro

abstract:
Background and PurposeRepurposing of currently available drugs is a valuable strategy to tackle the consequences of COVID-19. Recently, several studies have investigated the effect of psychoactive drugs on SARS-CoV-2 in cell culture models as well as in clinical practice. Our aim was to expand these studies and test some of these compounds against newly emerged variants.

Experimental ApproachSeveral antidepressant drugs and antipsychotic drugs with different primary mechanisms of action were tested in ACE2/TMPRSS2-expressing human embryonic kidney cells against the infection by SARS-CoV-2 spike protein-dependent pseudoviruses. Some of these compounds were also tested in human lung epithelial cell line, Calu-1, against the first wave (B.1) lineage of SARS-CoV-2 and the variants of concern, B.1.1.7 and B.1.351.

Key ResultsSeveral clinically used antidepressants, including fluoxetine, citalopram, reboxetine, imipramine, as well as antipsychotic compounds chlorpromazine, flupenthixol, and pimozide inhibited the infection by pseudotyped viruses with minimal effects on cell viability. The antiviral action of several of these drugs was verified in Calu-1 cells against the (B.1) lineage of SARS-CoV-2. By contrast, the anticonvulsant carbamazepine, and novel antidepressants ketamine and its derivatives as well as MAO and phosphodiesterase inhibitors phenelzine and rolipram, respectively, showed no activity in the pseudovirus model. Furthermore, fluoxetine remained effective against pseudo viruses with N501Y, K417N, and E484K spike mutations, and the VoC-1 (B.1.1.7) and VoC-2 (B.1.351) variants of SARS-CoV-2.

Conclusion and ImplicationsOur study confirms previous data and extends information on the repurposing of these drugs to counteract SARS-CoV-2 infection including different variants of concern.

category: pharmacology and toxicology


title: Inferring livestock movement networks from archived data to support infectious disease control in developing countries

abstract:
The use of network analysis to support livestock disease control in low middle-income countries (LMICs) has historically been hampered by the cost of generating empirical data in the absence of animal movement recording schemes. To fill this gap, methods which exploit freely available demographic and archived molecular data can be used to generate livestock networks based on gravity and phylogeographic modelling techniques, respectively. However, questions remain on the performance of these methods in capturing the topology of empirical networks. Here, we compare output from these network methodologies to a network constructed from either empirical data or randomly generated data. To facilitate this comparison, the spread of infectious diseases was simulated, it is this evaluation that demonstrates their potential utility to inform robust livestock disease control strategies.

The molecular network was the closest approximation to the empirical network, both in relation to topological and epidemic characteristics, whereas size of epidemics in the gravity network tended to be larger, better agreement across all three networks was observed when; a) total nodes infected, b) percentage infection take off were compared. These methods consistently identified the same important animal movement and trade hotspots as the empirical networks. We therefore consider this proof-of-concept that demographic data such as censuses and archived molecular data could be repurposed to inform livestock disease management in LMICs.

Author summaryLive animal movements in Africa represent a significant risk of transmission and spread of infectious diseases in livestock populations, and therefore, have direct implications on the food security of the continent. Here we explore the potential utility of available data to support control strategies, by comparing movement networks inferred from such data i.e. census and pathogen molecular data using gravity modelling and phylogeography respectively. Their utility is evaluated by comparing their topology and disease spread characteristics to empirical live animal movement. Based on our results, we posit that archived data can be repurposed to support infectious disease control on the African continent.

category: scientific communication and education


title: Glucose-sensing in AgRP neurons integrates homeostatic energy state with dopamine signalling in the striatum.

abstract: Hunger increases the motivation of an organism to seek out and consume highly palatable energy dense foods. While hunger-sensing Agouti-related peptide (AgRP) neurons influence this process, whether metabolic detection of homeostatic state via metabolic sensing in AgRP neurons potentiates motivation through the midbrain dopamine system is unexplored. Here, we used the AgRP-specific deletion of carnitine acetyltransferase (Crat), a metabolic enzyme regulating glucose and fatty acid oxidation, as a model of impaired metabolic-sensing in AgRP neurons. We then tested the hypothesis that appropriate metabolic-sensing in AgRP neurons is required to increase food reward motivation by modulating accumbal or striatal dopamine release. Electrophysiological studies confirm that Crat deletion in AgRP neurons (KO) impairs normal ex vivo glucose-sensing, and in vivo photometry experiments show that AgRP neurons in KO mice do not exhibit normal responses to repeated palatable food presentation and consumption, highlighting that this model is appropriate to test the hypothesis. Fiber photometry experiments, using the dopamine sensor GRAB-DA, revealed that impaired metabolic-sensing reduces acute dopamine release (seconds) in the nucleus accumbens, but not the dorsal striatum, to palatable food consumption and during operant responding. Positron electron tomography (PET) methods indicated that impaired metabolic-sensing in AgRP neurons suppressed radiolabelled 18F-fDOPA accumulation after [~]30 minutes in the dorsal striatum but not the ventral striatum, suggesting a role for AgRP neurons to restrict a long term post-ingestive dopamine response in the dorsal striatum. Finally, impaired metabolic-sensing in AgRP neurons suppresses motivated operant responding for sucrose rewards. Notably, these behavioural effects are potentiated in the hungry state and therefore highlight that metabolic-sensing in AgRP neurons is required for the appropriate temporal integration and transmission of homeostatic hunger-sensing to dopamine signalling in the striatum.


title: Metabolic-sensing in AgRP neurons integrates homeostatic state with dopamine signalling in the striatum


title: Noise Effect on the Temporal Patterns of Neural Synchrony

abstract:
Neural synchrony in the brain is often present in an intermittent fashion, i.e., there are intervals of synchronized activity interspersed with intervals of desynchronized activity. A series of experimental studies showed that this kind of temporal patterning of neural synchronization may be very specific and may be correlated with behavior (even if the average synchrony strength is not changed). Prior studies showed that a network with many short desynchronized intervals may be functionally different from a network with few long desynchronized intervals as it may be more sensitive to synchronizing input signals. In this study, we investigated the effect of channel noise on the temporal patterns of neural synchronization. We employed a small network of conductance-based model neurons that were mutually connected via excitatory synapses. The resulting dynamics of the network was studied using the same time-series analysis methods as used in prior experimental and computational studies. While it is well known that synchrony strength generally degrades with noise, we found that noise also affects the temporal patterning of synchrony. Noise, at a sufficient intensity (yet too weak to substantially affect synchrony strength), promotes dynamics with predominantly short (although potentially very numerous) desynchronizations. Thus, channel noise may be one of the mechanisms contributing to the short desynchronization dynamics observed in multiple experimental studies.

