title: A Versatile Enhanced Freeze-Substitution Protocol for Volume Electron Microscopy

abstract: Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including 2% osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% OsO4. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues, nematodes, yeast, and bacteria. Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.


title: Sucrose rather than GA transported by AtSWEET13 and AtSWEET14 supports pollen fitness at late anther development stages

abstract:
SummaryO_LIBoth sugar and hormone gibberellin (GA) are essential for anther-enclosed pollen development and thus for plant productivity in flowering plants. Arabidopsis (Arabidopsis thaliana) AtSWEET13 and AtSWEET14, which are expressed in anthers and associated with seed yield, transport both sucrose and GA. However, it is still unclear which substrate transported by them directly affects anther development and seed yield.
C_LIO_LIHistochemical staining, cross-sectioning and microscopy imaging techniques were used to investigate and interpret the phenotypes of AtSWEET13 and AtSWEET14 double mutant during anther development. Genetic complementation of atsweet13;14 using AtSWEET9 that transports sucrose but not GA was conducted to test the substrate preference relevant to the biological process.
C_LIO_LIThe loss of AtSWEET13 and AtSWEET14 resulted in reduced pollen viability and therefore decreased pollen germination. AtSWEET9 fully rescued
C_LIO_LIthe defects in pollen fertility of atsweet13;14, indicating AtSWEET13/14 mediated sucrose rather than GA is essential to pollen fertility.
C_LIO_LIAtSWEET13 and AtSWEET14 mainly function at the anther wall during late anther development stages and are likely responsible for sucrose efflux into locules to support pollen development to maturation, which is vital for subsequent pollen viability and germination.
C_LI

category: plant biology


title: Dynamic regulation of neural variability during working memory reflects dopamine, functional integration, and decision-making

abstract: The regulation of moment-to-moment neural variability may permit effective responses to changing cognitive demands. However, the mechanisms that support variability regulation are unknown. In the context of working memory, we leverage the largest available PET and fMRI dataset to jointly consider three lenses through which neural variability regulation could be understood: dopamine capacity, network-level functional integration, and flexible decision processes. We show that with greater working memory load, upregulation of variability was associated with elevated dopamine capacity and heightened functional integration, effects dominantly expressed in the striato-thalamic system rather than cortex. Strikingly, behavioral modeling revealed that working memory load evoked substantial decision biases during evidence accumulation, and those who jointly expressed a more optimal decision bias and higher dopamine capacity were most likely to upregulate striato-thalamic variability under load. We argue that the ability to align striato-thalamic variability to level of demand may be a hallmark of a well-functioning brain.

category: neuroscience


title: Periodic Time Cells in Human Entorhinal Cortex

abstract: The representation of time in the brain is a fundamental component of cognition. Here we investigated how the human brain represents time during a temporally continuous uninterrupted experience by presenting fifteen neurosurgical patients with an audiovisual video while recording neurons activity from multiple brain regions. We report on a set of units that modulate their activity in a strikingly periodic manner across different timescales--from seconds to many minutes. These cells were most prevalent in the entorhinal cortex and time could be decoded from their population activity. Furthermore, these cells remapped their dominant periodicity to shorter timescales during a subsequent recognition memory task. When the audiovisual sequence was presented at two different speeds (regular and faster), a significant percentage of these periodic time cells (PTCs) maintained their timescales, suggesting a degree of invariance with respect to the narrative content. The temporal periodicity of PTCs may complement the spatial periodicity of grid cells, thereby providing scalable spatiotemporal metrics for encoding and retrieval of human experience.


title: Developmental dynamics of the postsynaptic proteome to understand synaptic maturation and dysmaturation

abstract: The postsynaptic density (PSD) is a protein condensate composed of [~]1,000 proteins beneath the postsynaptic membrane of excitatory synapses. The number, shape, and plasticity of synapses are altered during development. However, the dynamics of synaptic protein composition across development have not been fully understood. Here we show alterations of PSD protein composition in mouse and primate brains during development. Proteins involved in synapse regulation are enriched in the differentially expressed (288 decreased and 267 increased) proteins on mouse PSD after a 2-week-old. We find that the changes in PSD protein abundance in mouse brains correlate with gene expression levels in postnatal mice and perinatal primates. This alteration of PSD composition is likely to be defective in the brains of mouse models or patients with autism spectrum disorder (ASD). Finally, we demonstrate that the brain of the common marmoset (Callithrix jacchus) changes PSD composition after the juvenile period. The alteration of PSD composition after 2-month-old is distinct from that observed in mice. Our results provide a comprehensive architecture of the remodeling of PSD composition across development, which may explain the molecular basics of synapse maturation and the pathology of psychiatric disorders, such as ASD.


title: Spinal cord extracts from sporadic ALS induce and propagate TDP-43 pathology in cerebral organoids

abstract:
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally.

Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue.

We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks.

Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS.


title: Interacting cortical gradients of neural timescales and functional connectivity and their relationship to perceptual behavior

abstract: Cognitive acts take place over a large range of temporal scales. Numerous corresponding gradients in neurodynamic timescales and long-range cortical interactions are believed to provide organizational constraints to the brain and influence neural populations' roles in cognition. However, it is unclear if gradients in various types of neural timescales and functional connectivity arise from related or distinct neurophysiological processes and if they influence behavior. Here, intracranial recordings from 4,090 electrode contacts in 35 individuals were used to systematically map gradients of multiple aspects of neurodynamics, neural timescales, and functional connectivity, and assess their interactions along category-selective ventral temporal cortex. Opposing functional connectivity gradients, with decreasing connectivity to visually responsive regions and increasing connectivity to regions that were not visually responsive, were observed along the ventral visual hierarchy. Endogenous neural timescales were correlated with functional connectivity to visually responsive regions after removing the effects of shared anatomical gradients, suggesting that these properties influence one another. Different stimulus evoked and endogenous timescales exhibited gradients with longer dynamics along the ventral visual hierarchy, but none of these timescales were significantly correlated with one another. This suggests that local neural timescales depend on neural and cognitive context and different timescales may arise through distinct neurophysiological processes. Furthermore, activity from neural populations with faster endogenous timescales and stronger functional connectivity to visually responsive regions was more predictive of perceptual behavior during a visual repeat detection task. These results reveal interrelationships and key distinctions among neural timescale and functional connectivity gradients that together can influence behavior.


title: The modulation of acute stress on Model-Free and Model-Based reinforcement learning in Gambling Disorder

abstract:
Background and aimsExperiencing acute stress is common in behavioral addictions such as gambling disorder. Additionally, like most substance-induced addictions, aberrant decision-making wherein a reactive habit-induced response (conceptualized as a Model-free [MF] in reinforcement learning) suppresses a flexible goal-directed response (conceptualized as a Model-based [MB]) is also common in gambling disorder. In the current study we investigated the influence of acute stress on the balance between habitual response and the goal-directed system.

MethodsA sample of N = 116 pathological gamblers (PG) and healthy controls (HC) performed an acute stress task - the Socially Evaluated Cold pressure task (SECPT) - or a control task. Self-reported stress and salivary cortisol were collected as measures of acute stress. Following the SECPT, participants performed the Two-Step Markov Task to account for the relative contribution of MB and MF strategies. Additionally, verbal working-memory and IQ measures were collected to account for their mediating effects on the orchestration between MB/MF and the impact of stress.

ResultsBoth groups had comparable baseline and stress-induced cortisol response to the SECPT. Non-stressed PG displayed lower MB learning than HC. MANOVA and regression analyses showed a deleterious effect of stress-induced cortisol response on the orchestration between MB and MF learning in HC but not in PG. Neither working memory nor IQ mediated these effects.

Discussion and ConclusionsDespite normal cortisol response to stress, we found an abnormal pattern of modulation of stress on the orchestration between MB and MF learning among PG.


title: TMEM106B is increased in Multiple Sclerosis plaques, and deletion causes accumulation of lipid after demyelination

abstract:
During inflammatory, demyelinating diseases such as multiple sclerosis (MS) axonal damage is prevalent early in the disease course. Axonal damage includes swellings, defects in transport, and failure to clear damaged intracellular proteins, all of which affect recovery and compromise the integrity of neurons and remyelination. Autophagy and the clearance of damaged cell components by the proteasome are important for the maintenance of normal cellular turnover; and the restoration of cellular homeostasis. The gradual accumulation of insoluble proteins in the brain is known to impair recovery from several neurodegenerative diseases. In this study, we used mass spectrometry to identify insoluble proteins within high-speed, mercaptoethanol/sarcosyl-insoluble pellets from purified white matter plaques isolated from the brains of individuals with MS. We determined that insoluble transmembrane protein106B (TMEM106B), expressed in neurons and glia and normally lysosomal-associated, is increased in MS plaques relative to normal-appearing white matter from individuals with Alzheimers disease and non-neurologic controls. We found that decreased TMEM106B protein in mice results in significant axonal damage and lipid droplet accumulation in the spinal cord following chronic myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. When TMEM106Bt/t mice were treated with cuprizone to experimentally induce demyelination, a significant increase in lipid deposition was observed in the corpus callosum of TMEM106Bt/t mice post-cuprizone withdrawal. Our study shows that the brain and spinal cord from challenged TMEM106Bt/t mice accumulate OilRedO+/Perilipin2+ lipid droplets. We postulate that increased insolubility of TMEM106B in MS plaques limits debris clearance by the lysosome which over time contributes to failed remyelination and axonal defects.

