title: A Deep Learning Framework for Prediction of Clinical Drug Response of Cancer Patients and Identification of Drug Sensitivity Biomarkers using Preclinical Samples

abstract:
BackgroundPrediction of the response of cancer patients to different treatments and identification of biomarkers of drug sensitivity are two major goals of individualized medicine. In this study, we developed a deep learning framework called TINDL, completely trained on preclinical cancer cell lines, to predict the response of cancer patients to different treatments. TINDL utilizes a tissue-informed normalization to account for the tissue and cancer type of the tumours and to reduce the statistical discrepancies between cell lines and patient tumours. In addition, this model identifies a small set of genes whose mRNA expression are predictive of drug response in the trained model, enabling identification of biomarkers of drug sensitivity.

ResultsUsing data from two large databases of cancer cell lines and cancer tumours, we showed that this model can distinguish between sensitive and resistant tumours for 10 (out of 14) drugs, outperforming various other machine learning models. In addition, our siRNA knockdown experiments on 10 genes identified by this model for one of the drugs (tamoxifen) confirmed that all of these genes significantly influence the drug sensitivity of the MCF7 cell line to this drug. In addition, genes implicated for multiple drugs pointed to shared mechanism of action among drugs and suggested several important signaling pathways.

ConclusionsIn summary, this study provides a powerful deep learning framework for prediction of drug response and for identification of biomarkers of drug sensitivity in cancer.


title: Recovering Escherichia coli plasmids in the absence of long-read sequencing data

abstract:
The incidence of infections caused by multidrug-resistant E. coli strains has risen in the past years. Antibiotic resistance in E. coli is often mediated by acquisition and maintenance of plasmids. The study of E. coli plasmid epidemiology and genomics often requires long-read sequencing information, but recently a number of tools that allow plasmid prediction from short-read data have been developed.

Here, we reviewed 25 available plasmid prediction tools and categorized them into binary plasmid/chromosome classification tools and plasmid reconstruction tools. We benchmarked six tools that aim to reliably reconstruct distinct plasmids, with a special focus on plasmids carrying antibiotic resistance genes (ARGs) such as extended-spectrum beta-lactamase genes. They use either assembly graph information (plasmidSPAdes, gplas), reference databases (MOB-Suite, FishingForPlasmids) or both (HyAsP and SCAPP) to produce plasmid predictions. The benchmark data set consisted of 240 E. coli strains, harboring 631 plasmids, which were representative for the diversity of E. coli in public databases. Notably, these strains were not used for training any of the tools.

We found that two thirds (n=425, 66.3.%) of all plasmids were correctly reconstructed by at least one of the six tools, with a range of 92 (14.58%) to 317 (50.23%) correctly predicted plasmids. However, the majority of plasmids that carried antibiotic resistance genes (n=85, 57.8%) could not be completely recovered as distinct plasmids by any of the tools. MOB-suite was the only tool that was able to correctly reconstruct the majority of plasmids (n=317, 50.23%), and performed best at reconstructing large plasmids (n=166, 46.37%) and ARG-plasmids (n=41, 27.9%), but predictions frequently contained chromosome contamination (40%). In contrast, plasmidSPAdes reconstructed the highest fraction of plasmids smaller than 18 kbp (n=168, 61.54%). Large ARG-plasmids, however, were recovered with small precision values (median=0.47, IQR=0.61), indicating that plasmidSPAdes frequently merged sequences derived from distinct replicons. Additionally, only 63% of all plasmid-borne ARGs were correctly predicted by plasmidSPAdes.

The remaining four tools (FishingForPlasmids, HyAsP, SCAPP and gplas) were able to correctly reconstruct a combined total of 18 plasmids that were missed by MOB-suite and plasmidSPAdes.

Available bioinformatic tools can provide valuable insight into E. coli plasmids, but also have important limitations. This work will serve as a guideline for selecting the most appropriate plasmid reconstruction tool for studies focusing on E. coli plasmids in the absence of long-read sequencing data.

category: bioinformatics


title: Galleria mellonella as a novel eco-friendly in vivo approach for the assessment of the toxicity of medicinal plants

abstract: The evaluation of medicinal plants toxicity is a prerequisite prior their usage. The vertebrate models used for this purpose are often the object of ethical consideration. Though invertebrate models including Galleria mellonella have shown their ability to be used to assess various products toxicity, to our knowledge, G. mellonella has never been exploited to determine the toxicity of medicinal plants. In this study, the toxicity of hydroalcoholic and aqueous extracts of seven (7) Cameroonian medicinal plants namely leaves of Cymbopogon citratus (DC.) Stapf, Moringa oleifera Lam and Vernonia amygdalina Delile; barks of Cinchona officinalis and Enantia chloranta Oliv; barks and seeds of Garcinia lucida Vesque and leaves and seeds of Azadirachta indica (Neem) were evaluated using the larval form of the Greater Wax Moth (Galleria mellonella). The median lethal doses (LD50), 90% lethal doses (LD90) and 100% lethal doses were successfully determined using the spline cubic survival curves and equations from the data obtained on the survival rate of G. mellonella 24h after the injection with the extracts. The LD50 values varied from 3.90 g/kg bw to >166.67 g/kg bw and the pattern of toxicity observed was in accordance with previous investigations on the plant materials concerned. The results obtained in this study suggest that G. mellonella can be used as a sensitive, reliable, and robust eco-friendly model to gauge the toxicity of medicinal plants. Thus, avoid the sacrifice of vertebrate models often used for this purpose to limit ethical concerns.