HighlightsO_LIChannel noise alters the temporal pattern of intermittent neural synchrony
C_LIO_LINoise may alter this pattern without significant change in average synchrony strength
C_LIO_LIThe resulting patterning is similar to that observed in multiple experiments
C_LI


title: Failure of the brain glucagon-like peptide-1-mediated control of intestinal redox homeostasis in a rat model of sporadic Alzheimer's disease

abstract: The gastrointestinal system may be involved in the etiopathogenesis of the insulin-resistant brain state (IRBS) and Alzheimers disease (AD). Gastrointestinal hormone glucagon-like peptide-1 (GLP-1) is being explored as a potential therapy as activation of brain GLP-1 receptors (GLP-1R) exerts neuroprotection and controls peripheral metabolism. Intracerebroventricular administration of streptozotocin (STZ-icv) is used to model IRBS and GLP-1 dyshomeostasis seems to be involved in the development of neuropathological changes. The aim was to explore i) gastrointestinal homeostasis in the STZ-icv model ii) assess whether the brain GLP-1 is involved in the regulation of gastrointestinal redox homeostasis and iii) analyze whether brain-gut GLP-1 axis is functional in the STZ-icv animals. Acute intracerebroventricular treatment with exendin-3(9-39)amide was used for pharmacological inhibition of brain GLP-1R in the control and STZ-icv rats, and oxidative stress was assessed in plasma, duodenum and ileum. Acute inhibition of brain GLP-1R increased plasma oxidative stress. TBARS were increased, and LMWT, SH, and superoxide dismutase (SOD) were decreased in the duodenum, but not in the ileum of the controls. In the STZ-icv, TBARS and CAT were increased, LMWT and SH were decreased at baseline, and no further increment of oxidative stress was observed upon central GLP-1R inhibition. The presented results indicate that i) oxidative stress is increased in the duodenum of the STZ-icv rat model of AD, ii) brain GLP-1R signaling is involved in systemic redox regulation, iii) brain-gut GLP-1 axis regulates duodenal, but not ileal redox homeostasis, and iv) brain-gut GLP-1 axis is dysfunctional in the STZ-icv model.


title: Passive clearing and 3D lightsheet imaging of the intact and injured spinal cord in mice

abstract: The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.


title: TCF-1 in CD4 T cells regulates GVHD severity and persistence

abstract: Graft-versus-host disease (GVHD) is a leading cause of mortality following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mature donor T cells in the graft mediate graft-versus-leukemia (GVL) responses against residual tumor cells, which may persist after pre-transplant conditioning regimens. Importantly, the same mature T cells also mediate GVHD. The transcription factor T Cell Factor-1 (TCF-1) is critical for T cell development in the thymus. Using a unique mouse model of allo-HSCT leading to GVHD, we investigated the role of TCF-1 in alloactivated T cell functioning and in GVHD. Here, we report that loss of TCF-1 in mature CD4 T cells reduces GVHD severity and persistence, improving survival of recipient mice. This was due to reduced proliferation, survival, and cytokine production of T cells, as well as increased exhaustion. Gene pathways involved in cytokine response, immune signaling, chemokine signaling, cell cycle, and T cell differentiation were altered by loss of TCF-1 in donor cells. Our companion paper shows that regulation of alloactivated CD4 T cells by TCF-1 differs from regulation of CD8 T cells, suggesting that TCF-1 plays a unique role in each subset. Therefore, targeting of TCF-1 or downstream signaling pathways may be an effective strategy for reducing GVHD following allo-HSCT.

category: immunology


title: The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection

abstract: Summary ParagraphWe identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone1, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications2, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry3. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir4. Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginones translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

category: microbiology


title: Catalase Activity is Critical for Proteus mirabilis Biofilm Development, EPS Composition, and Dissemination During Catheter-Associated Urinary Tract Infection

abstract: Proteus mirabilis is a leading uropathogen of catheter-associated urinary tract infections (CAUTIs), which are among the most common healthcare-associated infections worldwide. A key factor that contributes to P. mirabilis pathogenesis and persistence during CAUTI is the formation of catheter biofilms, which provide increased resistance to antibiotic treatment and host defense mechanisms. Another factor that is important for bacterial persistence during CAUTI is the ability to resist reactive oxygen species (ROS), such as through the action of the catalase enzyme. Potent catalase activity is one of the defining biochemical characteristics of P. mirabilis, and its single catalase gene (katA) was recently identified as a candidate fitness factor for UTI, CAUTI, and bacteremia. Here we show that disruption of katA results in increased ROS levels, increased sensitivity to peroxide, and decreased biofilm biomass. The biomass defect was due to a decrease in extracellular polymeric substances (EPS) production by the {Delta}katA mutant, and specifically due to reduced carbohydrate content. Importantly, the biofilm defect resulted in decreased antibiotic resistance in vitro and a colonization defect during experimental CAUTI. The {Delta}katA mutant also exhibited decreased fitness in a bacteremia model, supporting a dual role for catalase in P. mirabilis biofilm development and immune evasion.


title: Genomic characterization of three novel Desulfobacterota classes expand the metabolic and phylogenetic diversity of the Phylum

abstract:
An overwhelming majority of bacterial life remains uncharacterized. Recent efforts to assemble genomes from metagenomes have provided invaluable insights into these yet-uncultured bacterial lineages. We report on the characterization of 30 genomes belonging to three novel classes within the phylum Desulfobacterota. One class (proposed name Candidatus "Anaeroferrophillalia") was characterized by the capacity for heterotrophic growth, either fermentatively or utilizing polysulfide, tetrathionate and thiosulfate as electron acceptors. Autotrophic growth using the Wood Ljungdahl pathway and hydrogen or Fe(II) as an electron donor could also occur in absence of organic carbon sources. The second class (proposed name Candidatus "Anaeropigmentia") was characterized by its capacity for fermentative or aerobic growth at low oxygen thresholds using a broad range of sugars and amino acids, and the capacity to synthesize the methyl/alkyl carrier CoM, an ability that is prevalent in the archaeal but rare in the bacterial domain. Pigmentation is inferred from the capacity for carotenoids (lycopene) production, as well as the occurrence of the majority of genes involved in bacteriochlorophyll a biosynthesis. The third class (proposed name Candidatus "Zymogenia") was characterized by the capacity for heterotrophic growth fermentatively using broad sugars and amino acids as carbon sources, and the adaptation of some of its members to hypersaline habitats. Analysis of the distribution pattern of all three classes showed their occurrence as rare community members in multiple habitats, with preferences for anaerobic terrestrial (e.g. hydrocarbon contaminated environments, wetlands, bioreactors), freshwater (e.g. ground water and gas-saturated temperate lakes), and marine (e.g. hydrothermal vents, marine sediments, and coastal sediments) environments, over oxygenated (e.g. pelagic ocean and agricultural land) settings. Special preference for some members of the class Candidatus "Zymogenia" to hypersaline environments, e.g. hypersaline microbial mats and lagoons was observed.

ImportanceCulture-independent diversity surveys conducted in the last three decades have clearly demonstrated that the scope of microbial diversity is much broader than that inferred from isolation efforts. Multiple reasons have been put forth to explain the refractiveness of a wide range of the earths microbiome to isolation efforts. Documenting the scope of high-rank phylogenetic diversity on earth, as well as deciphering and documenting the metabolic capacities, physiological preferences, and putative ecological roles of these yet-uncultured lineages represents one of the central goals in current microbial ecology research. Recent efforts to assemble genomes from metagenomes have provided invaluable insights into these yet-uncultured lineages. This study expands our knowledge of the phylum Desulfobacterota through the characterization of 30 genomes belonging to three novel classes. The analyzed genomes were either recovered from Zodletone Spring in southwestern Oklahoma in this study, or recently binned from public metagenomes as part of the Global Earth Microbiome initiative. Our results expand the high-rank diversity within the bacterial tree of life by describing three novel classes within the phylum Desulfobacterota, document the utilization of multiple metabolic processes, e.g. iron-oxidation, aromatic hydrocarbon degradation, reduction of sulfur-cycling intermediates, and features, e.g. coenzyme M biosynthesis, and pigmentation, as salient characteristics in these novel Desulfobacterota classes.


title: Diverse arsenic-containing lipids in the surface ocean

abstract:
Arsenic is present at nanomolar levels throughout the surface ocean, and microbes assimilate this toxic element due to its similarity to inorganic phosphorus. Although the concentration and characterization of dissolved arsenic has been a focus of ocean studies, the size of the particulate arsenic pool and its partitioning into organic molecules within the microbial community is not known. We measured the particulate pool of arsenic in five surface samples from the open ocean and determined the contribution of arsenic-containing lipids to this pool. Here we show that the accumulation of arsenic into lipids is a widespread phenomenon in the surface ocean. Particulate arsenic concentrations were 15 to 42 pmol L-1 with 7-20% of the particulate arsenic pool in the form of arsenolipids. We characterized these arsenolipids and found that arsenosugar phospholipids dominated the arsenolipid pools in our samples with a minor component of arsenohydrocarbons and other unidentified lipids. A significant portion of the arsenosugar phospholipids (up to 35%) were present as previously undescribed mixed acyl ether lipids, suggesting a bacterial source.

