title: SimSearch: A Human-in-the-Loop Learning Framework for Fast Detection of Regions of Interest in Microscopy Images

abstract:
ObjectiveLarge-scale microscopy-based experiments often result in images with rich but sparse information content. An experienced microscopist can visually identify regions of interest (ROIs), but this becomes a cumbersome task with large datasets. Here we present SimSearch, a framework for quick and easy user-guided training of a deep neural model aimed at fast detection of ROIs in large-scale microscopy experiments.

MethodsThe user manually selects a small number of patches representing different classes of ROIs. This is followed by feature extraction using a pre-trained deep-learning model, and interactive patch selection pruning, resulting in a smaller set of clean (user approved) and larger set of noisy (unapproved) training patches of ROIs and background. The pre-trained deep-learning model is thereafter first trained on the large set of noisy patches, followed by refined training using the clean patches.

ResultsThe framework is evaluated on fluorescence microscopy images from a large-scale drug screening experiment, brightfield images of immunohistochemistry-stained patient tissue samples, and malaria-infected human blood smears, as well as transmission electron microscopy images of cell sections. Compared to state-of-the-art and manual/visual assessment, the results show similar performance with maximal flexibility and minimal a priori information and user interaction.

ConclusionsSimSearch quickly adapts to different data sets, which demonstrates the potential to speed up many microscopy-based experiments based on a small amount of user interaction.

SignificanceSimSearch can help biologists quickly extract informative regions and perform analyses on large datasets helping increase the throughput in a microscopy experiment.


title: Direct evidence for pinning of single, ice-bound antifreeze proteins by subzero nanoscopy

abstract: Ice-binding by antifreeze proteins (AFPs) reduces freezing temperatures and arrests ice-crystal ripening, making AFPs essential for survival in ice-laden environments and attractive as biocompatible antifreezes. Whilst their activity was identified over 50 years ago, the physical mechanisms are still debated because experimental insights at the molecular scale remain elusive. Here we introduce optical nanoscopy to resolve the ice/water interfacial dynamics of single AFPs. Using this method, we demonstrate pinning of individual proteins. Surprisingly, this quasi-permanent pinning is lost when freezing point depression activity is inhibited by a single mutation in the ice-binding site. These findings provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of activity and offer new insights in the molecular mechanisms by which these biological antifreezes function.

category: biophysics


title: Hyperglycemia and Cancer. Human lung carcinoma by means of Raman spectroscopy and imaging

abstract:
Raman spectroscopy and Raman imaging allow to identify the biochemical and structural features of human cancer lung cell line (CCL-185) and the cell line supplemented with glucose and deuterated glucose in normal and hyperglycemia conditions. We found that isotope substitution of glucose by deuterated glucose allows to separate de novo lipid synthesis from exogenous uptake of lipids obtained from the diet. We demonstrated that glucose is largely utilized for de novo lipid synthesis. Our results provide a direct evidence that high level of glucose decreases the metabolism via oxidative phosporylation in mitochondria in cancer cells and shifts the metabolism to glycolysis via Wartburg effect.

Graphical Abstract

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category: cancer biology


title: Nucleation complex behaviour is critical for cortical microtubule array homogeneity and patterning

abstract:
Plant cell walls are versatile materials that can adopt a wide range of mechanical properties through controlled deposition of cellulose fibrils. Wall integrity requires a sufficiently homogeneous fibril distribution to cope effectively with wall stresses. Additionally, specific conditions, such as the negative pressure in water transporting xylem vessels, may require more complex wall patterns, e.g., bands in protoxylem. The orientation and patterning of cellulose fibrils is guided by dynamic cortical microtubules. New microtubules are predominantly nucleated from parent microtubules causing positive feedback on local microtubule density with the potential to yield highly inhomogeneous patterns. Inhomogeneity indeed appears in all current cortical array simulations that include microtubule-based nucleation, suggesting that plant cells must possess an as-yet unknown balancing mechanism to prevent it. Here, in a combined simulation and experimental approach, we show that the naturally limited local recruitment of nucleation complexes to microtubules can counter the positive feedback, whereas local tubulin depletion cannot. We observe that nucleation complexes are preferentially inserted at microtubules. By incorporating our experimental findings in stochastic simulations, we find that the spatial behaviour of nucleation complexes delicately balances the positive feedback, such that differences in local microtubule dynamics - as in developing protoxylem - can quickly turn a homogeneous array into a patterned one. Our results provide insight into how the plant cytoskeleton is wired to meet diverse mechanical requirements and greatly increase the predictive power of computational cell biology studies.

Significance statementThe plant cortical microtubule array is an established model system for self-organisation, with a rich history of complementary experiments, computer simulations, and analytical theory. Understanding how array homogeneity is maintained given that new microtubules nucleate from existing microtubules has been a major hurdle for using mechanistic (simulation) models to predict future wall structures. We overcome this hurdle with detailed observations of the nucleation process from which we derive a more "natural" nucleation algorithm. With this algorithm, we enable various new lines of quantitative, mechanistic research into how cells dynamically control their cell wall properties. At a mechanistic level, moreover, this work relates to the theory on cluster coexistence in Turing-like continuum models and demonstrates its relevance for discrete stochastic entities.

category: plant biology


title: Canonical Strigolactones Are Not the Tillering-Inhibitory Hormone but Rhizospheric Signals in Rice

abstract: The plant hormones strigolactones (SLs) regulate shoot branching and mediate the communication with symbiotic mycorrhizal fungi, but also with noxious root parasitic weeds, such as Striga spp. SLs derive from carlactone (CL) and are divided structurally into canonical and non-canonical SLs. However, the questions about particular biological functions of the two groups and the identification of the SL that inhibits shoot branching are still unanswered, hampering targeted modification of SL pattern towards improving plant architecture and resistance against Striga. Here, we reported that 4-deoxyorobanchol (4DO) and orobanchol, the two canonical SLs in rice, do not have major role in determining rice shoot architecture. CRISPR/Cas9 mediated Osmax1-900 mutants, lacking these two SLs, do not show the high tillering and dwarf phenotype typical for SL-deficient plants. However, the absence of 4DO and orobanchol in root exudates significantly decreased their capability in inducing Striga seed germination, while caused only a delay in root colonization by mycorrhizal fungi. To confirm the genetic evidence, we used the SL-biosynthesis inhibitor TIS108. Our results showed that TIS108 is a MAX1-specific inhibitor that lowers 4DO and orobanchol synthesis, conferring a resistance to Striga without a severe impact on rice architecture. Hence, our work uncovers the specific function of canonical SLs as rhizospheric signals and paves the way for establishing chemical and genetic based approaches for combating the root parasitic weeds, by targeted depletion of their release.


title: The pitfalls of measuring representational similarityusing representational similarity analysis

abstract: A core challenge in neuroscience is to assess whether diverse systems represent the world similarly. Representational Similarity Analysis (RSA) is an innovative approach to address this problem and has become increasingly popular across disciplines from machine learning to computational neuroscience. Despite these successes, RSA regularly uncovers difficult-to-reconcile and contradictory findings. Here we demonstrate the pitfalls of using RSA to infer representational similarity and explain how contradictory findings arise and support false inferences when left unchecked. By comparing neural representations in primate, human and computational models, we reveal two problematic phenomena that are ubiquitous in current research: a "mimic" effect, where confounds in stimuli can lead to high RSA scores between provably dissimilar systems, and a "modulation effect", where RSA-scores become dependent on stimuli used for testing. Since our results bear on existing findings and inferences, we provide recommendations to avoid these pitfalls and sketch a way forward.

category: neuroscience


title: MicroRNA inhibition using antimiRs in acute human brain tissue sections

abstract:
IntroductionAn emerging pre-clinical approach for the treatment of pharmacoresistant epilepsy is targeting the microRNA (miRNA) system. MiRNAs are short noncoding RNAs that suppress gene expression at the post-transcriptional level. Targeting miRNAs, which is possible using antisense oligonucleotide  antimiRs can produce broad effects on gene expression suited to the complex pathophysiology in temporal lobe epilepsy. Potent anti-seizure and disease- modifying effects have been reported for antimiRs targeting microRNA-134 (antimiR-134). To date, however, pre-clinical testing has been performed using in vitro cell cultures and rodent models. It is uncertain how well this approach will translate to the clinic. Here, we develop an antimiR testing platform in human brain tissue sections.

MethodologyHuman brain specimens were obtained with consent from patients undergoing resective surgery to treat focal drug-resistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF), dissected for clinical neuropathological examination, and unused material transferred for sectioning. Individual tissue sections were incubated in oxygenated ACSF, containing either antimiR-134 or a non-targeting control antimiR, for 24 hours at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels measured using RT-qPCR.

ResultsACSF transport had no obvious impact on any clinical neurosurgical or neuropathological procedure and specimens were confirmed to be viable following this process. RNA was well- preserved by transportation of specimens in ACSF, with RNA integrity scores significantly higher than tissue transported without ACSF. AntimiR-134 mediated a specific and dose- dependent knockdown of miR-134 in human neocortical sections, with approximately 75% reduction of miR-134 at 1 {micro}M and 90% reduction at 3 {micro}M. These doses did not have off- target effects on expression of a selection of three other miRNAs (miR-10, miR-129 or miR- 132).

SignificanceThis is the first demonstration of antimiR-134 effects in live human brain tissues. The findings lend further support to the preclinical development of miR-134 and offer a flexible platform for the pre-clinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain.