category: pharmacology and toxicology


title: Automatic decomposition of electrophysiological data into distinct non-sinusoidal oscillatory modes

abstract:
Neurophysiological signals are often noisy, non-sinusoidal, and consist of transient bursts. Extraction and analysis of oscillatory features (such as waveform shape and cross-frequency coupling) in such datasets remains difficult. This limits our understanding of brain dynamics and its functional importance. Here, we develop Iterated Masking Empirical Mode Decomposition (itEMD), a method designed to decompose noisy and transient single channel data into relevant oscillatory modes in a flexible, fully data-driven way without the need for manual tuning. Based on Empirical Mode Decomposition (EMD), this technique can extract single-cycle waveform dynamics through phase-aligned instantaneous frequency. We test our method by extensive simulations across different noise, sparsity, and non-sinusoidality conditions. We find itEMD significantly improves the separation of data into distinct non-sinusoidal oscillatory components and robustly reproduces waveform shape across a wide range of relevant parameters. We further validate the technique on multi-modal, multi-species electrophysiological data. Our itEMD extracts known rat hippocampal theta waveform asymmetry and identifies subject-specific human occipital alpha without any prior assumptions about the frequencies contained in the signal. Notably, it does so with significantly less mode mixing compared to existing EMD-based methods. By reducing mode mixing and simplifying interpretation of EMD results, itEMD will enable new analyses into functional roles of neural signals in behaviour and disease.

New & NoteworthyWe introduce a novel, data-driven method to identify oscillations in neural recordings. This approach is based on Empirical Mode Decomposition and reduces mixing of components, one of its main problems. The technique is validated and compared with existing methods using simulations and real data. We show our method better extracts oscillations and their properties in highly noisy and non-sinusoidal datasets.

Running HeadDecomposition of data into non-sinusoidal oscillatory modes

category: neuroscience


title: The EEG spectral fingerprints of meditation and mind wandering differ between experienced meditators and novices

abstract: Previous literature suggests that meditation training is associated with changes in participants experience during meditation practice. In this study, we assess whether putative differences in the experience of meditation between meditators and non-meditators are reflected in EEG spectral modulations. For this purpose, we recorded electroencephalography (EEG) during rest and two breath focus meditations (with and without experience sampling) in a group of 29 adult participants with more than 3 years of meditation experience and a control group of 29 participants without any meditation experience. Experience sampling in one of the meditation conditions allowed us to disentangle periods of breath focus from mind wandering (i.e. moments of distraction driven by task-irrelevant thoughts) during meditation practice. Overall, meditators reported a greater level of focus and reduced mind wandering during meditation practice than controls. In line with these reports, EEG spectral modulations associated to meditation and mind wandering also differed significantly between meditators and controls. While meditators (but not controls) showed a significant decrease in individual alpha frequency and amplitude and a steeper 1/f slope during meditation relative to rest, controls (but not meditators) showed a relative increase in individual alpha amplitude during mind wandering relative to breath focus periods. Together, our results show that the experience of meditation changes with training and that this is reflected in oscillatory and non-oscillatory components of brain activity.


title: Investigating the Impact of Offer Frame Manipulations On Responders Playing The Ultimatum Game

abstract: The present study was designed to test the impact of frame manipulations on the decision-making of responders playing the ultimatum game. Experiment 1 investigated responders event-related potentials (ERPs) measured in response to the offers as a function of the frame (i.e., negative: "the proposer keeps" versus positive "the proposer offers"). While no difference in acceptation rate was found as a function of the offers frame, electrophysiological results suggest that the stronger negative affective response to the offers in the negative frame (N400) was successfully reappraised by the responders (P600), possibly explaining why the offer frame manipulation did not modulate acceptation rates. No framing effect was found when the ultimatum game was played in its one-shot version (Experiment 2), suggesting that repeated measurements did not affect responders behavior. However, an offer framing effect was found in female (but not in male) responders, when the players cognitive charge was increased using more complex game rules (Experiment 3), presumably reflecting womens greater affective responses to negative outcomes. Taken together, these results suggest that framing manipulations are associated with complex affective and cognitive processes, supporting the cognitive-affective tradeoff model.