Scientific significance statementMarine microbes experience constant exposure to the toxic element arsenic. However, there are no baseline measurements of how much arsenic accumulates in microbial communities nor do we know the full spectrum of arsenic containing biomolecules produced in marine systems. In culture-based studies there is strong evidence that phytoplankton synthesize arsenic-containing lipids, but these lipids have not been observed in natural communities of marine microbes. Here we make measurements of bulk particulate arsenic at five sites in the surface open ocean and show that a significant portion of this particulate arsenic is present as complex arsenic-containing lipids. We characterize this arsenolipid pool chemically and quantitatively to show a variety of chemically distinct and quantitatively significant lipids that expand our understanding of marine arsenic biogeochemistry.

Data availability statementMass spectrometry data and its associated metadata will be available on Dryad with publication. Scripts for data processing and figure generation found on github at https://github.com/kheal/particulate_As_data_analysis.

category: ecology


title: Influence of delayed density and ultraviolet radiation on caterpillar granulovirus infection and mortality

abstract:
O_LIInfectious disease is an important potential driver of population cycles, but this must occur through delayed density-dependent infection and resulting fitness effects. Delayed density-dependent infection by baculoviruses can be caused by environmental persistence of viral occlusion bodies, which can be influenced by environmental factors. In particular, ultraviolet radiation is potentially important in reducing the environmental persistence of viruses by inactivating viral occlusion bodies.
C_LIO_LIDelayed density-dependent viral infection has rarely been observed empirically at the population level although theory predicts that it is necessary for these pathogens to drive population cycles. Similarly, field studies have not examined the potential effects of ultraviolet radiation on viral infection rates in natural animal populations. We tested if viral infection is delayed density-dependent with the potential to drive cyclic dynamics and if ultraviolet radiation influences viral infection levels.
C_LIO_LIWe censused 18 Ranchmans tiger moth (Arctia virginalis) populations across nearly 9{degrees} of latitude over two years and quantified the effects of direct and delayed density and ultraviolet radiation on baculovirus infection rates, infection severity, and survival to adulthood. Caterpillars were collected from each population in the field and reared in the laboratory. Baculovirus has not previously been described infecting Arctia virginalis, and we used genetic methods to confirm the identity of the virus.
C_LIO_LIWe found that infection rate, infection severity, and survival to adulthood exhibited delayed density-dependence. Ultraviolet radiation in the previous summer decreased infection severity, and increased survival probability of the virus. Structural equation modelling indicated that the effect of lagged density on moth survival was mediated through infection rate and infection severity, and was 2.5 fold stronger than the effect of ultraviolet radiation on survival through infection severity. We successfully amplified polh, lef-8, and lef-9 viral genes from caterpillar samples, and BLAST search results confirmed that the virus was a nucleopolyhedrovirus.
C_LIO_LIOur findings provide clear evidence that delayed density dependence can arise through viral infection rate and severity in insects, which supports the role of viral disease as a potential mechanism, among others, that may drive insect population cycles. Furthermore, our findings support predictions that ultraviolet radiation can modify viral disease dynamics in insect populations, most likely through attenuating viral persistence in the environment.
C_LI


title: Influence of delayed density and ultraviolet radiation on caterpillar baculovirus infection and mortality


title: Spatiotemporal variations of surface water microplastics near Kyushu, Japan: A quali-quantitative analysis

abstract:
Microplastics in the ocean are threatening marine ecosystems. Although plastic contaminants are ubiquitous from rivers to polar oceans, their distribution is thought to be heterogeneous, implying that both spatial and temporal variability exist. Here, we elucidate the significant spatial and temporal (seasonal) variations in the quanti-qualitative characteristics of microplastics off the west coast of Kyushu, Japan in the East China Sea. Six surveys across nine stations (n = 54) were conducted over a 14-month period, and a total of 6131 plastic items were identified. The average microplastic abundance (items {middle dot}m-3) and size (mm) {+/-} S.D. were 0.49 {+/-} 0.92 (n = 54), and 1.71 {+/-} 0.93 (n = 6131), respectively. Differences between the highest and lowest abundances were 50-fold among monthly means (1.97 {+/-} 1.49, n = 9; 0.04 {+/-} 0.03, n = 9), and 550-fold across all net tows (5.50; 0.01). With respect to colour, polymer type, and shape, white and transparent (68.5%), polyethylene (80%) fragments (76.0%) were the dominant composition. There were statistically significant differences for each of the analytical microplastic parameters among survey months (p < 0.02). Our results provide baseline data, and lead to a more comprehensive understanding of the spatiotemporal characteristics of microplastic pollution.

HighlightsO_LISignificant spatiotemporal variability in microplastic litter was detected based on the quali-quantitative analyses.
C_LIO_LIAverage ({+/-} S.D.) microplastic abundance was 0.49 {+/-} 0.92 (items {middle dot}m-3) and size was 1.71 {+/-} 0.93 (mm).
C_LIO_LIDifferences between highest and lowest abundances were 50-fold among monthly means, and 550-fold across all net tows.
C_LIO_LIWhite and transparent polyethylene fragments were the dominant composition.
C_LI


title: A novel approach to comparative RNA-Seq does not support a conserved set of genes underlying animal regeneration

abstract: Molecular studies of animal regeneration typically focus on conserved genes and signaling pathways that underlie morphogenesis. To date, a holistic analysis of gene expression across animals has not been attempted, as it presents a suite of problems related to differences in experimental design and gene homology. By combining orthology analysis with a novel statistical method for testing gene enrichment across large datasets, we are able to test whether biological processes across organisms share transcriptional regulation. We applied this method to six publicly available RNA-seq datasets from diverse examples of animal regeneration. We recovered 160 conserved orthologous gene clusters, which are enriched in structural genes as opposed to those regulating morphogenesis. A breakdown of gene presence/absence provides only limited support for the conservation of pathways typically implicated in regeneration, such as Wnt signaling and cell pluripotency. Specifically, these pathways are only conserved if we allow gene paralogs to be interchangeable through evolution. Overall, our analysis does not support the hypothesis that a shared set of ancestral genes underlie regeneration mechanisms in animals. The methods described in this paper will be broadly applicable for studying the genetic underpinnings of traits across distantly related organisms.


title: Protein-primed RNA synthesis in SARS-CoVs and structural basis for inhibition by AT-527

abstract: How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and -2 Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP-Nsp8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 [A] resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and -2, representing a promising drug for COVID-19 treatment.