Key pointsO_LIASO antimiRs are promising treatments for pharmacoresistant epilepsy
C_LIO_LIWe developed a pipeline to preserve live human neocortical brain specimens from people undergoing resective surgery
C_LIO_LIRNA integrity was sufficient to measure miRNA levels in human brain tissues transported in modified ACSF
C_LIO_LIIncubation of acute human neocortical specimens in antimiR-134 resulted in potent and specific reduction in miR-134 levels
C_LIO_LIAcute human brain slices are a promising model for testing ASOs
C_LI


title: Effects of experiencing CS-US pairings on instructed fear reversal

abstract: Fear learning allows us to identify and anticipate aversive events, and adapt our behavior accordingly. This is often thought to rely on associative learning mechanisms where an initially neutral conditioned stimulus (CS) is repeatedly paired with an aversive unconditioned stimulus (US), eventually leading to the CS also being perceived as aversive and threatening. Importantly, however, humans also show verbal fear learning. Namely, they have the ability to change their responses to stimuli rapidly through verbal instructions about CS-US pairings. Past research on the link between experience-based and verbal fear learning indicated that verbal instructions about a reversal of CS-US pairings can fully override the effects of previously experienced CS-US pairings, as measured through fear ratings, skin conductance, and fear-potentiated startle. However, it remains an open question whether such instructions can also annul memory traces in the brain. Here, we used a fear reversal paradigm in conjunction with representational similarity analysis of fMRI data to test whether verbal instructions fully override the effects of experienced CS-US pairings in fear-related brain regions or not. Previous research suggests that only the right amygdala should show lingering representations of previously experienced threat (a so-called "Pavlovian trace"). Unexpectedly, we found evidence for the residual effect of prior CS-US experience to be much more widespread than anticipated, in the amygdala but also cortical regions like the dorsal anterior cingulate or dorso-lateral prefrontal cortex. This finding shines a new light on the interaction of different fear learning mechanisms, at times with unexpected consequences.


title: Concentration dependent dual effect of the endozepine ODN on neuronal spiking activity

abstract: Endozepines, known as the endogenous ligands of benzodiazepine-binding sites, include the diazepam binding inhibitor (DBI) and its processing products, the triakontatetraneuropeptide (TTN) and the octadecaneuropeptide (ODN). Despite indisputable evidences of the binding of ODN on GABAAR-BZ-binding sites, their action on this receptor lacks compelling electrophysiological observations, some studies reporting that ODN acts as a negative allosteric modulator (NAM) of GABAAR while others suggest the opposite (positive allosteric modulation, PAM effect). All these studies were carried out in vitro with various neuronal cell types. To further elucidate the role of ODN on neuronal excitability, we tested its effect in vivo in the cortex of the anesthetized mouse. Spontaneous neuronal spikes were recorded by the mean of an extracellular pipette in the vicinity of which ODN was micro-infused, either at high dose (10-5M) or low dose (10-11M). ODN at high dose induced a significant increase of neuronal spiking. This effect could be antagonized by the GABAAR-BZ-binding sites blocker flumazenil. In sharp contrast, at low concentration, ODN reduced neuronal spiking in a magnitude similar to GABA itself. Interestingly, this decrease of neuronal activity by low dose of ODN was not flumazenil dependent suggesting that this effect is mediated by another receptor. Finally, we show that astrocytes in culture, known to be stimulated by picomolar dose of ODN via a GPCR, increased their export of GABA when stimulated by low dose of ODN. Our results confirm the versatility of ODN in the control of GABA transmission, but suggest that its PAM-like effect is, at least in part, mediated via an astrocytic non-GABAAR ODN receptor.


title: Blood oxygenation-level dependent cerebrovascular reactivity imaging as strategy to monitor CSF-hemoglobin toxicity

abstract:
PurposeCell-free hemoglobin in the cerebrospinal fluid (CSF-Hb) may be one of the main drivers of secondary brain injury after aneurysmal subarachnoid hemorrhage. Haptoglobin scavenging of CSF-Hb has been shown to mitigate cerebrovascular disruption. Using digital subtraction angiography (DSA) and blood oxygenation-level dependent cerebrovascular reactivity imaging (BOLD-CVR) the aim was to assess the acute toxic effect of CSF-Hb on cerebral blood flow and autoregulation, as well as to test the protective effects of haptoglobin.

MethodsDSA imaging was performed in eight anesthetized and ventilated sheep (mean weight: 80.4 kg) at baseline, 15, 30, 45 and 60 minutes after infusion of hemoglobin (Hb) or co-infusion with haptoglobin (Hb:Haptoglobin) into the left lateral ventricle. Additionally, 10 ventilated sheep (mean weight: 79.8 kg) underwent BOLD-CVR imaging to assess the cerebrovascular reserve capacity.

ResultsDSA imaging did not show a difference in mean transit time or cerebral blood flow. Wholebrain BOLD-CVR compared to baseline decreased more in the Hb group after 15 minutes (Hb vs Hb:Haptoglobin: -0.03 {+/-}0.01 vs -0.01 {+/-}0.02) and remained diminished compared to Hb:Haptoglobin group after 30 minutes (Hb vs Hb:Haptoglobin: -0.03 {+/-}0.01 vs 0.0 {+/-}0.01), 45 minutes (Hb vs Hb:Haptoglobin: -0.03 {+/-}0.01 vs 0.01 {+/-}0.02) and 60 minutes (Hb vs Hb:Haptoglobin: -0.03 {+/-}0.02 vs 0.01 {+/-}0.01).

ConclusionIt is demonstrated that CSF-Hb toxicity leads to rapid cerebrovascular reactivity impairment, which is blunted by haptoglobin co-infusion. BOLD-CVR may therefore be further evaluated as a monitoring strategy for CSF-Hb toxicity after aSAH.


title: Single-neuron bursts encode pathological oscillations in Parkinson's disease and essential tremor

abstract:
Deep brain stimulation procedures offer an invaluable opportunity to study disease through intracranial recordings from awake patients. Herein, we address the relationship between singleneuron and aggregate-level (local field potential; LFP) activities in the subthalamic nucleus (STN) and thalamic ventral intermediate nucleus (Vim) of patients with Parkinsons disease (n=19) and essential tremor (n=16), respectively. Both disorders have been characterized by pathologically elevated LFP oscillations, as well as an increased tendency for neuronal bursting. Our findings suggest that periodic single-neuron bursts encode both pathophysiological beta (13-33Hz; STN) and tremor (4-10Hz; Vim) LFP oscillations, evidenced by strong time-frequency and phase-coupling relationships between the bursting and LFP signals. Spiking activity occurring outside of bursts had no relationship to the LFP. In STN, bursting activity most commonly preceded the LFP oscillation, suggesting that neuronal bursting generated within STN may give rise to an aggregate-level LFP oscillation. In Vim, LFP oscillations most commonly preceded bursting activity, suggesting that neuronal firing may be entrained by periodic afferent inputs. In both STN and Vim, the phasecoupling relationship between LFP and high-frequency oscillation (HFO) signals closely resembled the relationships between the LFP and single-neuron bursting. This suggests that periodic singleneuron bursting is likely representative of a higher spatial and temporal resolution readout of periodic increases in the amplitude of HFOs, which themselves may be a higher resolution readout of aggregate-level LFP oscillations. Overall, our results may reconcile "rate" and "oscillation" models of Parkinsons disease and shed light onto the single-neuron basis and origin of pathophysiological oscillations in movement disorders.

SignificanceIn surgical patients with Parkinsons disease and essential tremor, we leverage intracranial recordings to establish a link between pathophysiological phenomena across various scales of observation (spatio-temporal resolutions). We provide insights and reconcile theories about aberrant neurocircuit phenomena which underly theses debilitating, medically refractory movement disorders. Furthermore, our connectivity analyses between single-neuron and local field potential activities may shed light on the origin of the deleterious neural oscillations underlying these disorders. Ultimately, our findings may aid in the development or investigation of targeted therapies to address or correct underlying neurocircuit dysfunction, which can include neuropharmaceuticals, but also novel neuromodulatory strategies like closed-loop deep brain stimulation targeting pathophysiological oscillations and phase-dependent stimulation methods seeking to stimulate "at the right time/phase."


title: Attentional rhythms are generated in binocular cells

abstract: Visual attention is intrinsically rhythmic and oscillates based on the discrete sampling of either single or multiple objects. Recently, studies employing transcranial magnetic stimulation (TMS) and high-temporal behavioral approaches have found that the early visual cortex (V1/V2) modulates attentional rhythms. However, both monocular cells and binocular cells are in the early visual cortex, and whether the neural site of attentional rhythms is monocular cells or binocular cells remains poorly understood. In Experiment 1, we reset the phase of attentional rhythms in one monocular channel (left eye or right eye) by the dichoptic cue and tracked the temporal response function (TRF) of the monocular channel in the left and right eyes separately using time response tracking technology. We found no significant differences in the two TRFs of each monocular eye, suggesting that attention rarely switched between the two eyes, indicating that binocular cells, not monocular cells, are the neural site of attentional rhythms. These results were verified even when resetting the phases of attentional rhythms by a binocular cue in Experiment 2. These results constitute direct neural evidence supporting rhythmic attention theory.


title: Developmental switch in prediction and adaptation to pain in human neonates

abstract:
Habituation to recurrent non-threatening or unavoidable noxious stimuli is an important aspect of adaptation to pain and indicates the ability of the brain to encode expectation of imminent nociception. However, it is not known whether the newborn brain can predict and habituate to recurrent noxious inputs. We used electroencephalography to investigate changes in cortical microstates, which represent the complex sequential processing of noxious inputs, following repeated clinically-required heel lances in term and preterm infants. Noxious stimulus repetition decreased the engagement of early sensory-related microstates and associated behavioural and physiological responses in term infants, while preterm infants did not show signs of adaptation. Nevertheless, both groups displayed a switch between different microstates at longer latencies. These data suggests that the preterm brain is capable of encoding high-level contextual differences in pain, but cannot update its prediction, which allows for adaptation, emphasising the vulnerability of this population to recurrent pain.