Abbreviated AbstractTransmembrane protein106B (TMEM106B), a lysosome-associated protein, is significantly less soluble in multiple sclerosis plaques than in white matter controls. Decreased TMEM106B produces significant axonal damage and lipid accumulation in mouse models of demyelinating diseases. TMEM106B insolubility and likely loss of function may limit lysosome transport and contribute to CNS pathology.


title: Pathophysiology of Dyt1 dystonia is mediated by spinal cord dysfunction

abstract:
Dystonia, a neurological disorder defined by abnormal postures and disorganised movements, is thought to be a neural circuit disorder with dysfunction arising within and between multiple brain regions. Given that spinal circuits are the de facto final common pathway for motor control, we sought to determine their contribution to the movement disorder. We confined a dystonia-related mutation to the spinal cord, which led to behavioural and physiological recapitulation of a severe form of inherited, early-onset, generalised dystonia. These data challenge our current understanding of dystonia, and lead to broader insights into spinal cord function and its involvement in movement disorder pathophysiology.

One sentence summarySpinal Tor1a deletion causes severe early onset generalised dystonia, exposing spinal circuitopathy as a key factor in its pathophysiology.


title: Manipulating the rapid consolidation periods in a learning task affects general skills more than statistical learning

abstract: Memory consolidation processes have always been thought to cover up extended periods and have traditionally been investigated from the perspective of hours or days. However, the latest developments in memory research showed that memory stabilization processes could occur even within seconds. Here, we investigate this rapid form of consolidation during statistical learning. We aim to answer (a) whether this rapid offline improvement occurs in implicit statistical learning and general skill learning and (b) whether the duration of rest periods affects these two learning types differently. Participants performed a widely used statistical learning task - the Alternating Serial Reaction Time (ASRT) task - that enables us to measure implicit statistical and general skill learning separately. The ASRT task consisted of 25 learning blocks with a rest period between the blocks. In a between-subjects design, the length of the rest periods was fixed at 15 or 30 seconds, or the participants could control the length themselves. We found that the duration of rest periods does not affect the acquisition of statistical knowledge, but they do change the dynamics of learning in a way that learning is more likely to occur online in the case of certain fixed breaks. Moreover, we found weaker general skill learning in the self-paced group than in the fixed rest period groups (i.e., 15 and 30-second breaks). These results suggest that distinct learning processes are differently affected by the duration of short rest periods.


title: Inter-scanner brain MRI volumetric biases persist even in a harmonized multi-subject study of multiple sclerosis

abstract:
Background/PurposeMulticenter study designs involving a variety of MRI scanners have become increasingly common. However, these present the issue of biases in image-based measures due to scanner or site differences. To assess these biases, we imaged 11 volunteers with multiple sclerosis (MS) with scan and rescan data at 4 sites.

Materials and MethodsImages were acquired on Siemens or Philips scanners at 3-tesla. Automated white matter lesion detection and whole brain, gray and white matter, and thalamic volumetry were performed, as well as expert manual delineations of T1 and T2 (FLAIR) lesions. Random effect and permutation-based nonparametric modeling was performed to assess differences in estimated volumes within and across sites.

ResultsRandom effect modeling demonstrated model assumption violations for most comparisons of interest. Non-parametric modeling indicated that site explained > 50% of the variation for most estimated volumes. This expanded to > 75% when data from both Siemens and Philips scanners were included. Permutation tests revealed significant differences between average inter- and intra-scanner differences in most estimated brain volumes (P < .05). The automatic activation of spine coil elements during some acquisitions resulted in a shading artifact in these images. Permutation tests revealed significant differences between thalamic volume measurements from acquisitions with and without this artifact.

ConclusionDifferences in brain volumetry persisted across MR scanners despite protocol harmonization. These differences were not well explained by variance component modeling; however, statistical innovations for mitigating inter-scanner differences show promise in reducing biases in multi-center studies of MS.


title: Feature-space selection with banded ridge regression

abstract: Encoding models provide a powerful framework to identify the information represented in brain recordings. In this framework, a stimulus representation is expressed as a feature space and is used in a regularized linear regression to predict brain activity. To account for a potential complementarity of different feature spaces, a joint model is fit on multiple feature spaces simultaneously. To adapt regularization strength to each feature space, ridge regression is extended to banded ridge regression, which optimizes a different regularization hyperparameter per feature space. The present paper proposes a method to decompose over feature spaces the variance explained by a banded ridge regression model. It also describes how banded ridge regression performs a feature-space selection, effectively ignoring non-predictive and redundant feature spaces. This feature-space selection leads to better prediction accuracy and to better interpretability. Banded ridge regression is then mathematically linked to a number of other regression methods with similar feature-space selection mechanisms. Finally, several methods are proposed to address the computational challenge of fitting banded ridge regressions on large numbers of voxels and feature spaces. All implementations are released in an open-source Python package called Himalaya.


title: Multicomponent Pseudomonas aeruginosa vaccines eliciting Th17 cells and functional antibody responses confer enhanced protection against experimental acute pneumonia in mice

abstract: The Gram-negative pathogen Pseudomonas aeruginosa is a common cause of pneumonia in hospitalized patients. Its increasing antibiotic resistance and widespread occurrence present a pressing need for vaccines. We previously showed that a P. aeruginosa type III secretion system protein, PopB elicits a strong Th17 response in mice after intranasal (IN) immunization and confers antibody-independent protection against pneumonia in mice. In the current study, we evaluated the immunogenicity and protective efficacy in mice of the combination of PopB (purified with its chaperone protein PcrH) and OprF/I, an outer membrane hybrid fusion protein, compared to immunization with the proteins individually either by the intranasal (IN) or subcutaneous (SC) routes. Our results show that after vaccination, a Th17 recall response from splenocytes was detected only in mice vaccinated with PopB/PcrH, either alone or in combination with OprF/I. Mice that were immunized with the combination of PopB/PcrH and OprF/I had enhanced protection in an acute lethal P. aeruginosa pneumonia model, regardless of vaccine route, compared to the mice vaccinated the with either alone or adjuvant control. Immunization generated IgG titers against the vaccine proteins and whole P. aeruginosa cells. Interestingly, none of these antisera had opsonophagocytic killing activity, but antisera from mice immunized with vaccines containing OprF/I had the ability to block IFN-{gamma} binding to OprF/I, a known virulence mechanism. Hence, vaccines combining PopB/PcrH with OprF/I that elicit functional antibodies lead to a broadly and potently protective vaccine against P. aeruginosa pulmonary infections.

category: immunology


title: Non-invasive Fluorescence Imaging of Gut Commensal Bacteria in Live Mice

abstract: In mammals, gut commensal microbiota interact extensively with the host and the same interactions can be dysregulated in diseased states. The development of methods to monitor gut microbiota in vivo can lead to improved foundational understanding of the biological events underpinning these interactions. The current standard for non-invasive monitoring of gut bacteria entails classification by 16S rRNA sequencing from fecal samples. This method has many advantages but also has serious limitations, especially for monitoring dynamic changes in the gut of live animals. In recent years, several imaging techniques have been widely adopted that afford non-invasive assessment of animal subjects - most notably in cancer biology; however, these technical gains have not translated to the imaging of gut bacterial communities. Herein, we describe a method to non-invasively image commensal bacteria based on the specific metabolic labeling of bacterial cell walls to illuminate the gut bacteria of live mice. This tagging strategy may additionally provide unprecedented insight into cell wall turnover of gut commensals, which has implications for bacterial cellular growth and division, in a live animal.

category: microbiology


title: The microtubule minus end-binding protein CAMSAP2 does not regulate microtubule dynamics in primary pancreatic β-cells but facilitates insulin secretion

abstract: Glucose stimulation induces the remodeling of microtubules in Islet {beta}-cells to potentiate glucose-stimulated insulin secretion. CAMSAP2 is a microtubule minus-end binding protein and is reported to stabilize and position microtubules in several non-{beta}-cells, such as human retinal pigment epithelium cells. In immortalized insulinoma MIN6 cells, CAMSAP2 binds to and forms short stretches at microtubule minus ends in the cytoplasm, which is consistent with the reported subcellular localization and functions of CAMSAP2 in non-{beta}-cells. Surprisingly, we found that CAMSAP2 expressed in primary islet {beta}-cells does not form short stretches in the cytoplasm, but instead is localized to the Golgi apparatus. This novel localization is specific to {beta}-but not -cells in islets and it is independent of MT-binding. Knockdown of CAMSAP2 by shRNA impairs Golgi-ER trafficking, reduces total insulin content, and attenuates GSIS without affecting the MT dynamics or releasability of insulin granules in islet {beta}-cells. Corresponding to these results, we found that primary islets and MIN6 cells express different CAMSAP2 isoforms. We propose that primary islet {beta} cells use a novel CAMSAP2 isoform for a MT-independent non-canonical function, which is to promote Golgi-ER trafficking that supports efficient production of insulin secretory granules.