category: biochemistry


title: A non-invasive brain-machine interface via independent control of individual motor units

abstract: Brain-machine interfaces (BMIs) have the potential to augment human functions and restore independence in people with disabilities, yet a compromise between non-invasiveness and performance limits their relevance. Here, we demonstrate a BMI controlled by individual motor units non-invasively recorded from the biceps brachii. Through real-time auditory and visual neurofeedback of motor unit activity, 8 participants learned to skillfully and independently control three motor units in order to complete a two-dimensional center-out task, with marked improvements in control over 6 days of training. Concomitantly, dimensionality of the motor unit population increased significantly relative to naturalistic behaviors, largely violating recruitment orders displayed during stereotyped, isometric muscle contractions. Finally, participants demonstrated the potential of a motor unit BMI to power general applications by navigating a virtual keyboard in a spelling task, achieving performances comparable to spelling-tailored non-invasive BMIs that leverage less flexible control strategies to improve performance. These results highlight a largely unexplored level of flexibility of the sensorimotor system and show that this can be exploited to create a versatile, skillfully-controllable non-invasive BMI that has great potential to both provide translational benefit and augment motor functions.

category: bioengineering


title: Encapsulation and recovery of murine hematopoietic stem and progenitor cells in a thiol-crosslinked maleimide-functionalized gelatin hydrogel

abstract: Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective biophysical, biochemical, and cellular cues of the stem cell niche microenvironment play an important role in regulating stem cell fate decisions. Three-dimensional (3D) culture of stem cells within biomaterials provides a route to present biophysical and biochemical stimuli such as cell-matrix interactions and cell-cell interactions via secreted biomolecules. Herein, we describe a maleimide-functionalized gelatin (GelMAL) hydrogel that can be crosslinked via thiol-Michael addition click reaction for the encapsulation of sensitive stem cell populations. The maleimide functional units along the gelatin backbone enables gelation via the addition of a dithiol crosslinker without requiring external stimuli (e.g., UV light, photoinitiator), reducing reactive oxide species generation. Additionally, the versatility of crosslinker selection enables easy insertion of thiol-containing bioactive or bioinert motifs. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were encapsulated in GelMAL, with mechanical properties tuned to mimic the in vivo bone marrow niche. We report insertion of a cleavable peptide crosslinker that can be degraded by the proteolytic action of SortaseA, a mammalian-inert enzyme. Notably, SortaseA exposure preserves stem cell surface markers, an essential metric of hematopoietic activity used in immunophenotyping. This novel GelMAL system enables a route to producing artificial stem cell niches with tunable biophysical properties with intrinsic cell-interaction motifs and orthogonal addition of bioactive crosslinks.


title: Implementing QR Codes in Academia to Improve Sample Tracking, Data Accessibility, and Traceability in Multicampus Interdisciplinary Collaborations

abstract: With the growing number of multicampus interdisciplinary projects in academic institutions, there is an increasing need for tracking systems that make device and sample data and associated results instantly accessible to all collaborators involved. This need has become particularly salient with the COVID pandemic and consequent travel restrictions that have hampered in person meetings and laboratory visits. We report a Quick Response (QR) code tracking system that integrates project management tools for seamless communication and tracking of materials and devices between three manufacturing sites (cleanrooms) and six research laboratories across five universities. We use this system to track the design, fabrication, and quality control of bioelectronic devices (in the cleanroom and engineering laboratories), in vitro experimental results (in a biology laboratory), and in vivo testing (in the school of medicine). Setting up a tracking system for multisite interdisciplinary teams improves traceability, efficiency, and the quality of produced results making it easier to accomplish project milestones on a tight timeline. This tracking system is particularly useful to track device issues and ensure engineering device consistency when working with expensive biological samples in vitro and animals in vivo to reduce waste of biological and animal resources associated with device failure.


title: Optical and magnetic properties of particles containing guanine in strong basic solution

abstract: Guanine based particle is one of very efficient material to control light especially near the surface of animal body. Past studies reported that platelets of anhydrous guanin crystal are utilized by living creatures, and they can change their colour and light reflection intensity when the arrangement of platelets. Guanine has relatively high reflective index and its particle can exhibit a unique optical property. Under higher pH which can be provided by a high concentration of NaOH aqueous solution, guanine molecules formed a sodium salt particle which contain guanine. Here this work presents that particles containing guanine in basic aqueous solution with NaOH exhibited very strong shining and light reflection switching by magnetic fields. In addition, adding an extracted solution of a fish iridophore enhanced the formation of intensively light reflecting guanine particles floating in a strong basic solution.


title: HiCBin: Binning metagenomic contigs and recovering metagenome-assembled genomes using Hi-C contact maps

abstract: Recovering high-quality metagenome-assembled genomes (MAGs) from complex microbial ecosystems remains challenging. Conventional shotgun-based binning approaches may encounter barriers when multiple samples are scarce. Recently, high-throughput chromosome conformation capture (Hi-C) has been applied to simultaneously study multiple genomes in natural microbial communities. Several Hi-C-based binning pipelines have been put forward and yielded state-of-the-art results using a single sample. We conclude that normalization and clustering are two vital steps in the Hi-C-based binning analyses, and develop HiCBin, a novel open-source pipeline, to resolve high-quality MAGs utilizing Hi-C contact maps. HiCBin employs the HiCzin normalization method and the Leiden community detection algorithm based on the Potts spin-glass model and includes the spurious contact detection into binning pipelines for the first time. Using the metagenomic yeast sample with a perfect ground truth of contigs species identity, we comprehensively evaluate the impacts on the binning performance of different normalization methods and clustering algorithms from the HiCBin and other available metagenomic Hi-C analysis pipelines, demonstrate that the HiCzin and the Leiden algorithm achieve the best binning accuracy, and show that the spurious contact detection can improve the retrieval performance. We also validate our method and compare the capability to recover high-quality MAGs of HiCBin against other state-of-the-art Hi-C-based binning tools including ProxiMeta, bin3C, and MetaTOR, and one popular shotgun-based binning software MetaBAT2 on a human gut sample and a wastewater sample. HiCBin provides the best performance and applicability in resolving MAGs and is available at https://github.com/dyxstat/HiCBin.


title: Population-scale detection of non-reference sequence variants using colored de Bruijn Graphs

abstract:
MotivationWith the increasing throughput of sequencing technologies, structural variant (SV) detection has become possible across tens of thousands of genomes. Non-reference sequence (NRS) variants have drawn less attention compared to other types of SVs due to the computational complexity of detecting them. When using short-read data, the detection of NRS variants inevitably involves a de novo assembly which requires high-quality sequence data at high coverage. Previous studies have demonstrated how sequence data of multiple genomes can be combined for the reliable detection of NRS variants. However, the algorithms proposed in these studies have limited scalability to larger sets of genomes.

ResultsWe introduce PopIns2, a tool to discover and characterize NRS variants in many genomes, which scales to considerably larger numbers of genomes than its predecessor PopIns. In this article, we briefly outline the PopIns2 workflow and highlight our novel algorithmic contributions. We developed an entirely new approach for merging contig assemblies of unaligned reads from many genomes into a single set of NRS using a colored de Bruijn graph. Our tests on simulated data indicate that the new merging algorithm ranks among the best approaches in terms of quality and reliability and that PopIns2 shows the best precision for a growing number of genomes processed. Results on the Polaris Diversity Cohort and a set of 1000 Icelandic human genomes demonstrate unmatched scalability for the application on population-scale datasets.

AvailabilityThe source code of PopIns2 is available from https://github.com/kehrlab/PopIns2.