title: Parafoveal words can modulate sentence meaning: Electrophysiological evidence from an RSVP-with-flanker task

abstract: During natural reading, readers also take up some visual information from not-yet-fixated words to the right of the current fixation and it is well-established that this parafoveal preview facilitates the subsequent foveal processing of the word. However, the extraction and integration of word meaning from the parafoveal word and its possible influence on the semantic sentence context are controversial. In the current study, we recorded event-related potentials (ERPs) in the RSVP-with-flankers paradigm to test whether and how updates of sentential meaning that are based only on parafoveal information influence the subsequent foveal processing. Using Chinese sentences, the sentence congruency of parafoveal and foveal target words were orthogonally manipulated. In contrast to previous research, we also controlled for potentially confounding effects of parafoveal-to-foveal repetition priming (identity preview effects) on the N400. Crucially, we found that the classic effect of foveal congruency on the N400 component only appeared when the word in preview had been congruent with sentence meaning; in contrast, there was no N400 when the preview word had been incongruent. These results indicate that sentence meaning rapidly adapts to parafoveal preview, which already changes the context for the then fixated word. We also show that a correct parafoveal preview generally attenuates the N400 once a word is fixated, regardless of congruency. Taken together, our findings underline the highly generative and adaptive framework of language comprehension.


title: History of winning and hierarchy landscape influence stress susceptibility in mice

abstract: Social hierarchy formation is strongly evolutionarily conserved. Across species, rank within social hierarchy has large effects on health and behavior. To investigate the relationship between social rank and stress susceptibility, we exposed ranked male and female mice to social and non-social stressors and manipulated social hierarchy position. We found that rank predicts same sex social stress outcomes: dominance in males and females confers resilience while subordination confers susceptibility. Pre-existing rank does not predict non-social stress outcomes in females and weakly does so in males, but rank emerging under stress conditions reveals social interaction deficits in male and female subordinates. Both history of winning and rank of cage mates affect stress susceptibility in males: rising to the top rank through high mobility confers resilience and mice that lose dominance lose stress resilience, though gaining dominance over a subordinate animal does not confer resilience. Overall, we have demonstrated a relationship between social status and stress susceptibility, particularly when taking into account individual history of winning and the overall hierarchy landscape in male and female mice.


title: DACO: Distortion/artefact correction for diffusion MRI data in an integrated framework

abstract: In this paper we propose a registration-based algorithm to correct various distortions or artefacts (DACO) commonly observed in diffusion weighted (DW) magnetic resonance images (MRI). The registration in DACO is proceeded on the basis of a pseudo b0 image, which is synthesized from the anatomical images such as T1-weighted image or T2-weighted image, and a pseudo diffusion MRI (dMRI) data, which is derived from the Gaussian model of diffusion tensor imaging (DTI) or the Hermite model of MAP-MRI. DACO corrects (1) the susceptibility-induced distortions, (2) the intensity inhomogeneity, and (3) the misalignment between the dMRI data and anatomical images by registering the real b0 image to the pseudo b0 image, and corrects (4) the eddy current (EC)-induced distortions and (5) the head motions by registering each of the DW images in the real dMRI data to the corresponding image in the pseudo dMRI data. As the above artefacts interact with each other, DACO models each type of artefact in an integrated framework and estimates these models in an interleaved and iterative manner. The mathematical formulation of the models and the comprehensive estimation procedures are detailed in this paper. The evaluation using the human connectome project data shows that DACO could estimate the model parameters accurately. Furthermore, the evaluation conducted on the real human data acquired from clinical MRI scanners reveals that the method could reduce the artefacts effectively. The DACO method leverages the anatomical image, which is routinely acquired in clinical practice, to correct the artefacts, minimizing the additional acquisitions needed to conduct the algorithm. Therefore, our method is beneficial to most dMRI data, particularly to those without acquiring the field map or blip-up and blip-down images.


title: Ultrastructural readout of in vivo synaptic activity for functional connectomics.

abstract: Large-volume ultrastructural mapping approaches yield detailed circuit wiring diagrams but lack an integrated synaptic activity readout which is essential for functional interpretation of the connectome. Here we resolve this limitation by combining functional synaptic labelling in vivo with focused ion-beam scanning electron microscopy (FIBSEM) and machine learning-based segmentation. Our approach generates high-resolution near-isotropic three-dimensional readouts of activated vesicle pools across large populations of individual synapses in a volume of tissue, opening the way for detailed functional connectomics studies. We apply this method to measure presynaptic activity in an ultrastructural context in synapses activated by sensory input in primary visual cortex in awake head-fixed mice, showing that the numbers of recycling and non-recycling vesicles approximate to a lognormal distribution across a large number of synapses. We also demonstrate that neighbouring boutons of the same axon, which share the same spiking activity, can differ greatly in their presynaptic release probability.