category: cell biology


title: Testicular macrophages are recruited during a narrow time window by fetal Sertoli cells to promote organ-specific developmental functions

abstract: While macrophages are most commonly known for their roles in innate immunity, a growing body of evidence supports the idea that fetal-derived tissue-resident macrophages play developmental roles during organogenesis. In the testis, it has long been proposed that macrophages are important players in steroidogenesis and other testicular functions, but which macrophage populations are involved is unclear. We previously showed that macrophages play critical roles in fetal testis morphogenesis and reported the presence of 2 unique adult testicular macrophage populations, interstitial and peritubular. There has been some debate regarding the hematopoietic origins of testicular macrophages and whether distinct macrophage populations promote specific testicular functions. Here we have undertaken an extensive lineage-tracing study of mouse hematopoietic cells. We found that, while yolk-sac-derived macrophages comprise the earliest testicular macrophages, fetal hematopoietic stem cells (HSCs) give rise to monocytes that colonize the gonad during a narrow time window in mid-gestation, after which time HSCs no longer contribute to testicular macrophages. These long-lived monocytes, over the course of fetal and postnatal life, differentiate into testicular macrophages. Our data indicate that Sertoli cells, and not germ cells, are required for recruitment of immune cells and peritubular macrophage differentiation. Finally, we show that yolk-sac-derived macrophages and HSC-derived macrophages play distinct roles in testis cord morphogenesis, whereas interstitial macrophages promote adult Leydig cell proliferation and steroid production. Overall, our findings offer clarity regarding the origins of testicular macrophages and provide insight into the diversity of their tissue-specific developmental roles.

category: developmental biology


title: Field margins as substitute habitat for the conservation of birds in agricultural wetlands

abstract: Breeding birds in agricultural landscapes have declined considerably since the 1950s and the beginning of agricultural intensification in Europe. Given the increasing pressure on agricultural land, it is necessary to identify conservation measures that consume little productive land. We tested the hypothesis that field margins represent substitute habitats for bird species in agricultural wetlands. We monitored bird species in 86 crop fields in rice paddy landscapes of Camargue (southern France), a wetland of international importance for birds. We investigated whether the area of three types of field margins (grass strips, hedgerows and reed strips) within a 500 m buffer around each studied crop field had an effect on the abundance of three groups of birds defined based on their primary habitat (reedbeds, grasslands, and forest edge species). We controlled for the area of each semi-natural habitat (wetlands, grasslands, and woodlands), crop diversity and mean crop field size. Our study confirms that bird guilds are favored by the cover of their primary habitat but are also influenced by the cover of field margins. Reedbed birds are favored by the cover of wetlands and reed strips, grassland birds are favored by the cover of grasslands and wetlands and negatively impacted by the cover of woodlands and hedgerows, while forest edge birds are favored by the cover of hedgerows and negatively impacted by the cover of reed strips. These results suggest that field margins may represent substitute habitats for reedbed and forest edge bird species and highlight their importance for biodiversity conservation in wetland agricultural landscapes. However, our results also suggest that increasing the area of hedgerows and reed strips may have a negative effect on grassland birds and forest edge birds, respectively. Recommendations for field margin management in agricultural wetlands should therefore be tailored to local conservation priorities.

category: ecology


title: Association of variants in ACE, ACTN3, AGT, IL6 and BDKRB2 genes with athlete status and playing position in Colombian amateur rugby athletes

abstract:
Genetic polymorphisms are involved in different metabolic pathways that are manifested at the physiological level and have been associated with specific phenotypes in sport from anthropometric and functional characterizations that pose conditional and physiological demands for the rugby athlete. The identification of this type of polymorphisms in athletes represents a resource that contributes significantly to the processes of training, selection and sports orientation.

The purpose of this study was to describe type and frequencies of allelic and genotypic variants in ACTN3, ACE, AGT, BDRKB2 and IL6 genes in sub elite rugby athletes in Colombia. Additionally, the polymorphisms found were compared with a control population, as well as contrasted according to playing position backs and forwards.

In this research, 47 individuals from the Vallecaucana rugby league and 67 from a control group (non-athletes) were sampled. All were analyzed for polymorphisms in the ACE, AGT, ACTN3, IL6 and BDKRB2 genes, using the PCR RFLPs technique. The significance of the differences between the experimental and control groups was tested by the X2 test (p <0.05).

In rugby athletes we found a higher frequency of allele D (0.883) ACE gene, allele R (0.63) ACTN3 gene, allele G (0.819) IL6 gene, all associated with strength and power sports. There are significant genotypic differences between athletes and the control population in all the genes analyzed and significant allelic differences in the ACE, ACTN3, BDRKB2 and IL6 genes. When comparing the playing positions (backs vs. forwards), significant genotypic differences were observed in the ACTN3, BDRKB2, AGT and IL6 genes. At the allelic level, the R and X alleles of the ACTN3 gene and the I allele of the ACE gene show significant differences.

In conclusion, in the polymorphisms analyzed, an association with strength sports, explosive strength and rugby is observed. Significant genotypic and allelic differences were also recorded between the backs and forwards positions, as well as significant differences in the allelic and genotypic structure between the group of athletes and the control population.

category: genetics


title: Optical mapping compendium of structural variants across global cattle breeds

abstract:
Structural variants (SV) have been linked to several important bovine disease phenotypes, but due to the difficulty of their accurate detection with standard sequencing approaches, their role in shaping economically important traits across global cattle breeds is largely unexplored. Optical mapping is an alternative approach for mapping SVs that has been shown to have higher sensitivity than DNA sequencing approaches. The aim of this project was to use optical mapping to develop a high quality database of structural variation across cattle breeds from different geographical regions and origins, to enable further studies of the important roles of SV in cattle.

To do this we generated 100X Bionano optical mapping data for 18 cattle of nine different ancestries, three continents and covering both major cattle lineages. In total we identified 13,457 SVs, of which 1,200 putatively overlap a coding region. This resource therefore provides a high-quality set of optical mapping-based SV calls that can be used across studies, from validating DNA sequencing based SV calls to prioritising candidate functional variants in genetic association studies and expanding our understanding of the role of SVs in the evolution of cattle.

category: genomics


title: Promiscuous feeding across multiple honey bee hosts amplifies the vectorial capacity of Varroa destructor

abstract: Varroa destructor is a cosmopolitan pest and leading cause of colony loss of the European honey bee. Historically described as a competent vector of honey bee viruses, this arthropod vector is cause for the global pandemic of Deformed wing virus, now endemic in honeybee populations. Our work shows viral spread is driven by Varroa actively switching from one adult bee to another as they feed. Assays using fluorescent microspheres were used to show the movement of fluids in both directions between host and vector when Varroa feed. Therefore, Varroa could be in either an infectious or naive state dependent upon the disease status of their host. We tested this and confirm that the relative risk of a Varroa feeding was dependent on the infectiousness of their previous host. Varroa exhibit remarkable heterogeneity in their host switching behavior, with some Varroa switching infrequently while others switch at least daily. As a result, relatively few of the most active Varroa parasitize the majority of bees. This multiple feeding behavior has analogs in vectorial capacity models of other systems, where promiscuous feeding by individual vectors is a leading driver of vectorial capacity. We propose that the honeybee-Varroa relationship offers a unique opportunity to apply principles of vectorial capacity to a social organism, as virus transmission is both vectored and occurs through multiple host-to-host routes common to a crowded society.

category: animal behavior and cognition


title: Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism

abstract: Human cytomegalovirus (CMV) is a highly relevant pathogen, and its rodent counterparts serve as common infection models. Global proteome profiling of rat CMV-infected cells uncovered a pronounced loss of the transcription factor STAT2, which is crucial for interferon signalling. Deletion mutagenesis documented that STAT2 is targeted by the viral protein E27. Cellular and in vitro analyses showed that E27 exploits host-derived Cullin4-RING ubiquitin ligases (CRL4) to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopic structure determination revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors (DDB1- and Cullin4-associated factors, DCAFs) to displace them from the CRL4 catalytic core. Moreover, structural analyses elucidated the mechanism of STAT2 recruitment and indicate that E27-binding additionally disturbs STAT2 activation by occupying the IRF9 binding interface. For the first time, these data provide structural insights into cytomegalovirus-encoded interferon antagonism and establish an atomic model for STAT2 counteraction by CRL4 misappropriation with important implications for viral immune evasion.

category: biochemistry


title: Gaining insight into the allometric scaling of trees by utilizing 3d reconstructed tree models - a SimpleForest study

abstract:
Forestry utilizes volume predictor functions utilizing as input the diameter at breast height. Some of those functions take the power form Y = a * Xb. In fact this function is fundamental for the biology field of allometric scaling theories founded round about a century ago. The theory describes the relationships between organs/body parts and the complete body of organisms.

With digital methods we can generate 3d forest point clouds non destructively in short time frames. SimpleForest is one free available tool which generates fully automated ground and tree models from high resoluted forest plots. Generated topological ordered cylinder models are called commonly QSMs.

We use SimpleForest QSMs an build a function which estimates the total supported wood volume at any given point of the tree. As input we use the supported soft wood volume for those query points. Instead of measuring directly the soft wood volume we use as a proxy the number of supported twigs. We argue with the pipe model theory for the correctness of the proxy.