Contactthomas.krannich@bihealth.de or birte.kehr@klinik.uni-regensburg.de


title: scTenifoldKnk: a machine learning workflow performing virtual knockout experiments on single-cell gene regulatory networks

abstract:
Bigger PictureO_LIGene knockout (KO) experiments, using genetically altered animals, are a proven powerful approach to elucidate the role of a gene in a biological process. However, systematic KO experiments targeting many genes are usually prohibitive due to limited experimental and animal resources. Here, we present scTenifoldKnk, an efficient virtual KO tool that allows the systematic deletion of many genes individually. scTenifoldKnk uses single-cell RNA sequencing (scRNAseq) data from wild-type (WT) samples to predict gene function in a cell type-specific manner. We show that predictions made by scTenifoldKnk recapitulate findings from real-animal KO experiments. scTenifoldKnk has proven to be a powerful and effective approach for elucidating gene function, prioritizing KO targets, predicting experimental outcomes before real-animal KO experiments are conducted.
C_LI

HighlightsO_LIscTenifoldKnk performs virtual KO experiments using scRNAseq data.
C_LIO_LIscTenifoldKnk only requires data from WT samples; no data is needed from KO samples.
C_LIO_LIPredictions made by scTenifoldKnk recapitulate findings from real-animal KO experiments.
C_LI

Data Science Maturity Level

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=14 SRC="FIGDIR/small/436484v2_ufig1.gif" ALT="Figure 1">
View larger version (8K):
org.highwire.dtl.DTLVardef@157126aorg.highwire.dtl.DTLVardef@17a2e76org.highwire.dtl.DTLVardef@805c4dorg.highwire.dtl.DTLVardef@12bdc1b_HPS_FORMAT_FIGEXP  M_FIG C_FIG eTOC blurbscTenifoldKnk is a machine learning workflow performing virtual KO experiments to predict gene function. It constructs gene regulatory networks using single-cell RNA sequencing data from wild-type samples and then computationally deletes target genes. Real-data applications demonstrate that scTenifoldKnk recapitulates findings of real-animal KO experiments and accurately predicts gene function in analyzed cells.

SummaryGene knockout (KO) experiments are a proven, powerful approach for studying gene function. However, systematic KO experiments targeting a large number of genes are usually prohibitive due to the limit of experimental and animal resources. Here, we present scTenifoldKnk, an efficient virtual KO tool that enables systematic KO investigation of gene function using data from single-cell RNA sequencing (scRNAseq). In scTenifoldKnk analysis, a gene regulatory network (GRN) is first constructed from scRNAseq data of wild-type samples, and a target gene is then virtually deleted from the constructed GRN. Manifold alignment is used to align the resulting reduced GRN to the original GRN to identify differentially regulated genes, which are used to infer target gene functions in analyzed cells. We demonstrate that the scTenifoldKnk-based virtual KO analysis recapitulates the main findings of real-animal KO experiments and recovers the expected functions of genes in relevant cell types.


title: scTenifoldKnk: an efficient virtual knockout tool for gene function predictions via single-cell gene regulatory network perturbation


title: Deep neural network analysis employing diffusion basis spectrum imaging metrics as classifiers improves prostate cancer detection and grading

abstract:
Structural and cellular complexity of prostatic histopathology limits the accuracy of noninvasive detection and grading of prostate cancer (PCa). We addressed this limitation by employing a novel diffusion basis spectrum imaging (DBSI) to derive structurally-specific diffusion fingerprints reflecting various underlying prostatic structural and cellular components. We further developed diffusion histology imaging (DHI) by combining DBSI-derived structural fingerprints with a deep neural network (DNN) algorithm to more accurately classify different histopathological features and predict tumor grade in PCa. We examined 243 patients suspected with PCa using in vivo DBSI. The in vivo DBSI-derived diffusion metrics detected coexisting prostatic pathologies distinguishing inflammation, PCa, and benign prostatic hyperplasia. DHI distinguished PCa from benign peripheral and transition zone tissues with over 95% sensitivity and specificity. DHI also demonstrated over 90% sensitivity and specificity for Gleason score noninvasively. We present DHI as a novel diagnostic tool capable of noninvasive detection and grading of PCa.

One sentence summaryDiffusion histology imaging noninvasively and accurately detects and grades prostate cancer.


title: Off-resonance saturation as an MRI method to quantify ferritin-bound iron in the post-mortem brain

abstract:
PurposeTo employ an Off-Resonance Saturation (ORS) method to measure the ferritin-bound iron pool, which is an endogenous contrast agent which can give information on cellular iron status.

MethodsAn ORS acquisition protocol was implemented on a 7T preclinical scanner and the contrast maps were fitted to an established analytical model. The method was validated by correlation and Bland-Altman analysis on a ferritin-containing phantom. Ferritin-iron maps were obtained from post-mortem tissue of patients with neurological diseases characterized by brain iron accumulation, i. e. Alzheimers disease, Huntingtons disease and aceruloplasminemia, and validated with histology. Transverse relaxation rate and magnetic susceptibility values were also obtained for comparison.

ResultsIn post-mortem tissue, the ferritin-iron contrast strongly co-localizes with histological iron staining, in all the cases. Quantitative iron values obtained via the ORS method are in agreement with literature.

ConclusionsOff-resonance saturation is an effective way to detect iron in grey matter structures, while mitigating for the presence of myelin. If a reference region with little iron is available in the tissue, the method can produce quantitative iron maps. This method is applicable in the study of brain diseases characterized by brain iron accumulation and complement existing iron-sensitive parametric methods.

category: biophysics


title: A Differential Equation for Mutation Rates in Environmental Coevolution.

abstract: In their paper Natural selection for least action (Kaila and Annila 2008) they depict evolution as a process conforming to the Principle of Least Action (PLA). From this concept, together with the Coevolution model of Lewontin, an equation of motion for environmental coevolution is derived which shows that it is the time rate (frequency) of evolutionary change of the organism (mutations) that responds to changes in the environment. It is not possible to compare the theory with viral or bacterial mutation rates, as these are not measured on a time base. There is positive evidence from population level avian studies where the coefficient of additive evolvability (Cav) and its square (IA) change with environmental favourability in agreement with this model. Further analysis shows that the time rate of change of the coefficient of additive evolvability (Cav) and its square (IA) are linear with environmental favourability, which could help in defining the Lagrangian of the environmental effects.


title: The lifetime cost of reproductive potential, who spends the most?

abstract:
ObjectivesTo determine who spends more energy over a lifetime on maintaining their reproductive potential: men or women?

DesignAs a model and energetic equivalent, we set the mass of gametes supported over time from birth until exhaustion of fertility. We calculated gender-specific dynamics of gamete pool mass over time. To this purpose we collated data from existing literature, accounting for gamete volume over stages of development, time in each stage, mass density, and count. Our model generates the integral, or area under the curve (AUC) of the gamete pool mass over a lifetime as a proxy to energetic requirements.

Main outcome measuresThe area under gamete mass curve over a lifetime in men and women.

ResultsThe number of gametes over a lifetime is 600,000 in women and close to 1 trillion in men. Accounting for mass and time, women invest approximately 100 gram*days in maintaining the female oocyte pool. Women reach 50% of lifetime AUC by age 10, and 90% by age 25. Men invest approximately 30 Kg*days over a lifetime (300-fold more), reaching 50% of lifetime AUC at age 37 and 90% at age 62 years old.