title: Grizzly bear toe amputation due to seasonal overlap with furbearer trapping in southeast British Columbia

abstract: Science and adaptive management form crucial components of the North American model of wildlife management. Under this model, wildlife managers are encouraged to update management approaches when new information arises whose implementation could improve the stewardship and viability of wildlife populations and the well-being of animals. Here we detail a troubling observation of several grizzly bears with amputated toes in southeast British Columbia and assemble evidence to inform management strategies to remedy the issue. During the capture of 59 grizzly bears, we noticed that four individuals (~7%) had amputated toes on one of their front feet. The wounds were all healed and linear in nature. Further opportunistic record collection revealed that similar examples of amputated toes occurred beyond our study area, and that furbearer traps were frequently responsible for toe loss. We found evidence that seasonal overlap between the active season for grizzly bears and the fall trapping seasons for small furbearers with body grip traps and for wolves with foothold traps may explain the issue. Multiple options to reduce or eliminate this issue exist, but have varying degrees of expected efficacy and require differing levels of monitoring. The most certain approach to greatly reduce the issue is to delay the start of the marten trapping season until December 1, when most bears have denned, instead of opening the season on or prior to November 1, when more than 50% of bears are still active. Additionally, innovative solutions, such as narrowing trap entrances to exclude bear feet while still allowing entrance of target furbearers, have the potential to minimize accidental capture of bears, but the effectiveness of these approaches is unknown. Experimental evidence suggested that better anchoring traps was not a viable solution. Solutions that do not involve season changes will require monitoring of efficacy and compliance to ensure success.

category: ecology


title: Considerations for furbearer trapping regulations to prevent grizzly bear toe amputation and injury


title: Hurricane-induced population decrease in a Critically Endangered long-lived reptile

abstract: Catastrophic events, like hurricanes, bring lethal conditions that can have population-altering effects. The threatened Caribbean dry forest occurs in a region known for its high-intensity hurricane seasons and high species endemism, highlighting the necessity to better understand hurricane impacts as fragmentation and clearing of natural habitat continues. However, such studies remain rare, and for reptiles are mostly restricted to Anolis. Here we used single-season occupancy modeling to infer the impact of the intense 2017 Atlantic hurricane season on the critically endangered Lesser Antillean Iguana, Iguana delicatissima. We surveyed 30 transects across eight habitats on St. Eustatius during 2017-2019, which resulted in 344 individual surveys and 98 iguana observations. Analyses of abundance and site occupancy indicated both measures for 2018 and 2019 were strongly reduced compared to the pre-hurricane 2017 state. Iguanas at higher elevations were affected more profoundly, likely due to higher wind speeds, tree damage and extensive defoliation. Overall, our results indicate a decrease in population estimates (23.3-26.5%) and abundance (22-23.8%) for 2018 and 2019, and a 75% reduction in the number of opportunistic sightings of tagged iguanas between 2017-2018. As only small and isolated I. delicatissima populations remain, our study further demonstrates their vulnerability to stochastic events. Considering the frequency and intensity of hurricanes are projected to increase, our results stress the urgent need for population-increasing conservation actions in order to secure the long-term survival of I. delicatissima throughout its range.


title: Predictability and parallelism in the contemporary evolution of hybrid genomes

abstract: Hybridization between species is widespread across the tree of life. As a result, many species, including our own, harbor regions of their genome derived from hybridization. Despite the recognition that this process is widespread, we understand little about how the genome stabilizes following hybridization, and whether the mechanisms driving this stabilization tend to be shared across species. Here, we dissect the drivers of variation in local ancestry across the genome in replicated hybridization events between two species pairs of swordtail fish: Xiphophorus birchmanni x X. cortezi and X. birchmanni x X. malinche. We find unexpectedly high levels of repeatability in local ancestry across the two types of hybrid populations. This repeatability is attributable in part to the fact that the recombination landscape and locations of functionally important elements play a major role in driving variation in local ancestry in both types of hybrid populations. Beyond these broad scale patterns, we identify dozens of regions of the genome where minor parent ancestry is unusually low or high across species pairs. Analysis of these regions points to shared sites under selection across species pairs, and in some cases, shared mechanisms of selection. We show that one such region is a previously unknown hybrid incompatibility that is shared across X. birchmanni x X. cortezi and X. birchmanni x X. malinche hybrid populations.

category: evolutionary biology


title: Proteolytic Activation of Human-specific Olduvai Domains by the Furin Protease

abstract: Olduvai protein domains (formerly DUF1220) show the greatest human-specific increase in copy number of any coding region in the genome and are highly correlated with human brain evolution and cognitive disease. The majority of human copies are found within four NBPF genes organized in a variable number of a tandemly arranged three-domain blocks called Olduvai triplets. Here we show that these human-specific Olduvai domains are posttranslationally processed by the furin protease, with a cleavage site occurring once at each triplet. These findings suggest that all expanded human-specific NBPF genes encode proproteins consisting of many independent Olduvai triplet proteins which are activated by furin processing. The exceptional correlation of Olduvai copy number and brain size taken together with our new furin data, indicates the ultimate target of selection was a rapid increase in dosage of autonomously functioning Olduvai triplet proteins, and that these proteins are the primary active agent underlying Olduvais role in human brain expansion.