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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: Zebrafish Galectin 3 binding protein (Lgals3bp) is the target antigen of the microglial 4C4 monoclonal antibody

abstract:
BackgroundTwo decades ago, the fish-specific monoclonal antibody 4C4 was found to be highly reactive to zebrafish microglia, the macrophages of the central nervous system. This has resulted in 4C4 being widely used, in combination with available fluorescent transgenic reporters to identify and isolate microglia. However, the target protein of 4C4 remains unidentified, which represents a major caveat. In addition, whether the 4C4 expression pattern is strictly restricted to microglial cells in zebrafish has never been investigated.

ResultsHaving demonstrated that 4C4 is able to capture its native antigen from adult brain lysates, we used immunoprecipitation/mass-spectrometry, coupled to recombinant expression analyses, to identify its target. The cognate antigen was found to be a paralog of Galectin 3 binding protein (Lgals3bpb), known as MAC2-binding protein in mammals. Notably, 4C4 did not recognize other paralogs, demonstrating specificity. Moreover, our data show that Lgals3bpb expression, while ubiquitous in microglia, also identifies leukocytes in the periphery, including populations of gut and liver macrophages.

ConclusionsThe 4C4 monoclonal antibody recognizes Lgals3bpb, a predicted highly glycosylated protein whose function in the microglial lineage is currently unknown. Identification of Lgals3bpb as a new pan-microglia marker will be fundamental in forthcoming studies using the zebrafish model.

category: immunology


title: Kidins220 promotes thymic iNKT cell development by reducing TCR signals, but enhances TCR signals in splenic iNKT cells

abstract:
The stepwise development of thymic invariant natural killer T (iNKT) cells is controlled by the TCR signal strength. The scaffold protein Kinase D interacting substrate of 220 kDa (Kidins220) binds to the TCR regulating TCR signaling. T cell-specific Kidins220 knock-out (T-KO) mice contain severely decreased iNKT numbers. Very early in iNKT development TCR signals are reduced in the T-KO. In later steps, TCR signaling is increased in the T-KO leading to enhanced apoptosis of iNKT cells. Kidins220s absence affects the iNKT1 subset most as it requires the weakest TCR signals for development. We also show that in iNKT1 development, weak TCR signals promote the progressive loss of CD4. In the periphery, Kidins220 switches its role back to promoting TCR signaling as splenic T-KO iNKT cells produce less cytokines and show reduced TCR signaling after in vivo stimulation with -galactosylceramide. In conclusion, Kidins220 promotes or inhibits TCR signaling depending on the developmental context.

summary statementWe demonstrate that the transmembrane scaffold protein Kidins220 switches its role twice in iNKT cell biology: from a positive to a negative regulator of TCR signal strength during thymic development and back to a positive regulator in the periphery.


title: Fragmented habitat compensates for the adverse effects of genetic bottleneck

abstract: According to the population genetics theory, population subdivision helps organisms retain genetic diversity, crucial for adaptation in a changing environment. Habitat shape is thought to be important for generating and maintaining population subdivision, but empirical cases are needed to test this assumption. We studied Saimaa ringed seals, land-locked in a labyrinthine lake and recovering from a drastic bottleneck, by whole-genome sequencing 105 individuals and additional individuals from three other ringed seal subspecies. We analyzed the distribution of variation and the genetic relatedness among the individuals in relation to the habitat shape. Despite a severe history of a genetic bottleneck with prevalent homozygosity in Saimaa ringed seals, we found evidence for population structure mirroring the subregions of the lake. Highlighting the significance of habitat connectivity in conservation biology and the power of genomic tools in understanding its impact, the genome wide analyses showed the subpopulations to have retained unique variation and largely complementary patterns of homozygosity, giving hope to this endangered species.

category: evolutionary biology


title: The net-like heterotrophic amoeba Leukarachnion salinum sp. nov. (Ochrophyta, Stramenopiles) has a cryptic plastid

abstract: Numerous lineages of heterotrophs evolved from photosynthetic eukaryotic ancestors and usually have retained a plastid, although cases of secondary plastid loss are known, too. Based on a previous investigation by transmission electron microscopy (TEM), Leukarachnion sp. PRA-24, an amoeboid colourless protist related to the photosynthetic class Synchromophyceae within the algal phylum Ochrophyta, is a candidate for another case of a plastid loss. Here we provide a detailed characterisation of this organism and formally describe it as Leukarachnion salinum, sp. nov. While we could not find any unambiguous candidate for a plastid organelle by TEM, genome sequencing recovered a complete plastid genome with a reduced gene set similar to plastid genomes of other non-photosynthetic ochrophytes yet even more extreme in the sequence divergence. The presence in the L. salinum transcriptome assembly of homologs of hallmark plastid proteins, including components of the plastid protein import complexes, supported the notion that L. salinum has a cryptic plastid organelle. Based on an analysis of plastid-targeting signals in L. salinum proteins, the plastid presumably contains a unique combination of biosynthetic pathways producing haem, the folate precursor aminodeoxychorismate, and, surprisingly, tocotrienols. Our work thus uncovers the existence of a novel form of a relict plastid organelle.


title: Reduced injury risk links sociality to survival in a group-living primate

abstract: Affiliative social relationships and high social status predict longer lifespans in many mammal species, including humans. Yet, the mechanisms by which these components of sociality influence survival are still largely unknown. Using 10 years of long-term data on the incidence of injuries in a free-ranging population of rhesus macaques (Macaca mulatta), we investigated two possible mechanisms that could underpin the relationship between sociality and survival: sociality (1) reduces injury risk; and/or (2) increases the probability of survival after an injury. Our results showed that injured animals had a near three-fold increase in the probability of dying, confirming that injuries can be fatal in this population. Crucially, we found that sociality may reduce an individuals injury risk, but had no effect on the probability of injured individuals dying. Individuals with more affiliative partners and higher social status experienced fewer injuries compared to less socially integrated or lower status animals, with the effect of social status varying with age. Females living in bigger groups were more likely to be injured than those in smaller groups, while in males, smaller groups were associated with higher chances of being injured. Our study contributes novel evidence that sociality does not provide fitness benefits via socially-mediated promotion of injury recovery, but instead can enhance fitness by reducing the risk of being fatally injured in the first place.


title: Comparative genomics of Minnesotan barley-infecting Xanthomonas translucens shows overall genomic similarity but virulence factor diversity

abstract: Xanthomonas translucens pv. translucens (Xtt) is a global barley pathogen and a concern for resistance breeding and regulation. Long-read whole genome sequences allow in-depth understanding of pathogen diversity. We have completed long-read PacBio sequencing of two Minnesotan Xtt strains and an in-depth analysis of available Xtt genomes. We found that average nucleotide identity(ANI)-based approaches organize Xtt strains differently than the previously standard MLSA approach. According to ANI, Xtt forms a separate clade from Xanthomonas translucens pv. undulosa and consists of three main groups which are represented on multiple continents. The global distribution of Xtt groups suggests that regulation of seed is not important for prevention of Xtt spread. Some virulence factors, such as 17 Type III-secreted effectors, are highly conserved and offer potential targets for the elicitation of broad resistance. However, there is a high degree of variation in virulence factors meaning that germplasm should be screened for resistance with a diverse panel of Xtt.

category: genomics


title: Architectural experience: clarifying its central components and their relation to core affect with a set of first-person-view videos

abstract:
When studying architectural experience in the lab, it is of paramount importance to use a proxy as close to real-world experience as possible. Whilst still images visually describe real spaces, and virtual reality allows for dynamic movement, each medium lacks the alternative attribute. To merge these benefits, we created and validated a novel dataset of valenced videos of first-person-view travel through built environments. This dataset was then used to clarify the relationship of core affect (valence and arousal) and architectural experience. Specifically, we verified the relationship between valence and fascination, coherence, and hominess - three key psychological dimensions of architectural experience which have previously been shown to explain aesthetic ratings of built environments. We also found that arousal is only significantly correlated with fascination, and that both are embedded in a relationship with spatial complexity and unusualness. These results help to clarify the nature of fascination, and to distinguish it from coherence and hominess when it comes to core affect. Moreover, these results demonstrate the utility of a video dataset of affect-laden spaces for understanding architectural experience.