We can use the named relationship to also filter our QSMs made of an open data set of tree clouds. The filter corrects overestimated radii. And we compare the corrected QSM volume against the harvested reference data for 66 felled trees. We also found QSM data of TreeQSM, a competitive and broadly accepted QSM modeling tool. Our RMSE was less than 40% of the tree QSM RMSE. And for other error measures, the r2adj. and the CCC, the relative improvement looked even better with 27% and 21% respectively.

We consider this manuscript as highly impactful because of the magnitude of quality improvement we do. The relation between soft volume and total volume distributions seems to be really strong and tree data can easily also be used as example data for the generic field of allometric scaling.

category: bioinformatics


title: Auxin Plays a Role in the Adaptation of Rice to Anaerobic Germination and Seedling Establishment

abstract: Auxin is well known to stimulate coleoptile elongation and rapid seedling growth in the air. However, its role in regulating rice germination and seedling establishment under submergence is largely unknown. Previous studies have shown that excessive levels of IAA frequently cause the inhibition of plant growth and development. In the present study, the high-level accumulation of endogenous IAA is observed under submergence in the dark, stimulating rice coleoptile elongation but limiting the root and primary leaf growth during anaerobic germination (AG). We found that oxygen and light can reduce IAA levels, promote the seedling establishment and enhance rice AG tolerance. miRNA microarray profiling and RNA gel blot analysis results show that the expression of miR167 is negatively regulated by submergence; it subsequently modulates the accumulation of free IAA through the miR167-ARF-GH3 pathway. The OsGH3-8 encodes an IAA-amido synthetase that functions to prevent free IAA accumulation. Reduced miR167 levels or overexpressing OsGH3-8 increase auxin metabolism, reduce endogenous levels of free IAA and enhance rice AG tolerance. The present study reveals that poor seed germination and seedling growth inhibition resulting from excessive IAA accumulation would cause intolerance to submergence in rice, suggesting that a certain threshold level of auxin is essential for rice AG tolerance.


title: Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topology

abstract: The specific functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches. However, many spatial proteomics methods are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes. Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings. Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome in a single experiment. Biochemical and imaging-based follow-up studies demonstrate that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology data. This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, establishes a method for concomitant profiling of sub-organelle and membrane proteomes, and provides a resource for mitochondrial spatial biology.

category: systems biology


title: Commonality of Odorant Receptor Choice Mechanism Revealed by Analysis of a Highly Represented Odorant Receptor Transgene

abstract: In the mouse, more than 1,100 odorant receptors (ORs) are expressed in a monogenic and monoallelic fashion, referred to as singular gene expression. Using a 21bp singular-choice enhancer (x21), we radically increase representation of olfactory sensory neurons (OSNs) choosing a 5x21 enhanced OR transgene, but not overexpression of its mRNA on a per cell basis. RNA-sequencing and differential expression analysis identified 425 differentially expressed genes (DEGs). ORs make up 86% of all DEGs, of which 325 have decreased representation and 40 have increased representation. Underrepresented ORs include Class I, Class II and TAAR genes and within each of their respective olfactory bulb domains: DI, DII, and DIII (TAAR) we committedly observe multiple homogeneous glomeruli with an OR1A1-identity. The underrepresentation of endogenous, class-specific ORs across evolutionarily distinct cell types in favor of the expression of the 5x21-OR1A1 transgene argues that a common mechanism of singular gene choice is present for all OR-expressing OSNs.


title: Nitrogen palaeo-isoscapes: Changing spatial gradients of faunal δ15N in late Pleistocene and early Holocene Europe

abstract: Nitrogen isotope ({delta}15N) analysis of animal tissue is widely used in archaeology and palaeoecology to investigate diet and ecological niche. Data interpretations require an understanding of nitrogen isotope compositions at the base of the food web (baseline {delta}15N). Significant variation in animal {delta}15N has been recognised at various spatiotemporal scales and linked to changes both in baseline {delta}15N and animal ecology. Isoscapes (models of isotope spatial variation) have proved a useful tool for investigating spatial variability in biogeochemical cycles in present-day marine and terrestrial ecosystems, but so far, their application to palaeo-data has been limited. Here, we present time-sliced nitrogen isoscapes for late Pleistocene and early Holocene Europe (c. 50,000 to 10,000 years BP) using herbivore collagen {delta}15N data. This period covers the Last Glacial-Interglacial Transition, during which significant variation in the terrestrial nitrogen cycle occurred. Our results show clear changes in spatial gradients of {delta}15N through time. Prediction of the lowest faunal {delta}15N values in northern latitudes after, rather than during, the Last Glacial Maximum is consistent with the Late Glacial Nitrogen Excursion (LGNE). We consider the potential of incorporating climatic covariate data into isoscape models but find their inclusion does not improve model performance. These findings have implications for investigating the drivers of the LGNE, which has been linked to increased landscape moisture and permafrost thaw, and for understanding changing isotopic baselines, which are fundamental for studies investigating diets, niche partitioning, and migration of higher trophic level animals.


category: paleontology


title: The PCI domains are "winged" HEAT domains

abstract: The HEAT domains are a family of helical hairpin repeat domains, composed of four or more hairpins. HEAT is derived from the names of four family members: huntingtin, eukaryotic translation elongation factor 3 (eEF3), protein phosphatase 2 regulatory A subunit (PP2A), and mechanistic target of rapamycin (mTOR). HEAT domain-containing proteins play roles in a wide range of cellular processes, such as protein synthesis, nuclear transport and metabolism, and cell signaling. The PCI domains are a related group of helical hairpin domains, with a "winged-helix" (WH) subdomain at their C-terminus, which is responsible for multi-subunit complex formation with other PCI domains. The name is derived from the complexes, where these domains are found: the Proteasome "lid" regulatory subcomplex, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). We noted that in structure homology searches using HEAT domains, sometimes PCI domains appeared in the search results ahead of other HEAT domains, which indicated that the PCI domains could be members of the HEAT domain family, and not a related but separate group. Here, we report extensive structure homology analysis of HEAT and PCI domains, both within and between the two groups of proteins. We present evidence that the PCI domains as a group have greater structural homology with individual groups of HEAT domains than some of the HEAT domain groups have among each other. Therefore, our results indicate that the PCI domains have evolved from a HEAT domain that acquired a WH subdomain. The WH subdomain in turn mediated selfassociation into a multi-subunit complex, which eventually evolved into the common ancestor of the Proteasome lid/CSN/eIF3.


title: Comparative study between radiofrequency- and muscimol-induced inhibition of cultured networks of cortical neuron

abstract: Previous studies have shown that spontaneously active cultured networks of cortical neuron grown planar microelectrode array are sensitive to radiofrequency (RF) fields and exhibit an inhibitory response more pronounced as the exposure time and power increase. To better understand the mechanism behind the observed effects, we aimed at identifying similarities and dissimilarities between the inhibitory effect of RF fields (continuous wave, 1800 MHz) to the {gamma}-Aminobutyric acid type A (GABAA) receptor agonist muscimol (MU). Inhibition of network bursting activity in response to RF exposure became apparent at an SAR level of 28.6 W/kg and co-occurred with an elevation of the culture medium temperature of [~]1 {degrees}C. Differently to a pharmacological inhibition with MU, exposure to RF fields preferentially inhibits bursting over spiking activity and exerts less constrains on neural network bursting synchrony. Network rebound excitation, a phenomenon relying on intrinsic properties of cortical neurons, was observed consecutively to the removal of tonic hyperpolarization after washout of MU but not in response to cessation of RF exposure which implies that hyperpolarization is not the main driving force mediating the inhibitory effects of RF fields. At the level of single neurons, network inhibition induced by MU and RF fields occurred with reduced action potential (AP) half-width. As change in AP waveform tightly influence efficiency of synaptic transmission, the narrowing effect on AP seen under RF exposure might contribute to reduce network bursting activity. By pointing only to a partial overlap between the inhibitory hallmarks of these two forms of inhibition, our data suggest that the inhibitory mechanisms of action of RF fields differ from the ones mediated by the activation of GABAA receptor. The rapid onset of the inhibitory effect of RF fields and its reversibility strikingly similar to MU are in favour of a mechanism interacting with fast operating targets at the membrane such as ion channels.


title: RORβ modulates a gene program that is protective against articular cartilage damage

abstract: Osteoarthritis (OA) is the most prevalent chronic joint disease which increases in frequency with age eventually impacting most people over the age of 65. OA is the leading cause of disability and impaired mobility, yet the pathogenesis of OA remains unclear. Treatments have focused mainly on pain relief and reducing joint swelling. Currently there are no effective treatments to slow the progression of the disease and to prevent irreversible loss of cartilage. Here we demonstrate that stable expression of ROR{beta} in cultured cells results in alteration of a gene program that is supportive of chondrogenesis and is protective against development of OA. Specifically, we determined that ROR{beta} regulates the balance of FGFRs signaling on FGFR1/FGFR3 that ERK1/2-MAPK signaling was suppressed by FGFR1(cartilage destruction) and AKT signaling was enhanced by FGFR3 (cartilage protection). These results suggest a critical role for ROR{beta} in chondrogenesis and suggest that identification of mechanisms that control the expression of ROR{beta} in chondrocytes could lead to the development of disease modifying therapies for the treatment of OA.