title: MONI: A Pangenomics Index for Finding MEMs

abstract:
Recently, Gagie et al. proposed a version of the FM-index, called the r-index, that can store thousands of human genomes on a commodity computer. Then Kuhnle et al. showed how to build the r-index efficiently via a technique called prefix-free parsing (PFP) and demonstrated its effectiveness for exact pattern matching. Exact pattern matching can be leveraged to support approximate pattern matching but the r-index itself cannot support efficiently popular and important queries such as finding maximal exact matches (MEMs). To address this shortcoming, Bannai et al. introduced the concept of thresholds, and showed that storing them together with the r-index enables efficient MEM finding -- but they did not say how to find those thresholds. We present a novel algorithm that applies PFP to build the r-index and find the thresholds simultaneously and in linear time and space with respect to the size of the prefix-free parse. Our implementation called MONI can rapidly find MEMs between reads and large sequence collections of highly repetitive sequences. Compared to other read aligners - PuffAligner, Bowtie2, BWA-MEM, and CHIC - MONI used 2-11 times less memory and was 2-32 times faster for index construction. Moreover, MONI was less than one thousandth the size of competing indexes for large collections of human chromosomes. Thus, MONI represents a major advance in our ability to perform MEM finding against very large collections of related references.

AvailabilityMONI is publicly available at https://github.com/maxrossi91/moni.


title: MSNovelist: De novo structure generation from mass spectra

abstract: Structural elucidation of small molecules de novo from mass spectra is a longstanding, yet unsolved problem. Current methods rely on finding some similarity with spectra of known compounds deposited in spectral libraries, but do not solve the problem of predicting structures for novel or poorly represented compound classes. We present MSNovelist that combines fingerprint prediction with an encoder-decoder neural network to generate structures de novo from fragment spectra. In evaluation, MSNovelist correctly reproduced 61% of database annotations for a GNPS reference dataset. In a bryophyte MS2 dataset, our de novo structure prediction substantially outscored the best database candidate for seven features, and a potential novel natural product with a flavonoid core was identified. MSNovelist allows predicting structures solely from MS2 data, and is therefore ideally suited to complement library-based annotation in the case of poorly represented analyte classes and novel compounds.


title: DynaTMT: A user-friendly tool to process combined SILAC/TMT data

abstract: The measurement of protein dynamics by proteomics to study cell remodeling has seen increased attention over the last years. This development is largely driven by a number of technological advances in proteomics methods. Pulsed stable isotope labeling in cell culture (SILAC) combined with tandem mass tag (TMT) labeling has evolved as a gold standard for profiling protein synthesis and degradation. While the experimental setup is similar to typical proteomics experiments, the data analysis proves more difficult: After peptide identification through search engines, data extraction requires either custom scripted pipelines or tedious manual table manipulations to extract the TMT-labeled heavy and light peaks of interest. To overcome this limitation, which deters researchers from using protein dynamic proteomics, we developed a user-friendly, browser-based application that allows easy and reproducible data analysis without the need for scripting experience. In addition, we provide a python package that can be implemented in established data analysis pipelines. We anticipate that this tool will ease data analysis and spark further research aimed at monitoring protein translation and degradation by proteomics.


title: β-adrenergic stimulation synchronizes a broad spectrum of action potential firing rates of cardiac pacemaker cells towards a higher population average

abstract: The heartbeat is initiated by pacemaker cells residing in the sinoatrial node (SAN). SAN cells generate spontaneous action potentials (APs), i.e. normal automaticity. The sympathetic nervous system increases heart rate commensurate with cardiac output demand via stimulation of SAN {beta}-adrenergic receptors ({beta}AR). While SAN cells reportedly represent a highly heterogeneous cell population, the current dogma is that in response to {beta}AR stimulation all cells increase their spontaneous AP firing rate in a similar fashion. The aim of the present study was to investigate cell-to-cell variability in the responses of a large population of SAN cells. We measured {beta}AR responses among 166 single SAN cells isolated from 33 guinea pig hearts. In contrast to the current dogma, the SAN cell responses to {beta}AR stimulation substantially varied. In each cell, changes in AP cycle length highly correlated (R2=0.97) with the AP cycle length before {beta}AR stimulation. While, as expected, on average the cells increased their pacemaker rate, greater responses were observed in cells with slower basal rates, and vice versa, cells with higher basal rates showed smaller responses, no responses, or even decreased their rate. Thus, {beta}AR stimulation synchronizes the operation of the SAN cell population towards a higher average rate, rather than uniformly shifting the rate in each cell, creating a new paradigm of {beta}AR-driven fight-or-flight response among individual pacemaker cells.

category: biophysics


title: Breast invasive ductal carcinoma classification on whole slide images with weakly-supervised and transfer learning

abstract: AO_SCPLOWBSTRACTC_SCPLOWInvasive ductal carcinoma (IDC) is the most common form of breast cancer. For the non-operative diagnosis of breast carcinoma, core needle biopsy has been widely used in recent years which allows evaluation of both cytologic and tissue architectural features; so that it can provide a definitive diagnosis between IDC and benign lesion (e.g., fibroadenoma). Histopathological diagnosis based on core needle biopsy specimens is currently the cost effective method; therefore, it is an area that could benefit from AI-based tools to aid pathologists in their pathological diagnosis workflows. In this paper, we trained an Invasive Ductal Carcinoma (IDC) Whole Slide Image (WSI) classification model using transfer learning and weakly-supervised learning. We evaluated the model on a core needle biopsy (n=522) test set as well as three surgical test sets (n=1129) obtaining ROC AUCs in the range of 0.95-0.98.