Highlights- Developed a video database of first-person-view journeys through built environments
- We explored how core affect and architectural experience relate through the videos
- Previous results are supported: valence ties to fascination, coherence and hominess
- Arousal correlates only with fascination, and not coherence or hominess
- Arousal and fascination are tied to spatial complexity and unusualness

category: animal behavior and cognition


title: Bumblebees retrieve only the ordinal ranking of foraging options when comparing memories obtained in distinct settings

abstract: Are animals preferences determined by absolute memories for options (e.g., reward sizes) or by their remembered ranking (better/worse)? The only studies examining this question suggest humans and starlings utilize memories for both absolute and relative information. We show that bumblebees make decisions using only memories of ordinal comparisons. After learning to discriminate pairs of different flowers by sucrose concentration, bumblebees preferred flowers (in novel pairings) with 1) higher ranking over equal absolute reward, 2) higher ranking over higher absolute reward, and 3) identical qualitative ranking but different quantitative ranking equally. Despite these suboptimal choices, bumblebees found the optimal option in an ecologically relevant foraging task. Our results illuminate a divergent mechanism of learned preferences that may have arisen from adaptations to bumblebees natural environment.


title: Efficient Permutation-based Genome-wide Association Studies for Normal and Skewed Phenotypic Distributions

abstract:
MotivationGenome-wide Association Studies (GWAS) are an integral tool for studying the architecture of complex genotype and phenotype relationships. Linear Mixed Models (LMMs) are commonly used to detect associations between genetic markers and the trait of interest, while at the same time allowing to account for population structure and cryptic relatedness. Assumptions of LMMs include a normal distribution of the residuals and that the genetic markers are independent and identically distributed - both assumptions are often violated in real data. Permutation-based methods can help to overcome some of these limitations and provide more realistic thresholds for the discovery of true associations. Still, in practice they are rarely implemented due to its high computational complexity.

ResultsWe propose permGWAS, an efficient linear mixed model reformulation based on 4D-tensors that can provide permutation-based significance thresholds. We show that our method outperforms current state-of-the-art LMMs with respect to runtime and that a permutation-based threshold has a lower false discovery rate for skewed phenotypes compared to the commonly used Bonferroni threshold. Furthermore, using permGWAS we re-analysed more than 500 Arabidopsis thaliana phenotypes with 100 permutations each in less than eight days on a single GPU. Our re-analyses suggest that applying a permutation-based threshold can improve and refine the interpretation of GWAS results.

AvailabilitypermGWAS is open-source and publicly available on GitHub for download: https://github.com/grimmlab/permGWAS.

category: bioinformatics


title: Membrane interactions of mitochondrial lipid transfer proteins

abstract: The mitochondrial inner membrane is an integral part of the cellular lipid biosynthesis network. Intramitochondrial lipid transfer shuttles specific lipid species between the two mitochondrial membranes. This pathway is facilitated by designated protein complexes in the intermembrane space. A hetero-dimeric complex of Ups1 and Mdm35 has been identified in yeast to transfer phosphatidic acid from the outer to the inner membrane. Here, we define pH and lipid charge dependency of Ups1 membrane interaction. We show that Ups1 interacts with membranes in a membrane curvature dependent manner. Ups1 predominantly binds to membrane domains of positive curvature. Desorption of phosphatidic acid from positively curved membranes is energetically favorable over flat or negatively curved membranes. Hence, our results demonstrate that Ups1 specifically binds to membrane regions where extraction and insertion of lipids is enhanced.


title: Prime-pull immunization of mice with a BcfA-adjuvanted vaccine elicits mucosal immunity and prevents SARS CoV-2 infection and pathology

abstract:
Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. We show that intramuscular priming of mice with an alum and BcfA-adjuvanted Spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17 polarized tissue resident CD4+ T cells, and mucosal and serum antibodies. The serum antibodies efficiently neutralized SARS-CoV-2 and its Delta variant, suggesting cross-protection against a recent variant of concern (VOC). Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 and reduced viral replication in the nose and lungs. Histopathology showed a strong leukocyte and polymorphonuclear (PMN) cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. In contrast, viral load was not reduced in the upper respiratory tract of IL-17 knockout mice immunized with the same formulation, suggesting that the Th17 polarized T cell responses are critical for protection. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, protect against SARS-CoV-2 infection without causing enhanced respiratory disease.

SIGNIFICANCEThere remains a need for SARS CoV-2 booster vaccines that generate mucosal immunity and prevent transmission. We show that systemic priming followed by a mucosal booster with a BcfA-adjuvanted subunit vaccine generates neutralizing antibodies and Th17 polarized systemic and tissue-resident immune responses that provide sterilizing immunity against wildtype SARS CoV-2, and a variant of concern. Importantly, in contrast to alum alone, the addition of BcfA prevents respiratory pathology. These results suggest that a BcfA-adjuvanted mucosal booster may elicit mucosal immunity in individuals previously immunized systemically with approved vaccines. This foundational study in mice sets the stage for testing our vaccine regimen in larger animal models as a booster vaccine.


title: Antibody Resistance of SARS-CoV-2 Omicron BA.1, BA.1.1, BA.2 and BA.3 Sub-lineages

abstract: The SARS-CoV-2 Omicron variant has been partitioned into four sub-lineages designated BA.1, BA.1.1, BA.2 and BA.3, with BA.2 becoming dominant worldwide recently by outcompeting BA.1 and BA.1.1. We and others have reported the striking antibody evasion of BA.1 and BA.2, but side-by-side comparison of susceptibility of all the major Omicron sub-lineages to vaccine-elicited or monoclonal antibody (mAb)-mediated neutralization are urgently needed. Using VSV-based pseudovirus, we found that sera from individuals vaccinated by two doses of inactivated whole-virion vaccines (BBIBP-CorV) showed very weak to no neutralization activity, while a homologous inactivated vaccine booster or a heterologous booster with protein subunit vaccine (ZF2001) markedly improved the neutralization titers against all Omicron variants. The comparison between sub-lineages indicated that BA.1.1, BA.2 and BA.3 had comparable or even greater antibody resistance than BA.1. We further evaluated the neutralization profile of a panel of 20 mAbs, including 10 already authorized or approved, against these Omicron sub-lineages as well as viruses with different Omicron spike single or combined mutations. Most mAbs lost their neutralizing activity completely or substantially, while some demonstrated distinct neutralization patterns among Omicron sub-lineages, reflecting their antigenic difference. Taken together, our results suggest all four Omicron sub-lineages threaten the efficacies of current vaccines and antibody therapeutics, highlighting the importance of vaccine boosters to combat the emerging SARS-CoV-2 variants.

category: microbiology


title: Distinct evolutionary trajectories of SARS-CoV-2 interacting proteins in bats and primates identify important host determinants of COVID-19

abstract:
The COVID-19 pandemic is caused by SARS-CoV-2, a novel coronavirus that spilled from the bat reservoir. Despite numerous clinical trials and vaccines, the burden remains immense, and the host determinants of SARS-CoV-2 susceptibility and COVID-19 severity remain largely unknown. Signatures of positive selection detected by comparative functional-genetic analyses in primate and bat genomes can uncover important and specific adaptations that occurred at virus-host interfaces. Here, we performed high-throughput evolutionary analyses of 334 SARS- CoV-2 interacting proteins to identify SARS-CoV adaptive loci and uncover functional differences between modern humans, primates and bats. Using DGINN (Detection of Genetic INNovation), we identified 38 bat and 81 primate proteins with marks of positive selection. Seventeen genes, including the ACE2 receptor, present adaptive marks in both mammalian orders, suggesting common virus-host interfaces and past epidemics of coronaviruses shaping their genomes. Yet, 84 genes presented distinct adaptations in bats and primates. Notably, residues involved in ubiquitination and phosphorylation of the inflammatory RIPK1 have rapidly evolved in bats but not primates, suggesting different inflammation regulation versus humans. Furthermore, we discovered residues with typical virus-host arms-race marks in primates, such as in the entry factor TMPRSS2 or the autophagy adaptor FYCO1, pointing to host-specific in vivo important interfaces that may be drug targets. Finally, we found that FYCO1 sites under adaptation in primates are those associated with severe COVID-19, supporting their importance in pathogenesis and replication. Overall, we identified functional adaptations involved in SARS- CoV-2 infection in bats and primates, critically enlightening modern genetic determinants of virus susceptibility and severity.

Key findingsO_LIEvolutionary history of 334 SARS-CoV-2 interacting proteins (VIPs) in bats and primates identifying how the past has shaped modern viral reservoirs and humans - results publicly-available in an online resource.
C_LIO_LIIdentification of 81 primate and 38 bat VIPs with signatures of adaptive evolution. The common ones among species delineate a core adaptive interactome, while the ones displaying distinct evolutionary trajectories enlighten host lineage-specific determinants.
C_LIO_LIEvidence of primate specific adaptation of the entry factor TMPRSS2 pointing to its host- specific in vivo importance and predicting molecular interfaces.
C_LIO_LIFYCO1 sites associated with severe COVID-19 in human (GWAS) display hallmarks of ancient adaptive evolution in primates, highlighting its importance in SARS-CoV-2 replication or pathogenesis and differences with the bat reservoir.
C_LIO_LIIdentification of adaptive evolution in the bats multifunctional RIPK1 at residues that may differentially regulate inflammation.
C_LI


title: CK2 signaling from TOLLIP-dependent perinuclear endosomes is an essential feature of KRAS mutant cancers

abstract: Oncogenic RAS induces perinuclear translocation of the oncogenic kinases ERK1/2 and CK2 and the MAPK scaffold KSR1, forming endosomal signaling hubs termed perinuclear signaling centers (PSCs). PSCs are found in nearly all cancer cell lines and tumor tissues, suggesting that compartmentalization of oncogenic kinases drives tumorigenesis. We show here that the endosomal adaptor, TOLLIP, tethers RAB11A+ signaling endosomes containing CK2 and KSR1, but not ERK, to the perinuclear ER. TOLLIP binds to the KSR1 CA5 pseudo-kinase domain through a conserved region predicted to form a {beta}-hairpin, thus anchoring PSCs to perinuclear endosomes. TOLLIP is perinuclear in cancer cells and KRasG12D-driven mouse tumors but is pan-cytoplasmic in non-transformed cells, correlating with the presence of PSCs. TOLLIP is required for proliferation/survival of KRAS mutant tumor cells but not HRAS, NRAS and BRAF cancers or non-transformed cells. KRasG12D-induced lung lesions in TollipKO/KOmice showed reduced numbers of carcinomatous lesions, implicating TOLLIP in progression to malignant cancer. Phosphoproteomics studies revealed that perinuclear CK2 phosphorylates specific substrates, particularly proteins involved in ribosome biogenesis and translation such as RIOK1 and eIF4B. In summary, our findings identify TOLLIP as a key signaling adaptor in KRAS tumors whose inhibition is a potential vulnerability of these cancers.