ConclusionsThe study quantifies for the first time the area under gamete mass in men and women through a nuanced calculation accounting for all components of post-natal gamete dynamics. We found a 300-fold excess is supported male gamete mass over a lifetime (100g*days vs. 30 Kg*days in females vs. males, respectively). Our methodology offers a framework for assessing other components of the reproductive system in a similar quantitative manner.


title: Symptomatic and Disease-Modifying Effects of GABAA Receptor Positive Allosteric Modulation in a Mouse Model of Chronic Stress

abstract: Chronic stress is a risk factor for Major depressive disorder (MDD), and in rodents, it recapitulates human behavioral, cellular and molecular changes. In MDD and after chronic stress, neuronal dysfunctions and deficits in GABAergic signaling are observed and responsible for symptom severity. GABA signals predominantly through GABAA receptors (GABAA-R) composed of various subunit types that relate to downstream outcomes. Activity at 2-GABAA-Rs contributes to anxiolytic properties, 5-GABAA-Rs to cognitive functions, and 1-GABAA-Rs to sedation. Therefore, a therapy aiming at increasing 2- and 5-GABAA-Rs activity, but devoid of 1-GABAA-R activity, has potential to address several symptomologies of depression while avoiding side effects. This study investigated the activity profiles and behavioral efficacy of two molecules enantiomers of each other (GL-II-73 and GL-I-54), separately and as a racemic mixture (GL-RM), and potential disease-modifying effects on neuronal morphology. Results confirm GL-I-54 and GL-II-73 exert positive allosteric modulation at the 2-, 3-, 5-GABAA-Rs and 5-containing GABAA-Rs, respectively, and have anti-depressant and pro-cognitive effects independently. Using unpredictable chronic mild stress (UCMS) in male and female mice (n=12/group), we show that acute and chronic administration of GL-RM combined the anti-depressant and pro-cognitive effects of each enantiomer, although at lower doses avoiding sedation. Morphology studies showed reversal of spine density loss caused by UCMS after chronic GL-RM treatment at apical and basal dendrites of the PFC and CA1. Together, these results support using a racemic mixture with combined 2-, 3-, 5-GABAA-R profile to reverse chronic stress-induced mood symptoms, cognitive deficits, and with anti-stress neurotrophic effects.


title: Adult neurogenesis mediates forgetting of multiple types of memory in the rat

abstract:
The formation and retention of hippocampus-dependent memories is impacted by neurogenesis, a process that involves the production of new neurons in the dentate gyrus of the hippocampus. Recent studies demonstrate that increasing neurogenesis after memory formation induces forgetting of previously acquired memories. Neurogenesis-induced forgetting was originally demonstrated in mice, but a recent report suggests that the same effect may be absent in rats. Although a general species difference is possible, other potential explanations for these incongruent findings are that memories which are more strongly reinforced become resilient to forgetting or that perhaps only certain types of memories are affected. Here, we investigated whether neurogenesis-induced forgetting occurs in rats using several hippocampal dependent tasks including contextual fear conditioning (CFC), the Morris Water Task (MWT), and touchscreen paired associates learning (PAL). Neurogenesis was increased following training using voluntary exercise for 4 weeks before recall of the previous memory was assessed. We show that voluntary running causes forgetting of context fear memories in a neurogenesis-dependent manner, and that neurogenesis-induced forgetting is present in rats across behavioral tasks despite differences in complexity or reliance on spatial, context, or object memories. In addition, we asked whether stronger memories are less susceptible to forgetting by varying the strength of training. Even with a very strong training protocol in the CFC task, we still observed enhanced forgetting related to increased neurogenesis. These results suggest that forgetting due to neurogenesis is a conserved mechanism that aids in the clearance of memories.

Significance StatementRecent evidence indicates that hippocampal neurogenesis mediates forgetting of older memories and enhances encoding of new memories free of proactive interference. This evidence comes from multiple rodent species, behavioral tasks, and methods of increasing neurogenesis. However, a recent paper by (Kodali et al. 2016) found that voluntary exercise-induced neurogenesis did not cause forgetting in the Morris Water Task in rats. The results call into question whether the phenomenon is a conserved function of neurogenesis across species. In the present study, we show that voluntary running causes robust forgetting in rats in a neurogenesis-dependent manner and that the effect is present across three different behavioral tasks, confirming the existence of the phenomenon in rats and adding to the growing evidence that forgetting is a conserved function of hippocampal neurogenesis.


title: Early life stress is associated with earlier emergence of permanent molars

abstract: Exposure to adversity can accelerate biological aging. However, existing biomarkers of early aging are either difficult to detect in individuals at scale, like epigenetic signatures, or cannot be detected until late childhood, like pubertal onset. We evaluated the hypothesis that early adversity is associated with earlier molar eruption, an easily assessed measure that has been used to track the length of childhood across primates. In a pre-registered analysis (N = 117, ages 4-7), we demonstrate that lower family income and exposure to adverse childhood experiences (ACEs) are significantly associated with earlier eruption of the first permanent molars, as rated in T2-weighted magnetic resonance images (MRI). We replicate relationships between income and molar eruption in a population-representative dataset (NHANES; N = 1,973). These findings suggest that the impact of stress on the pace of biological development is evident in early childhood, and detectable in the timing of molar eruption.


title: TCF-1 in CD8 T cells separates GVHD from GVL

abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for hematological malignancy due to graft-versus-leukemia (GVL) effects, mediated by alloreactive donor T cells. However, these same donor cells cause graft-versus-host disease (GVHD), a life-threatening complication. During GVHD, donor T cells proliferate, migrate, and produce cytokines, leading to damage of healthy host tissues. Separating the linked processes of GVHD and GVL is critical to improving outcomes. Here we show that TCF-1, a major T cell developmental factor, plays a role in regulation of these processes. Using a mouse model of allo-HSCT leading to GVHD, we found that conditional loss of TCF-1 in mature CD8 T cells separates GVHD from GVL, providing an optimal clinical phenotype. This occurred due to changes in cytokine production, proliferation, cell survival, chemokine receptor expression, and gene expression. Thus, modulation of TCF-1 signaling reduces GVHD severity and persistence, but maintains GVL effects, and could be useful for allo-HSCT or other T cell-mediated disorders. Furthermore, here and in our companion manuscript we show that regulation of mature, alloactivated T cells by TCF-1 differs between CD4 and CD8 T cells, suggesting that TCF-1 is critical for regulation of mature alloactivated T cells, with unique programs in each subset.


title: The Dual-Antigen Ad5 COVID-19 Vaccine Delivered as an Intranasal Plus Subcutaneous Prime Elicits Th1 Dominant T-Cell and Humoral Responses in CD-1 Mice

abstract: In response to the need for an efficacious, thermally-stable COVID-19 vaccine that can elicit both humoral and cell-mediated T-cell responses, we have developed a dual-antigen human adenovirus serotype 5 (hAd5) COVID-19 vaccine in formulations suitable for subcutaneous (SC), intranasal (IN), or oral delivery. The vaccine expresses both the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins using an hAd5 platform with E1, E2b, and E3 sequences deleted (hAd5[E1-, E2b-, E3-]) that is effective even in the presence of hAd5 immunity. In the vaccine, S is modified (S-Fusion) for enhanced cell-surface display to elicit humoral responses and N is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal pathway to increase MHC I and II presentation. Initial studies using subcutaneous (SC) prime and SC boost vaccination of CD-1 mice demonstrated that the hAd5 S-Fusion + N-ETSD vaccine elicits T-helper cell 1 (Th1) dominant T-cell and humoral responses to both S and N. We then compared SC to IN prime vaccination with either an SC or IN boost post-SC prime and an IN boost after IN prime. These studies reveal that IN prime/IN boost is as effective at generating Th1 dominant humoral responses to both S and N as the other combinations, but that the SC prime with either an IN or SC boost elicits greater T cell responses. In a third study to assess the power of the two routes of delivery when used together, we used a combined SC plus IN prime with or without a boost and found the combined prime alone to be as effective as the combined prime with either an SC or IN boost in generating both humoral and T-cell responses. The findings here in CD-1 mice demonstrate that combined SC and IN prime-only delivery has the potential to provide broad immunity - including mucosal immunity - against SARS-CoV-2 and supports further testing of this delivery approach in additional animal models and clinical trials.