category: pathology


title: BAM15 treats mouse sepsis and sepsis-AKI, linking circulating mitochondrial DNA and tubule reactive oxygen species

abstract: The pathogenesis of sepsis is complex and heterogeneous; hence, a precision medicine strategy may be required. Acute kidney injury (AKI) following sepsis portends higher mortality. Overproduction of mitochondrial reactive oxygen species (mtROS) is a potential mediator of sepsis and sepsis-induced AKI. BAM15 is a chemical uncoupler that dissipates the mitochondrial proton gradient without generating mtROS, and improves experimental renal ischemic injury. We injected BAM15 into mice at 0 or 6 hours after cecal ligation and puncture (CLP) treated with fluids and antibiotics. BAM15 reduced mortality, even when started at 6 hours, when mice were ill, and reduced kidney damage but did not affect other organs. Serial plasma and urinary levels of mitochondrial DNA (mtDNA) were increased following CLP, and decreased after BAM15 (at 0 and at 6 hours). In vitro BAM15 prevented mtROS overproduction and mtDNA release from septic kidney tubule cells; mtROS generation correlated with mtDNA release. BAM15 also promotes mitochondrial biogenesis signaling. We conclude that BAM15 is an effective preventive and therapeutic candidate in experimental sepsis, and that BAM15 and mtDNA are mechanistically linked via mtROS, which may form a drug-companion diagnostic pair to improve precision medicine approaches to diagnosing and treating clinical sepsis.


title: Mapping circuit dynamics during function and dysfunction

abstract: Neural circuits can generate many spike patterns, but only some are functional. The study of how circuits generate and maintain functional dynamics is hindered by a poverty of description of circuit dynamics across functional and dysfunctional states. For example, although the regular oscillation of a central pattern generator is well characterized by its frequency and the phase relationships between its neurons, these metrics are ineffective descriptors of the irregular and aperiodic dynamics that circuits can generate under perturbation or in disease states. By recording the circuit dynamics of the well-studied pyloric circuit in C. borealis, we used statistical features of spike times from neurons in the circuit to visualize the spike patterns generated by this circuit under a variety of conditions. This unsupervised approach captures both the variability of functional rhythms and the diversity of atypical dynamics in a single map. Clusters in the map identify qualitatively different spike patterns hinting at different dynamical states in the circuit. State probability and the statistics of the transitions between states varied with environmental perturbations, removal of descending neuromodulation, and the addition of exogenous neuromodulators. This analysis reveals strong mechanistically interpretable links between complex changes in the collective behavior of a neural circuit and specific experimental manipulations, and can constrain hypotheses of how circuits generate functional dynamics despite variability in circuit architecture and environmental perturbations.


title: Disentangling Age and Schooling Effects on Inhibitory Control Development: An fNIRS Investigation

abstract:
Children show marked improvements in executive functioning (EF) between 4 and 7 years of age. In many societies, this time period coincides with the start of formal school education, in which children are required to follow rules in a structured environment, drawing heavily on EF processes such as inhibitory control. This study aimed to investigate the longitudinal development of two aspects of inhibitory control, namely response inhibition and response monitoring and their neural correlates. Specifically, we examined how their longitudinal development may differ by schooling experience, and their potential significance in predicting academic outcomes. Longitudinal data was collected in two groups of children at their homes. At T1, all children were roughly 4.5 years of age and neither group had attended formal schooling. One year later at T2, one group (P1, n = 40) had completed one full year of schooling while the other group (KG, n = 40) had stayed in kindergarten. Behavioural and brain activation data (measured with functional near-infrared spectroscopy, fNIRS) in response to a Go/No-Go task and measures of academic achievement were collected. We found that P1 children, compared to KG children, showed a greater change over time in activation related to response monitoring in the bilateral frontal cortex. The change in left frontal activation difference showed a small positive association with mathematical ability, suggesting certain functional relevance of response monitoring for academic performance. Overall, the school environment is important in shaping the development of the neural network underlying monitoring of one owns performance.