title: Deposit-feeding worms control subsurface ecosystem functioning in intertidal sediment with strong physical forcing

abstract:
Intertidal sands are global hotspots of terrestrial and marine carbon cycling with strong hydrodynamic forcing by waves and tides and high macrofaunal activity. Yet, the relative importance of hydrodynamics and macrofauna in controlling these ecosystems remains unclear. Here we compare bacterial, archaeal, and eukaryotic communities in upper intertidal sands dominated by subsurface deposit-feeding worms (Abarenicola pacifica) to adjacent worm-free areas. We show that hydrodynamic forcing controls organismal assemblages in surface sediments, while in deeper layers selective feeding by worms on fine, algae-rich particles strongly decreases the abundance and richness of all three domains. In these deeper layers, bacterial and eukaryotic network connectivity decreases, while percentages of taxa involved in degradation of refractory organic macrostructures, oxidative nitrogen and sulfur cycling, and macrofaunal symbioses, increase. Our findings reveal macrofaunal activity as the key driver of ecosystem functioning and carbon cycling in intertidal sands below the mainly physically controlled surface layer.

Significance StatementHydrodynamics and bioturbation are the main forces controlling chemical exchanges between sediment and seawater in coastal environments. However, little is known about the relative impact of both processes on sediment biological communities. We show that intertidal sand ecosystems dominated by lugworms can be divided into vertically distinct hydrodynamically and biologically controlled layers. Hydrodynamic forcing controls biological communities in surface layers by regulating organic carbon and electron acceptor inputs. By contrast, lugworms structure subsurface ecosystems through the selective consumption of fine particles, which diminishes microbial and eukaryotic populations and weakens ecological networks, while promoting the burial of, mostly terrestrial, macrodetritus. Our study demonstrates that globally distributed marine invertebrates control intertidal sand ecosystems below the physically controlled surface layer.


title: A Lysozyme Murein Hydrolase with Broad-spectrum Antibacterial Activity from Enterobacter Phage myPSH1140

abstract: Bacteriophages and bacteriophage-derived peptidoglycan hydrolases (endolysins) present promising alternatives for the treatment of infections caused by multi-drug resistant Gram-negative and Gram-positive pathogens. In this study, Gp105, a putative lysozyme murein hydrolase from Enterobacter phage myPSH1140 was characterized in silico, in vitro as well as in vivo using the purified protein. Gp105 contains a T4-type lysozyme-like domain (IPR001165) and belongs to Glycoside hydrolase family 24 (IPR002196). The putative endolysin indeed had strong antibacterial activity against Gram-negative pathogens including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp. and A. baumannii. Also, an in vitro peptidoglycan hydrolysis assay showed strong activity against purified peptidoglycans. This study demonstrates the potential of Gp105 to be used as an antibacterial protein to combat Gram-negative pathogens.


title: Human Milk Oligosaccharide Utilization in Intestinal Bifidobacteria is Governed by a Global Transcriptional Regulator NagR

abstract:
Bifidobacterium longum subsp. infantis (B. infantis) is a prevalent beneficial bacterium that colonizes the human neonatal gut and is uniquely adapted to efficiently use human milk oligosaccharides (HMOs) as a carbon and energy source. Multiple studies have focused on characterizing the elements of HMO utilization machinery in B. infantis; however, the regulatory mechanisms governing the expression of these catabolic pathways remain poorly understood. A bioinformatic regulon reconstruction approach used in this study implicated NagR, a transcription factor from the ROK family, as a negative global regulator of genomic loci encoding lacto-N-biose/galacto-N-biose (LNB/GNB), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT) utilization pathways in B. infantis. This conjecture was corroborated by transcriptome profiling upon nagR genetic inactivation and experimental assessment of binding of recombinant NagR to predicted DNA operators. The latter approach also implicated N-acetylglucosamine (GlcNAc), a universal intermediate of LNT and LNnT catabolism, and its phosphorylated derivatives as plausible NagR transcriptional effectors. Reconstruction of NagR regulons in various Bifidobacterium lineages revealed multiple regulon expansion events, suggesting evolution from a local regulator of GlcNAc catabolism in ancestral bifidobacteria to a global regulator controlling foraging of mixtures of GlcNAc-containing host-derived glycans in mammalian gut-colonizing B. infantis and Bifidobacterium bifidum.

ImportanceThe predominance of bifidobacteria in the gut of breastfed infants is attributed to the ability of these bacteria to utilize human milk oligosaccharides (HMOs). Thus, individual HMOs such as lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) are considered promising prebiotics that would stimulate the growth of bifidobacteria and confer multiple health benefits to preterm and malnourished children suffering from impaired (stunted) gut microbiota development. However, the rational selection of HMO-based prebiotics is hampered by the incomplete knowledge of regulatory mechanisms governing HMO utilization in target bifidobacteria. This study describes NagR-mediated transcriptional regulation of LNT and LNnT utilization in Bifidobacterium longum subsp. infantis. The elucidated regulatory network appears optimally adapted to simultaneous utilization of multiple HMOs, providing a rationale to add HMO mixtures (rather than individual components) into infant formulas. The study also provides insights into the evolutionary trajectories of complex regulatory networks controlling carbohydrate metabolism in bifidobacteria.


title: Phylogenetic and Ecological Trends in Specialization: Disentangling the Drivers of Ectoparasite Host Specificity

abstract:
O_LIEcological specialization reflects both evolutionary and ecological processes. For parasitic taxa, ecological specialization can be assessed as the degree to which a parasite species will associate with certain host species, a property known as host specificity.
C_LIO_LIEctoparasitic bat flies have been previously reported as highly host specific, presumably due to a history of coevolution with their bat hosts. However, there is conflicting evidence of coevolution between bats and bat flies. Resource-driven competition between parasite individuals and between species may also be important in explaining patterns of bat fly specificity.
C_LIO_LITo test the importance of evolutionary and ecological factors on bat fly specificity, we collected and identified 21 bat fly species from 16 host bat species from the State of Rio de Janeiro in Brazil. We generated a bat fly species phylogeny from molecular data, estimated d specialization values (a metric of specificity), and used linear and cophylogenetic models to compare the importance of various drivers of parasite ecological specialization.
C_LIO_LIWe found that bat fly co-occurrence frequency (a proxy for interspecific competition) and mean infection intensity (a proxy for intraspecific competition) best predicted patterns of bat fly specialization. Co-occurrence frequency had a significantly negative association with specialization, while mean infection intensity has a significantly positive association with specialization. Coevolutionary congruence had a small effect size and did not significantly predict parasite specialization.
C_LIO_LIWe found multiple shifts toward more generalized host niches across the bat fly phylogeny. Our results suggest that ecological processes such as resource-driven competition may be more important than evolutionary processes in shaping bat fly host specialization networks.
C_LIO_LIBat flies showed variable degrees of host specialization, parasitized phylogenetically distant host species, and showed low phylogenetic congruence to their hosts. This suggests that as a group, bat flies may show flexibility in their host preference phenotypes and may change their host associations in the face of environmental disturbance.
C_LI

category: ecology


title: A full spectrum PNGase activity analysis of R328 mutations on NGLY1

abstract: In humans, N-glycanase 1 (NGLY1; Peptide: N-glycanase, PNGase) is responsible for the deglycosylation of misfolded glycoproteins. Pathogenic mutations in NGLY1 cause a clinical condition known as congenital disorder of deglycosylation (NGLY1-CDDG), a rare autosomal recessive disease first reported in 2012. Although NGLY1-CDDG was diagnosed through whole-exome or whole-genome sequencing and by evaluating the expression levels of NGLY1, the clinical relevance of a detected mutation in NGLY1 needs to be further confirmed. In this study, an in vitro enzymatic assay system was established to evaluate the thermal stability and substrate specificity of NGLY1, as well as the optimum reaction conditions for its activity. A panel of all mutations at the amino acid site R328 in NGLY1 was subjected to this assay. The results revealed that R328A, R328D, R328E, R328F, R328G, R328I, R328P, R328V, R328W, and R328Y were dysfunctional mutations (10/19); NGLY1 mutations with R328H and R328T exhibited similar activity as wild-type NGLY1 (2/19); and NGLY1 mutations with R328C, R328K, R328L, R328M, R328N, R328Q, and R328S showed decreased activity (7/19) compared to wild-type NGLY1. In addition, the effect of potential regulatory compounds, including N-acetyl-L-cysteine and dithiothreitol, on NGLY1 was examined. This in vitro assay may serve as a standard protocol to facilitate rapid diagnosis of all mutations on NGLY1 and a practical screening method for drugs and compounds with potential therapeutic value for NGLY1-CDDG patients.