title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 pandemic, which has caused a devastating global health crisis. The emergence of highly transmissible novel viral strains that escape neutralizing responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2 biology and to develop additional therapeutic strategies. Using a comprehensive identification of RNA binding proteins (RBP) by mass spectrometry (ChIRP-M/S) approach, we identified 142 high-confidence cellular factors that bind the SARS-CoV-2 viral genome during infection. By systematically knocking down their expression in a human lung epithelial cell line, we found that the majority of the RBPs identified in our study are proviral factors that regulate SARS-CoV-2 genome replication. We showed that some of these proteins represented drug targets of interest for inhibiting SARS-CoV-2 infection. In conclusion, this study provides a comprehensive view of the SARS-CoV-2 RNA interactome during infection and highlights candidates for host-centered antiviral therapies.


title: Dry Swab Method of sample collection for SARS-CoV2 testing can be used for culturing virus

abstract:
Back groundEarlier studies suggested the use of dry swab method for SARS-CoV-2 detection as it does not need VTM and subsequent RNA extraction step making the process cheaper, safer and faster. In this study we explore whether the virus in the dry swab is viable and can be cultured and propagated.

MethodSwabs were spiked with SARS-CoV-2 and stored in three different conditions: a) as dry swab (SD, eluted in 1 mL DMEM), b) in 1 mL of Viral Transport Medium (SVTM), and c) in 1 mL of Tris-EDTA buffer (STE). The sample groups were stored either at room temperature (RT, 25{degrees}C{+/-}1{degrees}C) or at 4{degrees}C for 1, 4, 8, 12, 24, 48 and 72 hours before being used as viral inoculums for the propagation studies in Vero cells.

ResultsThe RT-qPCR data suggests that SD incubated both at RT and 4{degrees}C harbors viral particles that are viable and culturable at par with SVTM and STE.

ConclusionThe dry swab method, in addition to its advantages in detection of the virus, also renders viable viral particles that can be cultured and propagated.


title: Distinct domains of Me31B interact with different eIF4E isoforms in the male germ line of Drosophila melanogaster

abstract:
Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs where might play a role in mRNA storage and translational repression. We also demonstrate that the DEAD-box RNA helicase Me31B, a component of PBs, physically interacts with eIF4E-1 and eIF4E-3 both in the yeast two-hybrid system and FRET in Drosophila S2 cells.

Moreover, truncated and point mutated Me31B proteins indicate that the binding sites of Me31B for eIF4E-1 and eIF4E-3 are located in different domains. Residues Y401-L407 (at the carboxy-terminal) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminal) are critical for interaction with eIF4E-3. Thus, Me31B represents a novel type of eIF4E-interacting protein. Our observations suggest that Me31B might recognize different eIF4E isoforms in different tissues, which could be the key to silencing specific messengers. They provide further evidence that alternative forms of eIF4E and their interactions with various partners add complexity to the control of gene expression in eukaryotes.

category: molecular biology


title: Membrane insertion of Cavin1 facilitates caveolae assembly

abstract:
Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades and lipid sorting. The caveolae coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modelling to show that Cavin1 inserts into membranes. We establish that initial PI(4,5)P2-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of the HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Significance statementCaveolae are cholesterol enriched membrane invaginations coupled to severe muscle and lipid disorders. Their formation is dependent on assembly of the protein Cavin1 at the lipid membrane interface driving membrane curvature. In this work, we dissect the mechanism for how Cavin1 binds and inserts into membranes using a combination of biochemical and biophysical characterization as well as computational modelling. The proposed model for membrane assembly potentiates dynamic switching between shielded and exposed hydrophobic helices used for membrane insertion and clarifies how Cavin1 can drive membrane curvature and the formation of caveolae.

category: cell biology


title: Membrane insertion mechanism of the caveolae coat protein Cavin1


title: Ferroptotic stress promotes the accumulation of pro-inflammatory proximal tubular cells in maladaptive renal repair

abstract: Overwhelming lipid peroxidation induces ferroptotic stress and ferroptosis, a non-apoptotic form of regulated cell death that has been implicated in maladaptive renal repair in mice and humans. Using single-cell transcriptomic and mouse genetic approaches, we show that proximal tubular (PT) cells develop a molecularly distinct, pro-inflammatory state following injury. While these inflammatory PT cells transiently appear after mild injury and return to their original state without inducing fibrosis, they accumulate and contribute to persistent inflammation after severe injury. This transient inflammatory PT state significantly downregulates glutathione metabolism genes, making them vulnerable to ferroptotic stress. Genetic induction of high ferroptotic stress in these cells after mild injury leads to the accumulation of the inflammatory PT cells, enhancing inflammation and fibrosis. Our study broadens the roles of ferroptotic stress from being a trigger of regulated cell death to include the promotion and accumulation of proinflammatory cells that underlie maladaptive repair.


title: Dispersal across habitat boundaries: uncovering the demographic fates of populations in unsuitable habitat

abstract: Patchy landscapes are characterized by abrupt transitions among distinct habitat types, forcing species to cross habitat boundaries in order to spread. Since seed dispersal is a probabilistic process, with a kernel that decays with distance, most individuals will fail to reach new, suitable habitat. Although failed dispersers are presumed dead in population models, their demographic fates may not be so simple. If transient survival is possible within unsuitable habitat, then through time, individuals may be able to reach distant, suitable habitat, forming new populations and buffering species from extinction. In a fragmented Californian grassland, we explored the fates of individuals that crossed habitat boundaries, and if those fates differed among specialists dispersing from two habitat types: serpentine habitat patches and the invaded non-serpentine matrix. We surveyed the diversity of seedbank and adult life stages along transects that crossed boundaries between patches and the matrix. First, we considered how patch specialists might transiently survive in the matrix via seed dormancy or stepping-stone populations. Second, we investigated the dispersal of an invasive matrix specialist (Avena fatua) into patches, to assess if sink populations existed across the habitat boundary. We found that dormancy maintained populations of patch specialists deep into the matrix, as abundances of seedbanks and of adult plant communities differed with distance into the matrix. We found evidence that these dormant seeds disperse secondarily with vectors of material flows in the landscape, suggesting that they could eventually reach suitable patches even if they first land in the matrix. We found that A. fatua were largely absent deep in patches, where reproductive outputs plummeted and there was no evidence of a dormant seedbank. Our results not only reveal the demographic fates of individuals that land in unsuitable habitat, but that their ecological consequences differ depending on the direction by which the boundary is crossed (patch [->] matrix = matrix [->] patch). Dormancy is often understood as a mechanism for persisting in face of temporal variability, but it may serve as a means of traversing unsuitable habitat in patchy systems, warranting its consideration in estimates of habitat connectivity.


title: Genes identified in rodent studies of alcohol intake are enriched for heritability of human substance use

abstract:
BackgroundRodent paradigms and human genome-wide association studies (GWASs) on drug use have the potential to provide biological insight into the pathophysiology of addiction.

MethodsUsing GeneWeaver, we created rodent alcohol and nicotine gene-sets derived from 19 gene expression studies on alcohol and nicotine outcomes. We partitioned the SNP-heritability of these gene-sets using four large human GWASs: 1) alcoholic drinks per week, 2) problematic alcohol use, 3) cigarettes per day and 4) smoking cessation. We benchmarked our findings with curated human alcoholism and nicotine addiction gene-sets and performed specificity analyses using other rodent gene-sets (e.g., locomotor behavior) and other human GWASs (e.g., height).