Research HighlightsO_LIUsing a school cut-off design, we collected longitudinal home assessments of two aspects of inhibitory control, namely response inhibition and response monitoring, and their neural correlates.
C_LIO_LIFor response monitoring, P1 children showed a greater difference over time in activation between correct and incorrect responses in the bilateral frontal cortex.
C_LIO_LIThe left frontal activation difference in P1 children showed a small association with mathematical ability, suggesting some functional relevance of response monitoring for academic performance.
C_LIO_LIThe school environment plays an important role in shaping the development of the neural network underlying monitoring of one owns performance.
C_LI


title: Spontaneous pauses in firing of external pallidum neurons are associated with exploratory behavior

abstract: Spontaneous pauses in firing are the hallmark of external pallidum (GPe) neurons. However, their role in the basal ganglia (BG) network remains unknown. Pupil size and saccades have been linked to attention and exploration. Here, we show that pauses are associated with dilated pupils and increased saccade rate. Our results suggest that pauses are a powerful mechanism by which the GPe may influence BG downstream structures and affect behavioral output.


title: Dynamics in Fip1 regulate eukaryotic mRNA 3'-end processing

abstract: Cleavage and polyadenylation factor (CPF/CPSF) is a multiprotein complex essential for mRNA 3-end processing in eukaryotes. It contains an endonuclease that cleaves pre-mRNAs, and a polymerase that adds a poly(A) tail onto the cleaved 3-end. Several CPF subunits, including Fip1, contain intrinsically-disordered regions (IDRs). IDRs within multiprotein complexes can be flexible, or can become ordered upon interaction with binding partners. Here, we show that yeast Fip1 anchors the poly(A) polymerase Pap1 onto CPF via an interaction with zinc finger 4 of another CPF subunit, Yth1. We also reconstitute a fully recombinant 850-kDa CPF. By incorporating selectively-labelled Fip1 into recombinant CPF, we could study the dynamics of this single protein within the megadalton complex using nuclear magnetic resonance spectroscopy (NMR). This reveals that a Fip1 IDR that connects the Yth1- and Pap1-binding sites remains highly dynamic within CPF. Together, our data suggest that Fip1 dynamics mediate conformational transitions within the 3-end processing machinery to coordinate cleavage and polyadenylation.

category: molecular biology


title: Oxidative phosphorylation safeguards pluripotency via UDP-N-acetylglucosamine

abstract:
The roles of mitochondrial respiration in pluripotency remain largely unknown. We show here that mouse ESC mitochondria possess superior respiration capacity compared to somatic cell mitochondria, and oxidative phosphorylation (OXPHOS) generates the majority of cellular ATP in ESCs. Inhibition of OXPHOS results in extensive pluripotency and metabolic gene expression reprogram, leading to disruption of self-renewal and pluripotency. Metabolomics profiling identifies UDP-N-acetylglucosamine (UDP-GlcNAc) as one of the most significantly decreased metabolites in response to OXPHOS inhibition. The loss of ESC identity induced by OXPHOS inhibition can be ameliorated by directly adding GlcNAc both in vitro and in vivo. This work demonstrates that mitochondrial respiration, but not glycolysis, produces the majority of ATP in ESCs, and uncovers a novel mechanism whereby mitochondrial respiration is coupled with the hexosamine biosynthesis pathway to generate UDP-GlcNAc for ESC identity maintenance.

SIGNIFICANCEOxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for generating ATP in mammalian cells. It is widely assumed that somatic cells utilize OXPHOS, whereas embryonic stem cells (ESCs) utilize glycolysis with low mitochondrial respiration rates even under aerobic conditions. However, the relative contribution of OXPHOS and glycolysis to ATP generation in ESCs, and the role of mitochondrial respiration in regulating ESC identity, have remained unclear. In this study, Cao et al demonstrate that mouse ESC mitochondria have a significantly higher respiration capacity than somatic cell mitochondria. Oxidative phosphorylation produces the majority of cellular ATP in mESCs and is coupled with the hexosamine biosynthesis pathway to generate UDP-GlcNAc for pluripotency maintenance. These findings define the function and mechanism of OXPHOS in regulating pluripotency, and challenge the traditional concept that mESCs rely on glycolysis over OXPHOS for their major supply of energy.

HIGHLIGHTSO_LIESC mitochondria have a significantly higher respiration capability than somatic cell mitochondria
C_LIO_LIOXPHOS, but not glycolysis, produces the majority of cellular ATP in ESCs
C_LIO_LIOXPHOS inhibition induces a decrease in O-GlcNAcylation and the expression of pluripotency genes in blastocysts that can be partially rescued by adding GlcNAc
C_LIO_LIOXPHOS is coupled with the hexosamine biosynthesis pathway for UDP-GlcNAc biosynthesis to maintain ESC identity
C_LI

category: cell biology


title: Nucleolar NOC1 controls protein synthesis and cell competition in Drosophila

abstract:
NOC1 is a nucleolar protein necessary in yeast for both transport and maturation of ribosomal subunits. Here we show that in Drosophila NOC1 is essential for the correct animal development, and that its ubiquitous downregulation results in small larvae with reduced polysome abundance and decreased protein synthesis. NOC1 expression in multiple organs, such as the prothoracic gland and the fat body, is necessary for proper organ functioning. Reduction of NOC1 in clones from the imaginal discs results in small clones with cells that die by apoptosis, an event that is partially rescued using a M/+ background, suggesting that reduction of NOC1 causes the cells to acquire a loser state. This event was supported also by an increase in the transcription of Xrp1 and by activation of the pro-apoptotic eiger-JNK pathway, resulting in the upregulation of DILP8 as an indication of cellular damage. Here, we show that Drosophila NOC1 is important in the control of pre-rRNAs maturation and essential step in the regulation of ribosome biogenesis and its downregulation results in defects in growth and in cell competition, highlighting its novel function in this field.