title: Single cell transcriptomics of ferrets reveal a common temporal pattern of progenitors in brain development of gyrencephalic mammals

abstract: The diversity of neural stem cells is a hallmark of gyrencephalic brains, including that in humans. Ferrets are an excellent model to study the complex brain development in gyrencephalic mammals, but information on their neural progenitor subtypes is fragmentary. Here, we investigated the temporal series of single-cell transcriptomes of progenitors in developing cortices in ferrets for comparison with human datasets. We found that the diversity and temporal trajectory of neural progenitors, termed radial glia (RG), are well conserved between ferrets and humans. Truncated RG (tRG), a progenitor subtype previously described in humans, and outer RG-like cells were assigned to ferret transcriptomes. In vivo and transcriptome analyses indicated that ferret tRG are generated via asymmetric RG divisions during late neurogenesis, and suggested that tRG is eventually fated to ependymal and glial populations. Therefore, the combined analyses of human and ferret transcriptomes enable the determination of progenitor fate sequences in vivo.


title: Histone deacetylase 1 maintains lineage integrity through histone acetylome refinement during early embryogenesis

abstract: Histone acetylation is a pivotal epigenetic modification that controls chromatin structure and regulates gene expression. It plays an essential role in modulating zygotic transcription and cell lineage specification of developing embryos. While the outcomes of many inductive signals have been described to require enzymatic activities of histone acetyltransferases and deacetylases (HDACs), the mechanisms by which HDACs confine the utilization of the zygotic genome remain to be elucidated. Here, we show that histone deacetylase 1 (Hdac1) progressively binds to the zygotic genome from mid blastula and onward. The recruitment of Hdac1 to the genome at blastula is instructed maternally. Cis-regulatory modules (CRMs) bound by Hdac1 possess epigenetic signatures underlying distinct functions. We highlight a dual function model of Hdac1 where Hdac1 not only represses gene expression by sustaining a histone hypoacetylation state on inactive chromatin, but also maintains gene expression through participating in dynamic histone acetylation-deacetylation cycles on active chromatin. As a result, Hdac1 maintains differential histone acetylation states of bound CRMs between different germ layers and reinforces the transcriptional program underlying cell lineage identities, both in time and space. Taken together, our study reveals a comprehensive role for Hdac1 during early vertebrate embryogenesis.


title: Spermine inhibits PAMP- elicited ROS and Ca2+ burst and reshapes the transcriptional landscape of PTI in Arabidopsis

abstract: Polyamines are small polycationic amines which levels increase during defense. Previous studies support the contribution of the polyamine spermine (Spm) to the establishment of the hypersensitive response (HR) during incompatible plant-pathogen interactions. However, the potential contribution of Spm to other layers of defense, and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in particular, was not completely established. Here we compared the contribution of Spm and putrescine (Put) to early and late PTI responses. We find that Put and Spm show opposite effects on PAMP-elicited reactive oxygen species (ROS) production, with Put increasing whereas Spm lowering flg22-stimulated ROS burst. Through genetic and pharmacological approaches, we find that the inhibitory effect of Spm on flg22-elicited ROS is independent of polyamine oxidation and EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1), PAD4 (PHYTOALEXIN DEFICIENT 4), salicylic acid and NPR1 (NONEXPRESSER OF PR GENES 1) defense components but resembles chemical inhibition of RBOHD (RESPIRATORY BURST OXIDASE HOMOLOG D) function. Remarkably, Spm can also suppress ROS elicited by FLS2-independent but RBOHD-dependent pathways, thus pointing to compromised RBOHD function. Consistent with this, we find that Spm dampens flg22-stimulated cytosolic Ca2+ influx necessary for RBOHD function and reshapes the transcriptional landscape of PTI and defense responses against Pseudomonas syringae pv. tomato DC3000. Overall, we provide molecular evidence for the differential contribution of Put and Spm to PTI with an impact on plant defense.


title: Chemotherapy drugs induce different gut microbiota disorder pattern and NODs/RIP2/NF-κB signaling pathway activation that lead to different degrees of intestinal injury

abstract:
5-Fluorouracil (5-FU), irinotecan (CPT-11), oxaliplatin (L-OHP) and calcium folinate (CF) are the widely used chemotherapy drugs to treat colorectal cancer. However, the use of chemotherapy is often accompanied by intestinal inflammation and gut microbiota disorder. Moreover, the change of gut microbiota may lead to destruction of the intestinal barrier, which contributes to the severity of intestinal injury. There was no detailed comparison of intestinal injury and gut microbiota disorder among 5-FU, CPT-11, L-OHP and CF, which is not benefit for the development of targeted detoxification therapy after chemotherapy. In this project, a model of chemotherapy-induced intestinal injury in tumor-bearing mice was established by intraperitoneal injection of chemotherapy drugs at a clinically equivalent dose. 16S rDNA sequencing was used to detect gut microbiota. We found that 5-FU, CPT-11 and L-OHP caused intestinal injury, inflammatory cytokine (IFN-{gamma}, TNF-, IL-1{beta}, and IL-6) secretion, and gut microbiota disorder. Importantly, we established a complex but clear network between the gut microbiota change pattern and intestinal damage degree induced by different chemotherapy drugs. L-OHP caused the most severe damage in intestine and disorder of gut microbiota, and showed considerable overlap of the microbiota change pattern with 5-FU and CPT-11. The phylogenetic investigation of communities by reconstruction of unobserved states, V1.0 (PICRUSt) analysis showed that the microbiota disorder pattern induced by 5-FU, CPT-11 and L-OHP was related to the NOD like signaling pathway. Therefore, we detected the protein expression of the NODs/RIP2/NF-{kappa}B signaling pathway and found that L-OHP activated that pathway highest. Furthermore, by RDA/CCA analysis, we found that Bifidobacterium, Akkermansia, Allobaculum, Catenibacterium, Mucispirillum, Turicibacter, Helicobacter, Proteus, Escherichia Shigella, Alloprevotealla, Vagococcus, Streptococcus and Candidatus Saccharimonas were highly correlated with the NODs/RIP2/NF-{kappa}B signaling pathway, and influenced by chemotherapy drugs.

IMPORTANCEThe chemotherapy-induced intestinal injury limit drugs clinical use. Intestinal injury involves multiple signaling pathways and the disruption of microbiota. Our results suggest that the degree of intestinal injury caused by different drugs of the first-line colorectal chemotherapy regimen is related to the change pattern of microbiota. Moreover, the NODs/RIP2/NF-{kappa}B signaling pathway was activated in different degrees is also related to the change pattern of microbiota. We found L-OHP caused the most severe change of gut microbiota, and showed considerable overlap of the microbiota changes pattern with 5-FU and CPT-11. Here, we have established a network of different chemotherapy drugs, gut microbiota and NODs/RIP2/NF-{kappa}B signaling pathway, which may provide a new basis for further elucidating the mechanism and clinical treatment of intestinal injury caused by chemotherapy.

category: molecular biology


title: Semi-automated protocol to quantify and characterize fluorescent, 3D vascular images

abstract: The microvasculature facilitates gas exchange, provides nutrients to cells, and regulates blood flow in response to stimuli. Vascular abnormalities are an indicator of pathology for various conditions, such as compromised vessel integrity in small vessel disease and angiogenesis in tumors. Traditional immunohistochemistry enables visualization of tissue cross-sections containing exogenously labeled vasculature. Although this approach can be utilized to quantify vascular changes within small fields-of-view, it is not a practical way to study the vasculature on the scale of whole organs. Three-dimensional (3D) imaging presents a more appropriate method to visualize the vascular architecture in tissue. Here we describe the complete protocol that we use to characterize the vasculature of different organs in mice encompassing the methods to fluorescently label vessels, optically clear tissue, collect 3D vascular images, and quantify these vascular images with a semi-automated approach. To validate the automated segmentation of vascular images, one user manually segmented fifty random regions of interest across different vascular images. The automated segmentation results had an average sensitivity of 80{+/-}8% and an average specificity of 90{+/-}5% when compared to manual segmentation. Applying this procedure of image analysis presents a method to reliably quantify and characterize vascular networks in a timely fashion. This procedure is also applicable to other methods of tissue clearing and vascular labels that generate 3D images of microvasculature.

category: bioengineering


title: Next generation sequencing analysis of gastric cancer identifies the leukemia inhibitory factor receptor (LIFR) as a driving factor in gastric cancer progression and as a predictor of poor prognosis

abstract: Gastric cancer (GC) is the third cause of cancer-related-death worldwide. Nevertheless, because GC screening programs are not cost-effective, most patients receive diagnosis in the advanced stages, when surgical options are limited because the presence of diffuse disease. Peritoneal dissemination occurs in approximately one third of patients with GC and is a strong predictor of poor outcome. Despite the clinical relevance, biological and molecular mechanisms underlying the formation of peritoneal metastasis in GC remain poorly defined. To investigate this point, we conducted a high-throughput sequencing of transcriptome expression in paired samples of normal and neoplastic gastric mucosa in 31 GC patients with or without peritoneal carcinomatosis. The RNAseq analysis led to the discovery of a group of highly upregulated or downregulated genes that were differentially modulated in patients with peritoneal disease in comparison to GC patients without peritoneal involvement. Among these genes the leukemia inhibitory factor receptor (LIFR) and the one cut domain family member (ONECUT)2 were the only two genes that predicted survival at univariate statistical analysis. Because LIFR was the highest regulated gene we have further assessed whether this receptor plays a mechanistic role in GC dissemination. For this purpose, we have first assessed the expression of LIF, a member of IL-6 cytokine family, and LIFR in GC cell lines. Our results demonstrate that exposure of MKN45 cells to LIF, promoted a concentration-dependent proliferation and epithelial-mesenchymal transition (EMT) as shown by modulation of E-cadherin/vimentin gene expression along with JAK and STAT 3 phosphorylation and acquisition of a migratory phenotype. These features were reversed by in vitro treatment with a LIFR antagonist. Together, these data provide support to the notion that development of LIF/LIFR inhibitors might have a role in the treatment of GC.