category: biochemistry


title: Structural and biochemical basis of FANCI-FANCD2 interdependent ubiquitination

abstract: The Fanconi Anaemia pathway operates for the repair of interstrand crosslinks and the maintenance of genomic stability upon replication stalling. Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step in this pathway. Evidence suggests that FANCD2 ubiquitination precedes FANCI ubiquitination, and that both the FANCD2-ubiquitinated (ID2Ub) and the di-monoubiquitinated (IUbD2Ub) complex clamp on DNA. However, FANCD2 is deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUbD2). Here, we present a 4.1 [A] cryo-EM structure of IUbD2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUbD2Ub, is also in the closed ID2 conformation and clamps on DNA. While the target lysine of FANCD2 (K561) is partially buried in the non-ubiquitinated ID2-DNA complex, it becomes fully exposed in the IUbD2-DNA structure, and thus can be ubiquitinated at a faster rate. The IUbD2-DNA complex cannot easily revert to the non-ubiquitinated ID2 state, due to USP1-UAF1-resistance, conferred by the presence of DNA and FANCD2. ID2Ub-DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. FANCI ubiquitination also progresses at a faster rate in ID2Ub-DNA over ID2-DNA complex, and results in partial DNA-dependent protection from FANCD2 deubiquitination. Taken together, our results suggest that, while FANCD2 ubiquitination promotes FANCI ubiquitination, FANCI ubiquitination in turn maintains FANCD2 ubiquitination by two mechanisms: it prevents excessive FANCD2 deubiquitination within an IUbD2Ub-DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUbD2 complex.


title: Impairments in SHMT2 expression or cellular folate availability reduce oxidative phosphorylation and pyruvate kinase activity

abstract:
BackgroundSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the proteins active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cells models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression.

MethodsPrimary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6 dam crossed with a heterozygous Shmt2+/- male to generate Shmt2+/+ (wild-type) or Shmt2+/- (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression ({Delta}SHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.

ResultsHomozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not {Delta}SHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in {Delta}SHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/- MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.

ConclusionsThe results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.


title: Structure-based discovery of a non-canonical prototype long-chain monoacylglycerol lipase through an endogenous catalysis intermediate complex

abstract: The identification and characterization of enzyme function is lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study where the high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacyl catalysis intermediate allowed its functional characterization as long-chain monacylglycerol lipase. Different from most other lipases, this enzyme uses a minimal helix-{beta}-hairpin-helix lid domain that positions the substrate through a hydrophobic tunnel directly to the enzymes active site. Knowledge about the molecular details of the substrate binding site and hydrolysis allowed us to boost the enzymatic activity by adjusting protein/substrate interactions. This enzyme serves as a prototype for non-conventional esterases and lipases that share similar helix-{beta}-hairpin-helix lid domains of variable size. Taken together, our data provide crucial insight to advance our knowledge of unusual esterases and lipases, facilitating future biotechnology applications.


title: Characterizing a New Fluorescent Protein for Low Limit of Detection Sensing in the Cell-Free System

abstract: Cell-free protein synthesis-based biosensors have been developed as highly accurate, low- cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensors dynamic range must be increased in the system to achieve the low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein - mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder Green Fluorescent Protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce false positives proportion.

category: synthetic biology


title: Mechanical morphotype switching as an adaptive response in mycobacteria

abstract: Bacterial stress tolerance during infection is commonly thought to be encoded by discrete biochemical stress-specific mechanisms 1. However, increasing evidence suggests that fluctuating conditions of stress drive biomechanical changes to bacterial components on varying cellular scales 2-5. Here, we use atomic-force microscopy (AFM) 6 to demonstrate that during macrophage infection, intracellular mycobacteria diverge into two mechanically distinct subpopulations exhibiting either a two-fold increase or decrease in mean cell surface stiffness. We describe this process as "mechanical morphotype switching", which we define as a phenotypic differentiation into two distinct, semi-stable mechanical cell states. Mycobacterial mechanical morphotype states are enriched by gene mutation and cytokine modulation of host macrophage responses, suggesting a molecular basis for switching and a role for host immunity in driving selection of discrete mechanical morphotypes. Crucially, mycobacterial mechanical morphotype switching enhances survival in macrophages of both non-pathogenic Mycobacterium smegmatis and pathogenic, M. abscessus. These results reveal that mycobacteria mechanically adapt to enhance intracellular viability. Our study highlights the importance of bacterial mechanical properties for optimised survival in host intracellular environments during pathogenesis.


title: Collateral sensitivity as a strategy to suppress resistance emergence: the challenge of diverse evolutionary pathways

abstract: Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.


title: Antibiotic persistence of Brucella abortus in its protective intracellular niche

abstract:
BackgroundHuman Brucellosis caused by the facultative intracellular pathogen Brucella spp. is an endemic bacterial zoonosis manifesting as acute or chronic infections associated with high morbidity. Treatment typically involves a combination therapy of two antibiotics administered for several weeks to month, but despite this harsh treatment relapses occur at a rate of 5-15%. Although poor compliance and reinfection may account for a fraction of the observed relapse cases, it is apparent that the properties of the infectious agent itself may play a decisive role in this phenomenon.

Methodology/Principal findingsWe used B. abortus carrying a dual reporter in a macrophage infection model to gain a better understanding of the efficacy of recommended therapies in cellulo. For this we used automated fluorescent microscopy as a prime read-out and developed specific CellProfiler pipelines to score infected macrophages at the population and the single cell level. Combining microscopy of constitutive and induced reporters with classical CFU determination, we quantified the protective nature of the Brucella intracellular niche to various antibiotics and the ability of B. abortus to persist in cellulo despite harsh antibiotic treatments.

Conclusion/SignificanceWe demonstrate that treatment of infected macrophages with antibiotics at recommended concentrations fails to fully prevent growth and persistence of B. abortus in cellulo, which may be explained by a protective nature of the intracellular niche. Moreover, we show the presence of bona fide intracellular persisters upon antibiotic treatment, which are metabolically active and retain the full infectious potential, therefore constituting a plausible reservoir for reinfection and relapse. In conclusion, our results highlight the need to extend the spectrum of models to test new antimicrobial therapies for Brucellosis to better reflect the in vivo infection environment, and to develop therapeutic approaches targeting the persister subpopulation.

Authors SummaryBrucellosis is a zoonosis endemic to many low- and middle-income countries around the world. Recommended therapies by the WHO are comprised of at least two antibiotics for several weeks, sometimes month. Despite these harsh treatments relapses are frequent. The underlying reasons for these relapses, besides reinfection and non-compliance to treatment, are unknown. Our study shows that Brucella abortus can form so called "persisters" under recommended treatments in rich broth but also inside macrophages. This small bacterial subpopulation survives antibiotic treatment and resumes growth after removal of the antibiotics and could therefore serve as a reservoir for relapses in human Brucellosis. Furthermore, we show that the macrophage intracellular niche of Brucella has protective properties against recommended antibiotics as observed for other intracellular pathogens, highlighting the necessity to develop new infection models to assess antibiotic efficacy.


title: Distinct cohorts of Aspergillus fumigatus transcription factors are required for epithelial damage occurring via contact- or soluble effector-mediated mechanisms

abstract:
Damage to the lung epithelium is a unifying feature of disease caused by the saprophytic fungus Aspergillus fumigatus. However, the mechanistic basis and the regulatory control of such damage is poorly characterized. Previous studies have identified A. fumigatus mediated pathogenesis as occurring at early ([≤] 16 hours) or late (>16 hours) phases of the fungal interaction with epithelial cells, and respectively involve direct contact with the host cell or the action of soluble factors produced by mature fungal hyphae. Both early and late phases of epithelial damage have been shown to be subject to genetic regulation by the pH-responsive transcription factor PacC. This study sought to determine whether other transcriptional regulators play a role in modulating epithelial damage. In particular, whether the early and late phases of epithelial damage are governed by same or distinct regulators. Furthermore, whether processes such as spore uptake and hyphal adhesion, that have previously been documented to promote epithelial damage, are governed by the same cohorts of epithelial regulators. Using 479 strains from the recently constructed library of A. fumigatus transcription factor null mutants, two high-throughput screens assessing epithelial cell detachment and epithelial cell lysis were conducted. A total of 17 transcription factor mutants were found to exhibit reproducible deficits in epithelial damage causation. Of these, 10 mutants were defective in causing early phase damage via epithelial detachment and 8 mutants were defective in causing late phase damage via epithelial lysis. Remarkably only one transcription factor, PacC, was required for causation of both phases of epithelial damage. The 17 mutants exhibited varied and often unique phenotypic profiles with respect to fitness, epithelial adhesion, cell wall defects, and rates of spore uptake by epithelial cells. Strikingly, 9 out of 10 mutants deficient in causing early phase damage also exhibited reduced rates of hyphal extension, and culture supernatants of 7 out of 8 mutants deficient in late phase damage were significantly less cytotoxic. Our study delivers the first high-level overview of A. fumigatus regulatory genes governing lung epithelial damage, suggesting highly coordinated genetic orchestration of host-damaging activities that govern epithelial damage in both space and time.