ResultsThe rodent alcohol gene-set was enriched for heritability of drinks per week, cigarettes per day, and smoking cessation, but not problematic alcohol use. However, the rodent nicotine gene-set was not significantly associated with any of these traits. Both rodent gene-sets showed enrichment for several non-substance use GWASs, and the extent of this relationship tended to increase as a function of trait heritability. In general, larger gene-sets demonstrated more significant enrichment. Finally, when evaluating human traits with similar heritabilities, both rodent gene-sets showed greater enrichment for substance use traits.

ConclusionOur results suggest that rodent gene expression studies can help to identify genes that capture heritability of substance use traits in humans, yet the specificity to human substance use was less than expected due to various factors such as the genetic architecture of a trait. We outline various limitations, interpretations and considerations for future research.

category: genetics


title: A New Monocyte Epigenetic Clock Reveals Effects of Alcohol Consumption on Epigenetic Aging in Three Independent Cohorts

abstract:
BackgroundExcessive alcohol consumption increases the risk of aging-related comorbidities and mortality. Assessing the impact of alcohol consumption on biological age is important for clinical decision-making and prevention. Evidence shows that alcohol alters monocyte function, and age is associated with DNA methylome and transcriptomic changes among monocytes. However, no monocyte-based epigenetic clock is currently available. In this study, we developed a new monocyte-based DNA methylation clock (MonoDNAmAge) by using elastic net regularization. The MonoDNAmAge was validated by benchmarking using epigenetic age acceleration (EAA) in HIV infection. Using MonoDNAmAge clock as well as four established clocks (i.e., HorvathDNAmAge, HannumDNAmAge, PhenoDNAmAge, GrimDNAmAge), we then evaluated the effect of alcohol consumption on biological aging in three independent cohorts (N=2,242).

ResultsMonoDNAmAge, comprised of 186 CpG sites, was highly correlated with chronological age (rtraining=0.96, p<2.20E-16; rtesting=0.86, p=1.55E-141). The MonoDNAmAge clock predicted an approximately 10-year age acceleration from HIV infection in two cohorts. Quadratic regression analysis showed a nonlinear relationship between MonoDNAmAge and alcohol consumption in the Yale Stress Center Community Study (YSCCS, pmodel=4.55E-08, px2 =7.80E-08) and in the Veteran Aging Cohort Study (VACS, pmodel=1.85E-02, px2 =3.46E-02). MonoDNAmAge and light alcohol consumption showed a negative linear relationship in the Womens Interagency HIV Study (WIHS, {beta}=-2.63, px=2.82E-06). Heavy consumption increased EAAMonoDNAmAge up to 1.60 years in the VACS while light consumption decreased EAAMonoDNAmAge to 2.66 years in the WIHS. These results were corroborated by the four established epigenetic clocks.

ConclusionsWe observed a nonlinear effect of alcohol consumption on epigenetic age that is estimated by a novel monocyte-based "clock" in three distinct cohorts, highlighting the complex effects of alcohol consumption on biological age.

category: genomics


title: Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies

abstract: Lipopolysaccharides (LPS) are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria 1. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex 2. Despite detailed insights from X-ray crystallography into the architecture of LptDE transporter complexes 3-5, no structure of a laterally open LptD transporter has been described, a transient state that occurs during LPS release 6. To facilitate the acquisition of hitherto unknown conformations we subjected LptDE of N. gonorrhoeae to cryo-EM analyses. In complex with newly designed rigid chaperones derived from nanobodies (Pro-Macrobodies, PMbs) we obtained a map of a partially opened LptDE transporter at 3.4 [A] resolution and in addition we captured a laterally fully opened LptDE complex from a subset of particles. Our work offers new insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a novel, highly rigid and widely applicable chaperone scaffold to enable structural biology of challenging protein targets.


title: Glycosaminoglycan content of a mineralized collagen scaffold promotes mesenchymal stem cell secretion of factors to modulate angiogenesis and monocyte differentiation

abstract: Effective design of biomaterials to aid regenerative repair of craniomaxillofacial (CMF) bone defects requires approaches that modulate the complex interplay between exogenously added progenitor cells and cells in the wound microenvironment, such as osteoblasts, osteoclasts, endothelial cells, and immune cells. We are exploring the role of the glycosaminoglycan (GAG) content in a class of mineralized collagen scaffolds recently shown to promote osteogenesis and healing of craniofacial bone defects. We previously showed that incorporating chondroitin-6-sulfate or heparin improved mineral deposition by seeded human mesenchymal stem cells (hMSCs). Here, we examine the effect of varying scaffold GAG content on hMSC behavior, and their ability to modulate osteoclastogenesis, vasculogenesis, and the immune response. We report the role of hMSC-conditioned media produced in scaffolds containing chondroitin-6-sulfate (CS6), chondroitin-4-sulfate (CS4), or heparin (Heparin) GAGs on endothelial tube formation and monocyte differentiation. Notably, endogenous production by hMSCs within Heparin scaffolds most significantly inhibits osteoclastogenesis via secreted osteoprotegerin (OPG), while the secretome generated by CS6 scaffolds reduced pro-inflammatory immune response and increased endothelial tube formation. All conditioned media down-regulated many pro- and anti-inflammatory cytokines, such as IL6, IL-1{beta}, and CCL18 and CCL17 respectively. Together, these findings demonstrate that modifying mineralized collagen scaffold GAG content can both directly (hMSC activity) and indirectly (production of secreted factors) influence overall osteogenic potential and mineral biosynthesis as well as angiogenic potential and monocyte differentiation towards osteoclastic and macrophage lineages. Scaffold GAG content is therefore a powerful stimulus to modulate reciprocal signaling between multiple cell populations within the bone healing microenvironment.


title: Microbiome Maps: Hilbert Curve Visualizations of Metagenomic Profiles

abstract:
MotivationAbundance profiles from metagenomic sequencing data synthesize information from billions of sequenced reads coming from thousands of microbial genomes. Analyzing and understanding these profiles can be a challenge since the data they represent are complex. Particularly challenging is their visualization, as existing techniques are inadequate when the taxa number is in the thousands. We present a technique, and accompanying software, for the visualization of metagenomic abundance profiles using a space-filling curve that transforms a profile into an interactive 2D image.

ResultsWe created JO_SCPLOWASPERC_SCPLOW, an easy to use tool for the visualization and exploration of metagenomic profiles from DNA sequencing data. It orders taxa using a space-filling Hilbert curve, and creates a "Microbiome Map", where each position in the image represents the abundance of a single taxon from a reference collection. JO_SCPLOWASPERC_SCPLOW can order taxa in multiple ways, and the resulting microbiome maps can highlight "hot spots" of microbes that are dominant in taxonomic clades or biological conditions.

We use JO_SCPLOWASPERC_SCPLOW to visualize samples from a variety of microbiome studies, and discuss ways in which microbiome maps can be an invaluable tool to visualize spatial, temporal, disease, and differential profiles. Our approach can create detailed microbiome maps involving hundreds of thousands of microbial reference genomes with the potential to unravel latent relationships (taxonomic, spatio-temporal, functional, and other) that could remain hidden using traditional visualization techniques. The maps can also be converted into animated movies that bring to life the dynamicity of microbiomes.

AvailabilityJO_SCPLOWASPERC_SCPLOW is freely available at microbiomemaps.org and via biorg.cs.fiu.edu/jasper

Contactcvaldes2@unl.edu; giri@fiu.edu

Supplementary informationSupplementary materials are available at microbiomemaps.org