category: cancer biology


title: A QuantCrit investigation of society's educational debts due to racism, sexism, and classism in biology student learning

abstract: We investigated the intersectional relationships between racism, sexism, and classism in inequities in student conceptual knowledge in introductory biology courses using a quantitative critical framework. Using Bayesian hierarchical linear models, we examined students conceptual knowledge as measured by the Introductory Molecular and Cell Biology Assessment. The data came from the LASSO database and included 6,547 students from 87 introductory courses at 11 institutions. The model indicated that students with marginalized identities by race, gender, and class tended to start with lower scores than continuing-generation, White men. We conceptualized these differences as educational debts society owed these students due to racism, sexism, and classism. Instruction added to these educational debts for most marginalized groups, with the largest increases for students with multiple marginalized identities. After instruction, society owed Black and Hispanic, first-generation women an educational debt equal to 60-80% of the average learning in the courses. These courses almost all (85/87) used collaborative learning and half (45/87) supported instruction with learning assistants. While research shows collaborative learning better serves students than lecture-based instruction, these results indicate it does not repay educational debts due to racism, sexism, and classism.

category: scientific communication and education


title: A unique epigenomic landscape defines CD8+ tissue-resident memory T cells

abstract:
Memory T cells provide rapid and long-term protection against infection and tumors. The memory CD8+ T cell repertoire contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. While these T cell populations have been well characterized in terms of differentiation potential and function, the epigenetic changes underlying memory T cell fate determination and tissue-residency remain largely unexplored. Here, we examined the single-cell chromatin landscape of CD8+ T cells over the course of acute viral infection. We reveal an early bifurcation of memory precursors displaying distinct chromatin accessibility and define epigenetic trajectories that lead to a circulating (TCIRC) or tissue-resident memory T (TRM) cell fate. While TRM cells displayed a conserved epigenetic signature across organs, we demonstrate that these cells exhibit tissue-specific signatures and identify transcription factors that regulate TRM cell populations in a site-specific manner. Moreover, we demonstrate that TRM cells and exhausted T (TEX) cells are distinct epigenetic lineages that are distinguishable early in their differentiation. Together, these findings show that TRM cell development is accompanied by dynamic alterations in chromatin accessibility that direct a unique transcriptional program resulting in a tissue-adapted and functionally distinct T cell state.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/490680v1_ufig1.gif" ALT="Figure 1">
View larger version (56K):
org.highwire.dtl.DTLVardef@10a866aorg.highwire.dtl.DTLVardef@1febe9corg.highwire.dtl.DTLVardef@1941dd2org.highwire.dtl.DTLVardef@131dc58_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIscATAC atlas reveals the epigenetic variance of memory CD8+ T cell subsets over the course of acute infection
C_LIO_LIEarly bifurcation of memory precursors leads to circulating versus tissue-resident cell fates
C_LIO_LIIntegrating transcriptional and epigenetic analyses identified organ-specific TRM cell regulators including HIC1 and BACH2
C_LIO_LIEpigenetic distinction of TRM cells and TEX cell subsets
C_LI


title: Identification of viral dose and administration time in simulated phage therapy occurrences

abstract:
The rise in multidrug-resistant bacteria has sprung a renewed interest in applying phages as antibacterial, a procedure Western practitioners eventually abandoned due to several downfalls, including poor understanding of the dynamics between phages and bacteria. A successful phage therapy needs to account for the loss of infective virions and the multiplication of the hosts. The parameters critical inoculation size (VF) and failure threshold time (TF) have been introduced to assure that the viral dose (v{phi}) and administration time (t{phi}) would lead to an effective treatment. The problem with the definition of VF and TF is that they are non-linear equations with two unknowns; thus, their solution is cumbersome and not unique. The current study used machine learning in the form of a decision tree algorithm to determine ranges for the viral dose and administration times required to achieve an effective phage therapy. Within these ranges, a Pareto optimal solution of a multi-criterial optimization problem (MCOP) provides values leading to effective treatment. The algorithm was tested on a series of microbial consortia that described allochthonous invasions (the outgrowing of a species at high cell density by another species initially present at low concentration) to inhibit the growth of the invading species. The present study also introduced the concept of  mediated phage therapy, where targeting a booster bacteria might decrease the virulence of a pathogen immune to phagial infection. The results demonstrated that the MCOP could provide pairs of v{phi} and t{phi} that could effectively wipe out the bacterial target from the considered micro-environment. In summary, the present work introduced a novel method for investigating the phage/bacteria interaction that could help increase the effectiveness of phage therapy.

Author summaryPhage therapy is a treatment that can help fight infections with bacteria resistant to antibiotics. However, several phage therapy application have failed, possibly because phages were administered at the wrong time or in insufficient amounts. The present study implemented a machine learning protocol to correctly calculate the administration time and viral load to obtain effective phage therapy. Four simulated microbial consortia, including one case where the pathogen was not directly a phages host, were employed to prove the procedures concept. The results demonstrated that the procedure is suitable to help the microbiologists to instantiate an effective phage therapy and clear infections.


title: Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation

abstract: Numerous cell biology studies have used high concentrations of 1,6-hexanediol to dissolve membraneless organelles and disordered protein biomolecular condensates. Yet, little is known about how alkanediols effect liquid-liquid phase separation (LLPS), and why certain alkanediol isomers are more effective. Here, we evaluate the effect of various alkanediols on the archetypal phase separating protein FUS. Low-complexity domain and full-length FUS LLPS is decreased varyingly, while LLPS of FUS RGG-RNA condensates is even enhanced by some alkanediols. NMR experiments show that all diols act similarly, correlating atomistic changes with LLPS-preventing effects. Furthermore, we find no evidence for specific residue interactions - the largest perturbations are seen at backbone and glutamine side-chain hydrogen bonding sites, not hydrophobic/aromatic residues. Furthermore, 1,6 hexanediol favors formation of protein-solvent hydrogen bonds and increases FUS local motions. These findings show how alkanediols affect water-disordered protein interactions, underscoring the difficulty in using alkanediol-derivatives to target dissolution of specific membraneless organelles.


title: Skull and scalp segmentation in neonatal cerebral MRI using subject-specific probability models

abstract: This study presents a new approach for the segmentation of cranial bones in magnetic resonance images (MRIs) acquired from neonates in the age range of 39 to 42 weeks, gestational age. This approach uses subject specific probability maps of the skull and scalp, which are created from atlas computed tomography (CT) images taken retrospectively from neonates in the same age range. Our method uses also a subject specific probability map of cerebrospinal fluid (CSF), which is constructed from retrospective atlas MRIs. To construct skull, scalp and CSF probability maps, a subject specific bimodal MR-CT neonatal head template is created (using atlas MR and CT images), and employed. In the next step, the subject specific probability maps are fed to expectation maximization algorithm in conjunction with Markov random field method implemented in FSL software to segment skull and scalp from the input MR image. The results of the proposed method were evaluated through various experiments. First, we computed the similarity between frontal and occipital sutures (reconstructed from segmented cranial bones) and the ground truth (created manually by an expert radiologist). For this purpose, modified versions of Dice similarity index (DSI) were adopted and used. Second, the size of anterior fontanel was compared to its normal size as reported for the neonates in the same age range. Third, the thickness of cranial bones was computed and compared to its normal values as reported for healthy neonates. Finally, a retrospective data including MRI and CT images was used which have been acquired from the same neonate within a short time interval. After aligning the two images, the similarity between cranial bones of the MR and CT image was compared using DSI and modified Hausdorff distance. The results of these experiments demonstrated the success of our segmentation method.


title: Systematic benchmarking of all-in-one microbial SNP calling pipelines

abstract:
Clinical and public health microbiology is increasingly utilising whole genome sequencing (WGS) technology and this has lead to the development of a myriad of analysis tools and bioinformatics pipelines. Single nucleotide polymorphism (SNP) analysis is an approach used for strain characterisation and determining isolate relatedness. However, in order to ensure the development of robust methodologies suitable for clinical application of this technology, accurate, reproducible, traceable and benchmarked analysis pipelines are necessary. To date, the approach to benchmarking of these has been largely ad-hoc with new pipelines benchmarked on their own datasets with limited comparisons to previously published pipelines.