Contribution to the Field StatementAspergillus fumigatus is a soil dwelling fungus that can cause lethal lung infections in individuals with a compromised immune system. Disease initiates with inhalation of the fungal spores, followed by growth of the fungus leading to destruction of the lung. Our understanding of the A. fumigatus genes and mechanisms driving lung damage leading to establishment of disease is limited. This study has identified the genes moderating lung damage by assessing 479 regulatory mutants of A. fumigatus for their ability to cause epithelial damage using a lung cell line model. We observed that distinct cohorts of transcriptional regulators are required for driving early, and late phases of epithelial damage and that early- and late-occurring damage are associated respectively with hyphal growth rates and secreted fungal products. This study is the first to reveal that mechanistically distinct programs of host damage elicited during early and late stage of fungal interaction with epithelial cells are genetically regulated via distinct cohorts of A. fumigatus transcription factors.


title: The production of diverse brGDGTs by an Acidobacterium allows a direct test of temperature and pH controls on their distribution

abstract:
Microbial lipid biomarkers preserved in geological archives can be used to explore past climate changes. Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are unique bacterial biomarkers that have been used as molecular tools for the quantitative determination of terrestrial temperatures and the pH of depositional environments over a range of geological timescales. However, the exact biological source organisms - especially of the entire suite of brGDGTs found in the environment - remains unclear; by extension, so do the mechanisms that govern these proxies. Here, we identified a brGDGT-producing strain Candidatus Solibacter usitatus Ellin6076, by identifying archaeal tetraether synthase homologs in bacterial genomes. This strain synthesizes diverse brGDGTs, including regular C5-methylated and cyclic brGDGTs, and brGDGTs comprise up to 66% of the major lipids, far exceeding the proportions found in previous studies. The degree of C5-methylation in cultured strain Ellin6076 is primarily determined by temperature, whereas cyclization appears to be influenced by multiple factors. Consequently, culture-derived paleoclimate indices are in agreement with the global soil-derived MBT5ME (methylation index of C5-methyl brGDGTs) proxy for temperature but not the CBT5ME (cyclization index of C5-methyl brGDGTs) proxy for pH. Our findings provide important insights from a physiological perspective into the underlying mechanism of brGDGT-based proxies.

Significance StatementBranched glycerol dialkyl glycerol tetraethers (brGDGTs) are biomarkers widely used for the quantitative estimation of past climatic changes due to their ubiquitous occurrence in the environment and the relationships between their distributions and temperature and pH. However, the ecophysiology of brGDGT-producing bacteria and the mechanistic basis for brGDGT-based climate proxies remain unknown. Here, we identify a brGDGT-producing Acidobacterium and present a physiological study of brGDGTs in response to cultivation variables, which provides pivotal insights into how brGDGT producers modulate methylation and cyclization under different culturing conditions. Our study represents a significant advancement in understanding the physiological role of lipid structures in microbial adaptation and helps us interpret the relationships between brGDGT-based proxies and environmental conditions of the geological environment.


title: Neutrophil IL-1β secretion induced by ExoS expressing Pseudomonas aeruginosa is dependent on NLRP3 and Gasdermin D

abstract: Macrophages infected with Gram-negative bacteria expressing flagellin or Type III secretion system (T3SS) structural proteins are known to activate the NLRC4 inflammasome, resulting in caspase-1 and Gasdermin D (GSDMD) cleavage, IL-1{beta} secretion and pyroptotic cell death. We examined the role of these mediators in IL-1{beta} secretion by neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the Type III secretion system (T3SS) effectors ExoS and ExoT. We found that IL-1{beta} secretion by neutrophils was dependent on expression of the T3SS needle and translocon proteins. Although pro-GSDMD and pro-GSDME were processed in PAO1 infected neutrophils, only GSDMD was required for IL-1{beta} secretion. PAO1 - induced IL-1{beta} secretion by macrophages was NLRC4 dependent, IL-1{beta} secretion by neutrophils utilized NLRC4 only in the absence of P. aeruginosa exoenzymes. Instead, PAO1 - induced IL-1{beta} secretion required NLRP3, which mediated by ExoS ADP ribosyl transferase activity. Overall, these findings reveal fundamental differences between neutrophils and macrophages in IL-1{beta} secretion in response to pathogenic bacteria.


title: Niclosamide targets macrophages to rescue the disrupted peritoneal homeostasis in endometriosis

abstract:
Due to the vital roles of macrophages in the pathogenesis of endometriosis, targeting macrophages could be a new therapeutic direction. Here, we investigated the efficacy of niclosamide for the resolution of perturbed microenvironment caused by dysregulated macrophages in a mouse model of endometriosis. Single-cell transcriptomic analysis revealed the heterogeneity of macrophage subpopulations including three newly identified intermediate subtypes with sharing characteristics of traditional "small" or "large" peritoneal macrophages (SPMs and LPMs) in the peritoneal cavity. Endometriosis-like lesions (ELL) enhanced the differentiation of recruited macrophages, promoted the replenishment of resident LPMs, and increased ablation of embryo-derived LPMs, which were stepwise suppressed by niclosamide. In addition, niclosamide reversed intercellular communications between macrophages and B cells which were disrupted by ELL. Therefore, niclosamide rescued the perturbed microenvironment in endometriosis through its fine regulations on the dynamic progression of macrophages and could be a new promising therapy for endometriosis.

SummaryNiclosamide tunes the dynamic progression of peritoneal macrophages and their intercellular communications with B cells to rescue the disrupted microenvironment in the peritoneal cavity in a mouse model of endometriosis.

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category: molecular biology


title: Structures and comparison of endogenous 2-oxoglutarate and pyruvate dehydrogenase complexes from bovine kidney

abstract: The -keto acid dehydrogenase complex family catalyzes the essential oxidative decarboxylation of -keto acids to yield acyl-CoA and NADH. Despite performing the same overarching reaction, members of the family have different component structures and structural organization between each other and across phylogenetic species. While native structures of -keto acid dehydrogenase complexes from bacteria and fungi became available recently, the atomic structure and organization of their mammalian counterparts in their native states remain unknown. Here, we report the cryo electron microscopy (cryoEM) structures of the endogenous cubic 2-oxoglutarate dehydrogenase complex (OGDC) and icosahedral pyruvate dehydrogenase complex (PDC) cores from bovine kidney determined at 3.5 [A] and 3.8 [A] resolution, respectively. The structures of multiple protein were reconstructed from a single lysate sample, allowing direct structural comparison without the concerns of differences arising from sample preparation and structure determination. Although native and recombinant E2 core scaffold structures are similar, native structures are decorated with their peripheral E1 and E3 subunits. Asymmetric sub-particle reconstructions support heterogeneity in the arrangements of these peripheral subunits. Additionally, despite sharing a similar monomeric fold, OGDC and PDC E2 cores have distinct interdomain and intertrimer interactions, which suggests a means of modulating self-assembly to mitigate heterologous binding between mismatched E2 species. The lipoyl moiety lies near a mobile gatekeeper within the interdomain active site of OGDC E2 and PDC E2. Analysis of the two-fold related intertrimer interface identified secondary structural differences and chemical interactions between icosahedral and cubic geometries of the core. Taken together, our study provides direct structural comparison of OGDC and PDC from the same source and offers new insights into determinants of interdomain interactions and of architecture diversity among -keto acid dehydrogenase complexes.


title: Recycling of uridylated mRNAs in starfish embryos

abstract: In eukaryotes, mRNAs with long poly(A) tails are translationally active, whereas deadenylation of the tails decreases translation and uridylation of the short poly(A) tails causes the mRNA to be degraded. In this study, we confirmed that maternal cyclin B mRNAs with long poly(A) tails in blastula embryos of invertebrate starfish were deadenylated and uridylated, followed by decay. In starfish oocytes, however, cyclin B mRNAs with uridylated short poly(A) tails are stable. They are polyadenylated and translationally active immediately following hormonal stimulation for resumption of meiosis. Similarly, maternal ribosomal protein mRNAs, Rps29 and Rpl27a, which become uridylated following deadenylation upon hormonal stimulation, remain stable even after fertilisation and early development. At the morula stage, the uridylated maternal ribosomal protein mRNAs are modified to yield non-canonical poly (A) tails rich in U and G residues in the 5 region and in A residues at the 3 end, rendering them translationally active. These results indicate that the fates of uridylated mRNAs in starfish are decay and/or recycling.


title: Entropy change due to selection in replicator and recombinator systems with randomly chosen interaction matrix values

abstract:
Selection can be described as a process of information accumulation21. The present work extends this for frequency-dependent selection. For recombination, it is shown that the total entropy change can be calculated by summing the contributions of the individual gene loci in this case as well.

For more complex replicator systems with and without recombination with randomly selected interaction matrix values (defining the fitness landscape), a detailed investigation is made of how the number of replicators ("genotypes") surviving, the mean fitness, and the entropy change depend on the number of replicators initially present, on their initial frequencies, and on certain properties (symmetry: aij=aji versus aij=aji) of the interaction matrix. Survival of several to many replicators is the norm rather than the exception. The more survive, the smaller the entropy change due to selection, although this is no longer generally true for asymmetric interaction matrices. Cases deviating from the rule were studied, but their systematic classification is still lacking. Finally, how evolution alters replicator systems of greater complexity was analyzed. It is shown that different evolutionary algorithms can lead to an increase in mean fitness and/or an increase in the number of surviving replicators. In contrast to simple replicator systems, coexistence in more complex ones apparently results naturally as a consequence of selection and evolution. It results that the view that: "Evolution is based on a fierce competition between individuals and should therefore reward only selfish behavior"16 is merely an artifact that follows from the study of unrealistically simple systems.