In this study, Snpdragon, a fast and accurate SNP calling pipeline is introduced. Written in Nextflow, Snpdragon is capable of handling small to very large and incrementally growing datasets. Snpdragon is benchmarked using previously published datasets against six other all-in-one microbial SNP calling pipelines, Lyveset, Lyveset2, Snippy, SPANDx, BactSNP and Nesoni. The effect of dataset choice on performance measures is demonstrated to highlight some of the issues associated with the current available benchmarking approaches.

The establishment of an agreed upon gold-standard benchmarking process for microbial variant analysis is becoming increasingly important to aid in its robust application, improve transparency of pipeline performance under different settings and direct future improvements and development.

Snpdragon is available at https://github.com/FordeGenomics/SNPdragon.

Impact statementWhole-genome sequencing has become increasingly popular in infectious disease diagnostics and surveillance. The resolution provided by single nucleotide polymorphism (SNP) analyses provides the highest level of insight into strain characteristics and relatedness. Numerous approaches to SNP analysis have been developed but with no established gold-standard benchmarking approach, choice of bioinformatics pipeline tends to come down to laboratory or researcher preference. To support the clinical application of this technology, accurate, transparent, auditable, reproducible and benchmarked pipelines are necessary. Therefore, Snpdragon has been developed in Nextflow to allow transparency, auditability and reproducibility and has been benchmarked against six other all-in-one pipelines using a number of previously published benchmarking datasets. The variability of performance measures across different datasets is shown and illustrates the need for a robust, fair and uniform approach to benchmarking.

Data SummaryO_LIPreviously sequenced reads for Escherichia coli O25b:H4-ST131 strain EC958 are available in BioProject PRJNA362676. BioSample accession numbers for the three benchmarking isolates are:
O_LIEC958: SAMN06245884
C_LIO_LIMS6573: SAMN06245879
C_LIO_LIMS6574: SAMN06245880
C_LI
C_LIO_LIAccession numbers for reference genomes against the E. coli O25b:H4-ST131 strain EC958 benchmark are detailed in table 2.
C_LIO_LISimulated benchmarking data previously described by Yoshimura et al. is available at http://platanus.bio.titech.ac.jp/bactsnp (1).
C_LIO_LISimulated datasets previously described by Bush et al. is available at http://dx.doi.org/10.5287/bodleian:AmNXrjYN8 (2).
C_LIO_LIReal sequencing benchmarking datasets previously described by Bush et al. are available at http://dx.doi.org/10.5287/bodleian:nrmv8k5r8 (2).
C_LI

O_TBL View this table:
org.highwire.dtl.DTLVardef@3cb198org.highwire.dtl.DTLVardef@af9421org.highwire.dtl.DTLVardef@1261244org.highwire.dtl.DTLVardef@793b42org.highwire.dtl.DTLVardef@1217387_HPS_FORMAT_FIGEXP  M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONReference genomes used in the EC958 benchmarking dataset and the percent identity against the three included samples.

C_TABLECAPTION C_TBL


title: Automated systematic evaluation of cryo-EM specimens with SmartScope

abstract: Propelled by improvements in hardware for data collection and processing, single particle cryo-electron microscopy has rapidly gained relevance in structural biology. Yet, finding the conditions to stabilize a macromolecular target for imaging remains the most critical barrier to determining its structure. Attaining the optimal specimen requires the evaluation of multiple grids in a microscope as conditions are varied. While automation has significantly increased the speed of data collection, optimization is still carried out manually. This laborious process which is highly dependent on subjective assessments, inefficient and prone to error, often determines the success of a project. Here, we present SmartScope, the first framework to streamline, standardize, and automate specimen evaluation in cryo-electron microscopy. SmartScope employs deep-learning-based object detection to identify and classify features suitable for imaging, allowing it to perform thorough specimen screening in a fully automated manner. A web interface provides remote control over the automated operation of the microscope in real time and access to images and annotation tools. Manual annotations can be used to re-train the feature recognition models, leading to improvements in performance. Our automated tool for systematic evaluation of specimens streamlines structure determination and lowers the barrier of adoption for cryo-electron microscopy.

category: biophysics


title: An interactive mass spectrometry atlas of histone posttranslational modifications in T-cell acute leukemia

abstract: The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide and protein data for downstream analyses, exemplified by the PeptidoformViz tool. This data resource sets the stage for generalizing MS-based histone analysis and provides the first reusable histone dataset for epidrug development.


title: M2-polarized macrophages control LSC fate by enhancing stemness, homing, immune evasion and metabolic reprogramming

abstract: While it is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones, the tumor microenvironment (TME) in acute myeloid leukemias (AML) remains poorly understood. Here, we uncover the functional and prognostic relevance of an M2-polarized macrophage compartment. Intra bone marrow co-injection of M2d-macrophages together with leukemic blasts that fail to engraft on their own now induce fatal leukemia in mice. Even a short-term two-day in vitro exposure to M2d macrophages can "train" leukemic blasts after which cells are protected against phagocytosis, display increased mitochondrial metabolism and improved in vivo homing, resulting in full-blown leukemia. Single-cell RNAseq analysis of AML associated macrophages revealed metabolic-related pathways such as Fatty Acid Oxidation and NAD+ generation as therapeutical targetable vulnerabilities. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for effective targeting strategies.


title: Nav1.1 in mammalian sensory neurons is required for normal motor behaviors

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe mammalian voltage-gated sodium channel (NaV), NaV1.1, has been well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we report a new role for peripherally expressed NaV1.1 in murine motor behaviors. RNAscope analysis found 100% of proprioceptors express NaV1.1 transcript, consistent with in vitro patch clamp recordings showing this channel is required for repetitive firing in proprioceptors. Notably, genetic deletion of NaV1.1 in all sensory neurons caused profound motor coordination deficits in homozygous conditional knockout animals of both sexes, a phenotype similar to conditional Piezo2-knockout animals. Movement deficits were also observed in heterozygotes, demonstrating that NaV1.1 haploinsufficiency in sensory neurons leads to motor deficiencies. This behavioral phenotype was not due to reduced proprioceptor numbers or abnormal muscle spindle formation; however, we observed decreased proprioceptor innervation of motor neurons in the spinal cord in conditional knockouts, indicating loss of NaV1.1 in sensory neurons alters spinal cord circuitry. Ex vivo muscle afferent recordings also support the notion that loss of NaV1.1 leads to aberrant proprioceptor function. Collectively, these data provide the first evidence that NaV1.1 in mammalian sensory neurons is essential for motor coordination. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia. Thus, our data suggest sensory neuron dysfunction may contribute to the clinical manifestations and co-morbidities of neurological disorders in which NaV1.1 function is compromised.

category: neuroscience


title: Probabilistic Tracking U-fiber on the Superficial White Matter Surface

abstract: The short association fibers or U-fibers connect two neighboring gyri and travel in the superficial white matter (SWM) beneath the cortical layer. These U-fibers are essential for the understanding of neurodevelopment and neurodegeneration. However, the complex structures and the high curvature of the U-fibers lead to erroneous streamlines reconstruction of the traditional tractography since the volume-based tractography cannot use the biological characteristic of U-fibers that they tightly beneath the cortical layer. In this work, we proposed a surface-based framework for probabilistic tracking of the U-fibers on the triangular mesh of the SWM. We develop a novel approach to project the fiber orientation distributions (FODs) data onto the tangent space of the SWM surface. With the projected FODs, an advanced probabilistic tracking technique, which regularizes the streamlines based on the intrinsic geometry of the surface, is developed to reconstruct the highly bent U-fibers on the SWM surface. In the experiment, we demonstrate our method based on the high-resolution diffusion imaging data from the Human Connectome Project (HCP). We quantitatively compare the proposed method with state-of-the-art volume-based tractography from MRTrix and another surface-based tractography on the U-fibers of the central sulcus. Moreover, we show the reconstructed U-fibers on the parietal lobe and frontal lobe. The results show that our method outperforms the other two methods and successfully reconstructs the U-fibers on the cortical regions with high intersubject variability.


title: Bacterial association with metals enables in vivo tracking of microbiota using magnetic resonance imaging

abstract:
Bacteria constitute a significant part of the biomass of the human microbiota, but their interactions are complex and difficult to replicate outside the host. Exploiting the superior resolution of magnetic resonance imaging (MRI) to examine signal parameters of selected human isolates may allow tracking of their dispersion throughout the body. We investigated longitudinal and transverse MRI relaxation rates and found significant differences between several bacterial strains. Common commensal strains of lactobacilli display notably high MRI relaxation rates, partially explained by outstanding cellular manganese content, while other species contain more iron than manganese. Lactobacillus crispatus show particularly high values, 4-fold greater than any other species; over 10-fold greater signal than relevant tissue background; and a linear relationship between relaxation rate and fraction of live cells. Different bacterial strains have detectable, repeatable MRI relaxation rates that in future may enable tracking of their persistence in the human body for enhanced molecular imaging.

IMPORTANCE

To understand how spatial and temporal distribution of microbiota impact human health, dynamic tools for monitoring microbiota landscapes inside the host are needed. Particularly when considering the complexity of the gastrointestinal tract and the microbiota that dwell within, tools for monitoring deep segments of the gut non-invasively are required. Medical imaging provides solutions that enable the study of microorganisms in their preferred niche regardless of health status. To bootstrap this technology, we investigated the magnetic resonance imaging (MRI) properties of bacterial isolates and showed that outstanding signal detection is an inherent property of several strains. Among these, we showed that bacteria relying on manganese metabolism have an MRI characteristic that is distinct from mammalian cells. Our findings will lead to direct and safe imaging of bacteria; influence how we monitor both infection and gut health; and help direct the use of antibiotics to curtail the growing threat of antibiotic resistance.

category: microbiology


title: Acinetobacter baumannii and cefiderocol between cidality and adaptability

abstract:
Among the bacterial species included in the ESKAPE group, Acinetobacter baumannii is of great interest due to its intrinsic and acquired resistance to many antibiotic classes and its ability to infect different body districts. Cefiderocol is a novel cephalosporin active against Gram-negative bacteria with promising efficacy on A. baumannii infections, but some studies have reported therapeutic failures even in the presence of susceptible A. baumannii strains.

This study aims to investigate the interactions between cefiderocol and ten A. baumannii strains with different susceptibility profiles to this drug. We confirmed diverse susceptibility profiles in the strains, with resistance values close to the EUCAST-proposed breakpoints. MBC/MIC (minimal bactericidal concentration/minimal inhibitory concentration) ratios, demonstrated bactericidal activity of the drug; on the other hand, bacterial regrowth was evident after exposition to cefiderocol, as were changes in the shape of colonies and bacterial cells. A switch to a non-susceptible phenotype in the presence of high cefiderocol concentrations was found as adaptation mechanisms implemented by these A. baumannii strains to overcome the cidal activity of this antibiotic, also confirmed by the presence of heteroresistant, unstable subpopulations. As our isolates harbored numerous {beta}-lactamase genes, {beta}-lactamase inhibitors showed the ability to restore the antimicrobial activity of cefiderocol regardless of the different non-susceptibility levels of the tested strains.

These in vitro results, can sustain the concept of using combination therapy to eliminate drug-adapted subpopulations and regain full cefiderocol activity in this difficult-to-treat species.


title: Polyimidazolium protects against an invasive clinical isolate of Salmonella Typhimurium

abstract: Frequent outbreaks of Salmonella Typhimurium infection in both the animal and human population with potential for zoonotic transmission pose a significant threat to the public health sector. The rapid emergence and spread of more invasive multidrug-resistant clinical isolates of Salmonella further highlight the need for the development of new drugs with effective broad-spectrum bactericidal activities. Synthesis and evaluation of main-chain cationic polyimidazolium 1 (PIM1) against several gram-positive and gram-negative bacteria have previously demonstrated the efficacy profile of PIM1. The present study focuses on antibacterial and anti-biofilm activities of PIM1 against Salmonella both in vitro and in ovo setting. In vitro, PIM1 exhibited bactericidal activity against all tested three strains of Salmonella at a low dosage of 8 g/ml. Anti-biofilm activity of PIM1 was evident with complete inhibition for the initial attachment of biofilms at 16 g/ml and degradation of pre-formed biofilms in a dose-dependent manner. During the host cell infection process, PIM1 reduces extracellular bacterial adhesion and invasion rates to limit the establishment of infection. Once intracellular, the drug-resistant strain was tolerant and protected from PIM1 treatment. In a chicken egg infection model, PIM1 exhibited therapeutic activity for both Salmonella strains with stationary-phase and exponential-phase inocula. Moreover, PIM1 showed a remarkable efficacy against the stationary phase inocula of drug-resistant Salmonella by eliminating the bacteria burden in >50% of infected chicken egg embryos. Collectively, PIM1 has demonstrated its potential as a drug candidate for treatment of Salmonella infections, as well as a solution to tackle egg contamination issues on poultry farms.


title: Serine Deamination as a New Acid Resistance Mechanism in Escherichia coli

abstract:
Escherichia coli associates with humans early in life and can occupy several body niches either as a commensal in the gut and vagina, or as a pathogen in the urinary tract. As such, E. coli has an arsenal of acid response mechanisms that allow it to withstand the different levels of acid stress encountered within and outside the host. Here, we report the discovery of an additional acid response mechanism that involves the deamination of L-serine to pyruvate by the conserved L-serine deaminases SdaA and SdaB. L-serine is the first amino acid to be imported in E. coli during growth in laboratory media, as the culture senesces. However, there remains a lack in knowledge as to why L-serine is preferred and how it is utilized. We show that in acidified media, L-serine is brought into the cell via the SdaC transporter and deletion of both SdaA and SdaB renders E. coli susceptible to acid stress, with a phenotype similar to other acid stress deletion mutants. We also show that the pyruvate produced by L-serine de-amination activates the pyruvate sensor BtsS, which in concert with the non-cognate response regulator YpdB upregulates the putative transporter YhjX, similar to what has been reported for this system during transition of E. coli to stationary phase. Based on these observations, we propose that L-serine deamination constitutes another acid response mechanism in E. coli that may function to protect E. coli as it transitions to stationary phase of growth.

IMPORTANCEThe observation that L-serine uptake occurs as an E. coli culture senesces is well-established, yet the benefit E. coli garners from this uptake remains unclear. Here, we report a novel acid resistance mechanism, where L-serine is deaminated to pyruvate and ammonia, promoting acid tolerance in E. coli. This study is important as it provides evidence of the use of L-serine as an acid response strategy, not previously reported for E. coli.


title: Development of recombinant monoclonal antibodies targeting conserved VlsE epitopes in Lyme disease pathogens

abstract: VlsE (variable major protein-like sequence, expressed) is an outer surface protein of the Lyme disease pathogen (Borreliella species) and a key diagnostic biomarker of Lyme disease. However, the high sequence variability of VlsE poses a challenge to the development of consistent VlsE-based diagnostics and therapeutics. In addition, the standard diagnostic protocols detect immunoglobins elicited by the Lyme pathogen, not the presence of pathogen or its derived antigens. Here we describe the development of recombinant monoclonal antibodies (rMAbs) that bind specifically to conserved epitopes on VlsE. We first quantified amino-acid sequence variability encoded by the vls genes from thirteen B. burgdorferi genomes by evolutionary analyses. We showed broad inconsistencies of the sequence phylogeny with the genome phylogeny, indicating rapid gene duplications, losses, and recombination at the vls locus. To identify conserved epitopes, we synthesized peptides representing five long conserved invariant regions (IRs) on VlsE. We tested the antigenicity of these five IR peptides using sera from three mammalian host species including human patients, the natural reservoir white-footed mouse (Peromyscus leucopus), and VlsE-immunized New Zealand rabbits (Oryctolagus cuniculus). The IR4 and IR6 peptides emerged as the most antigenic and reacted strongly with both the human and rabbit sera, while all IR peptides reacted poorly with sera from natural hosts. Four rMAbs binding specifically to the IR4 and IR6 peptides were identified, cloned, and purified. Given their specific recognition of the conserved epitopes on VlsE, these IR-specific rMAbs are promising diagnostic and theragnostic agents for direct detection of Lyme disease pathogens regardless of strain heterogeneity.


title: Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion

abstract: Spatial accumulation of secretory vesicles underlies various cellular processes, such as neurotransmitter release at neuronal synapses [1], hyphal steering in filamentous fungi [2, 3], and local cell wall digestion preceding the fusion of yeast gametes [4]. Secretory vesicles transported on actin filaments by myosin V motors form clusters that serve as pool for local content release. During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes [5-7]. Focus position and coalescence are controlled by local signalling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis [6, 8-10], but the mechanisms of focusing have been elusive. Here, we show that the regulatory N-terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation in vivo and forms dense assemblies that exclude other macromolecules. Fus1 lacking its IDR fails to condense in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, replacement of Fus1 IDR with a heterologous low-complexity region that forms liquid condensates fully restores Fus1 condensation and function. By contrast, replacement of Fus1 IDR with a domain that forms more stable oligomers restores condensation but poorly supports cell fusion, suggesting that condensation is tuned to yield a structure selectively permeable for secretory vesicles. We propose that condensation of actin structures by an intrinsically disordered region may be a general mechanism for actin network organisation and the selective local concentration of secretory vesicles.

category: cell biology


title: Incorporating uniparental markers and demographic information in kinship analysis

abstract: Knowledge of kinship relations between members of wild populations is of great importance in ecological and conservation genetic studies. The bi-parentally inherited autosomal markers has been the Golden Standard in kinship analysis. However, analysis of kin relationship can be challenging in wild populations. The uni-parentally inherited markers and population demographic information can be helpful for identifying false-positive in kinship analysis. Here we showed how incorporating uniparental genetic and demographic information can improve the correct classification rate of kinship analyses by reanalyzing data of a recent study published in Science Advances. The application of next generation high-throughput sequencing to address fundamental ecological questions is of immense benefit to the field of molecular ecology, which could also generate uniparentally inherited organelle genomes together with nuclear data. We strongly recommended that uniparental genetic markers and demographic information be seriously considered in kinship analyses of wild populations.


title: Depth normalization for single-cell genomics count data

abstract: Single-cell genomics analysis requires normalization of feature counts that stabilizes variance while accounting for variable cell sequencing depth. We discuss some of the trade-offs present with current widely used methods, and analyze their performance on 526 single-cell RNA-seq datasets. The results lead us to recommend proportional fitting prior to log transformation followed by an additional proportional fitting.