title: Genotypic sex shapes maternal care in the African Pygmy mouse,  Mus minutoides

abstract: Parental care, one of the most sexually dimorphic behaviour in mammals, was long thought to be driven mostly, if not exclusively, by gonadal hormones. Over the past two decades, very few studies have challenged this view, highlighting the direct influence of the sex chromosome complement (XX vs XY). The African pygmy mouse, Mus minutoides, is a wild mouse species with a naturally occurring sex reversal due to a third, feminizing X* chromosome, leading to three female genotypes: XX, XX* and X*Y. Here, we show that the sex reversal in X*Y females shapes a divergent maternal care strategy from both XX and XX* females, rather than altering care quality. In addition, the sex reversal is likely to impact the dopaminergic system in the anteroventral periventricular nucleus of the hypothalamus, consistent with one component of maternal care: pup retrieval. Combining neurobiology and behavioural ecology in a wild mouse species subject to natural selection, we evaluate here the neural basis of maternal behaviours and strengthen the underestimated role of the sex chromosome in shaping sex differences in brain and behaviours. Furthermore, we highlight the emergence of a third sexual phenotype, challenging the binary view of sexes.


title: Detecting macroevolutionary genotype-phenotype associations using error-corrected rates of protein convergence

abstract: On macroevolutionary timescales, extensive mutations and phylogenetic uncertainty mask the signals of genotype-phenotype associations underlying convergent evolution. To overcome this problem, we extended the widely used framework of nonsynonymous-to-synonymous substitution rate ratios and developed the novel metric{omega} C, which measures the error-corrected convergence rate of protein evolution. While{omega} C distinguishes natural selection from genetic noise and phylogenetic errors in simulation and real examples, its accuracy allows an exploratory genome-wide search of adaptive molecular convergence without phenotypic hypothesis or candidate genes. Using gene expression data, we explored over 20 million branch combinations in vertebrate genes and identified the joint convergence of expression patterns and protein sequences with amino acid substitutions in functionally important sites, providing hypotheses on undiscovered phenotypes. We further extended our method with a heuristic algorithm to detect highly repetitive convergence among computationally nontrivial higher-order phylogenetic combinations. Our approach allows bidirectional searches for genotype-phenotype associations, even in lineages that diverged for hundreds of millions of years.


title: Demographic expansions and the emergence of host specialization in genetically distinct strains of the tick-transmitted bacterium Anaplasma phagocytophilum

abstract:
Bacteria species that must obligately replicate in vertebrate host cells make up a large proportion of the prokaryotic pathogens with human and veterinary health implications. In such bacterial taxa, extrinsic processes play an important role in influencing the phylogenetic diversity of viable hosts ( host range). These processes include both changes in host population densities and shifts in host geographic distributions. In Europe, distinct genetic strains of the tick-vectored bacterium Anaplasma phagocytophilum circulate among mammals in three discrete enzootic cycles. To date, the factors that contributed to the emergence of these strains have been poorly studied. Here we show that the strain which predominately infects roe deer (Capreolus capreolus) is evolutionarily derived. Its divergence from a likely host-generalist ancestor occurred after the last glacial maximum as mammal populations, including roe deer, recolonized the European mainland from southern refugia. We also provide evidence that this host-specialist strains effective population size (Ne) has tracked changes in the population of its roe deer host. Specifically, both host and bacterium appear to have undergone substantial increases in Ne over the past 1,500 years. In contrast, we show that while it appears to have undergone a major population expansion starting [~]3,500 years ago, in the past 500 years the contemporary host-generalist strain has experienced a substantial reduction in genetic diversity levels, possible as the result of reduced transmission opportunities between competent hosts.

IMPORTANCEThe findings of this study are some of the first to examine specific events in the evolution of host specialization in a naturally occurring, obligately intracellular bacterial species. They show that host range shifts and the emergence of host specialization may occur during periods of population growth in a host-generalist ancestor. The results discussed here also show the close correlation between demographic patterns in host and pathogen for a specialist system. These findings have important relevance for our understanding of the evolution of host-range diversity. They may inform future work on host-range dynamics and provide insights for understanding the emergence of pathogens which have human and veterinary health implications.


title: Novel Antibody Interfaces Revealed Through Structural Mining

abstract: Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class of therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, and elucidation of such interactions can provide insights into their biology and new opportunities for their optimization as drugs. Yet the transitory nature of weak interactions makes them difficult to investigate. Capitalizing on their rich structural data and high conservation, we have characterized all the ways that antibody Fab regions interact crystallographically. This approach led to the discovery of previously unrealized interfaces between antibodies. While diverse interactions exist, {beta}-sheet dimers and variable-constant elbow dimers are recurrent motifs. Disulfide engineering enabled interactions to be trapped and investigated structurally and functionally, providing experimental validation of the interfaces and illustrating their potential for optimization. This work provides first insight into previously undiscovered oligomeric interactions between antibodies, and enables new opportunities for their biotherapeutic optimization.


title: Biochemical characterization of AniA from Neisseria gonorrhoeae

abstract: AniA, the nitrite reductase from Neisseria gonorrhoeae, has been shown to play a crucial role in the infection mechanism of this microorganism by producing NO and abolishing epithelial exfoliation. This enzyme is a trimer with one type-1 copper center per subunit and one type 2 copper center in the subunits interface, with the latter being the catalytic site. The two centers were characterized by visible, EPR and CD spectroscopy for the first time, indicating that AniAs type 1 copper center has a high rhombicity, which is attributed to its tetrahedral geometry, and shorter Met-Cu bond, while type 2 copper center has the usual properties, though with a shorter hyperfine coupling constant (A//= 9.1 mT). The thermostability of AniA was analyzed by differential scanning calorimetry showing a single endothermic transition in the thermogram, with a maximum at 95 {degrees}C, while the CD spectra in the visible region indicates the presence of copper centers at 85-90 {degrees}C. The reoxidation rates of AniA in the presence of nitrite were analyzed by visible spectroscopy showing a pH dependence and being higher at pH 6.0. The high thermostability of this enzyme might be important for maintaining a high activity in the extracellular space and be less prone to denaturation and proteolysis, contributing to the proliferation of N. gonorrhoeae.


title: The structure of the F420-dependent sulfite-detoxifying enzyme from Methanococcales reveals a prototypical sulfite-reductase with assimilatory traits.

abstract: The coenzyme F420-dependent sulfite reductase (Fsr group I) protects hydrogenotrophic methanogens, one of the main contributors in worldwide methane emission, from toxic sulfite. Fsr is a single peptide composed of a F420H2-oxidase and a novel class of sulfite reductase. Both catalytic domains have been proposed to be the ancestors of modern F420-oxido/reductases and dissimilatory/assimilatory sulfite reductases. Here, we describe the X-ray crystal structures of Fsr natively isolated from Methanocaldococcus jannaschii (MjFsr) and Methanothermococcus thermolithotrophicus (MtFsr), respectively refined to 2.30 [A] and 1.55 [A] resolution. In both organisms, Fsr oligomerizes as a 280-kDa homotetramer, where each siroheme-[4Fe-4S] is catalytically active, in contrast to dissimilatory homologues. The siroheme-[4Fe-4S], embedded in the sulfite reductase domain, is electronically connected to the flavin in the F420H2-oxidase domain by five [4Fe-4S]-clusters. EPR spectroscopy determined the redox potentials of these [4Fe-4S]2+/1+ clusters (-435 to -275 mV), through which electrons flow from FAD to the siroheme-[4Fe-4S]2+/1+ (siroheme, -114 mV; [4Fe-4S] -445 mV). The electron relay is mainly organized by two inserted ferredoxin modules, which stabilize the higher degree of oligomerization. While the F420H2-oxidase part is similar to the {beta}-subunit of F420-reducing hydrogenases, the sulfite reductase domain is structurally analogous to dissimilatory sulfite reductases, whereas its siroheme-[4Fe-4S] cofactor is bound in the same way as in assimilatory ones. Accordingly, the reaction of MtFsr is unidirectional, reducing sulfite or nitrite with F420H2. Our results provide the first structural insights into this unique fusion, a snapshot of a primitive sulfite reductase that turns a poison into an elementary block of Life.


title: A multiscale atlas of the molecular and cellular architecture of the human kidney

abstract: The human kidney is composed of over 26 cell types that actively coordinate with each other to form higher-order structures, such as the nephron. It is not yet understood how these structures vary throughout a single organ or amongst the same organs within the human population. We have developed an extensive lipid and cellular atlas of the human kidney consisting of over 3 million cells comprising 75,000 functional tissue units (i.e., glomeruli, proximal tubules, distal tubules, and collecting ducts) from 13 human subjects. This atlas was developed using spatially registered and integrated technologies consisting of imaging mass spectrometry, multiplexed immunofluorescence, stained microscopy, and autofluorescence microscopy, to comprehensively probe large (i.e., centimeter-sized) areas of tissue. The cellular organization and lipid profiles of glomeruli, proximal tubules, distal tubules, and collecting ducts were discovered through these multimodal imaging data as well as their intra- and inter-subject variance. Relating the lipid profiles obtained from imaging mass spectrometry to distinct cell types obtained from immunofluorescence allowed us to hypothesize the functional role of specific phospholipids that have not previously been described. These hypotheses include subject characteristics, such as BMI and sex. The integrated data from the aforementioned datasets provide a valuable reference for kidney researchers, are publicly available through the NIH Human Biomolecular Atlas Program (https://portal.hubmapconsortium.org/), and discussed below.


title: A multiscale lipid and cellular atlas of the human kidney


title: Computational Design of Two New Vitamin K Epoxide Reductase Inhibitors

abstract: Vitamin K epoxide reductase (VKOR) inhibitors are commonly used to treat atrial fibrillation and ischemic stroke. However, due to the side effects and poor absorbance of existing drugs, there is urgent need for developing better VKOR inhibitor drugs. In this work, computational bioisosteric replacement and chemical intuition were used to design two novel VKOR inhibitors. The new candidates are predicted to have improved ADMET and binding properties compared to existing drugs.