summary statementNOC1 is a nucleolar protein necessary for protein synthesis and ribosomal assembling. Its modulation induces cell competition and affects animal growth.


title: Reduction of nucleolar NOC1 accumulates pre-rRNAs and induces Xrp1 affecting growth and resulting in cell competition in Drosophila


title: Distinct mitochondrial remodeling during early cardiomyocyte development in a human-based stem cell model

abstract:
Given the considerable interest in using stem cells for modelling and treating disease, it is essential to understand what regulates self-renewal and differentiation. Remodeling of mitochondria and metabolism, with the shift from glycolysis to oxidative phosphorylation (OXPHOS), play a fundamental role in maintaining pluripotency and stem cell fate. It has been suggested that metabolic  switch from glycolysis to OXPHOS is germ-layer specific as during early ectoderm commitment, glycolysis remains active while during the transition to mesoderm and endoderm lineages, it is downregulated. How mitochondria adapt during these metabolic changes and whether mitochondria remodeling is tissue specific remains unclear. Here we address the question of mitochondrial adaption by examining the differentiation of human pluripotent stem cells to cardiac progenitors and further to functional cardiomyocytes. Contrary to recent findings in neuronal differentiation, we found that mitochondrial content decreases continuously during mesoderm differentiation, despite clear mitochondrial remodeling giving increased mitochondrial activity and higher levels of ATP-linked respiration. Thus, our work both highlights similarities in mitochondrial remodeling during the transition from pluripotent to multipotent state in ectodermal and mesodermal lineages, while at the same time demonstrating cell-lineage-specific adaptions upon further differentiation. Our results improve understanding of how mitochondria remodeling and the metabolism interact during differentiation and show that it is erroneous to assume that increased OXPHOS activity during differentiation requires a simultaneous expansion of mitochondrial content.

Summary statementWe found that mitochondrial content decreases continuously during mesoderm differentiation, despite clear mitochondrial remodeling giving increased mitochondrial activity and higher levels of ATP-linked respiration during mesoderm differentiation.


title: Dynamic Apical-Basal Enrichment of the F-Actin during Cytokinesis in Arabidopsis Cells Embedded in Their Tissues

abstract: During the life cycle of any multicellular organism, cell division contributes to the proliferation of the cell in the tissues as well as the generation of specialized cells, both necessary to form a functional organism. Therefore, the mechanisms of cell division need to be tightly regulated, as malfunctions in their control can lead to tumor formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis is key for morphogenesis. In the green lineage, cell division is executed in radically different manners than animals, with the appearance of new structures (the preprophase band (PPB), cytokinetic the cell plate and phragmoplast), and the disappearance of ancestral mechanisms (cleavage, centrosomes). While F-actin and microtubules closely co-exist to allow the orientation and the progression of the plant cell division, recent studies mainly focused on the microtubules involvement in this key process. Here, we used our recently developed root tracking system to follow actin dynamics in dividing Arabidopsis meristematic root cells. In this study, we imaged in time and space the fluorescent-tagged F-actin reporter Lifeact together with cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulations in the phragmoplasts, we observed and quantified a dynamic apical-basal enrichment of the F-actin during cytokinesis. The role and the possible actors responsible for F-actin dynamics during cytokinesis are discussed.


title: Myeloid-to-mesenchymal NGF-p75 signaling coordinates skeletal cell migration during repair

abstract: Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to re-innervation and osteogenesis is unknown. Here, we found that NGF is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to impact their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.


title: NGF-p75 signaling coordinates skeletal cell migration during bone repair


title: S18-phosphorylation of USP7 regulates interaction with TCEAL4 that defines specific complexes and potentially distinct functions.

abstract: USP7 is a nuclear deubiquitylase (DUB) with multiple cancer-associated substrates for which selective inhibitors are available, yet it remains unclear how the pleiotropic effects of USP7 are regulated. We report that S18-phosphorylation does not influence USP7 catalytic activity but instead confers selectivity for protein interactions. In particular, non-S18-phosphorylatable USP7 preferentially interacts with USP11 and TRIM27, together with TCEAL1 and TCEAL4 whose functions are unknown. Intriguingly, USP7 can interact with two cellular forms of TCEAL4, but USP11 only interacts with a lower abundance K142 mono-ubiquitylated form (TCEAL4-Ub), which can scaffold a complex containing both DUBs. Whilst USP11 and TCEAL4 are both USP7 substrates, TCEAL4-Ub levels are specifically maintained by USP11 with their levels positively correlated in cancer cell lines. Together these data illustrate how USP7 phosphorylation and TCEAL4 ubiquitylation combine to define distinct USP7 complexes. As TCEAL4 itself interacts with proteins involved in ubiquitylation and various forms of DNA regulation, these complexes may direct cellular activity of USP7.


title: Bayesian estimation of population size and overlap from random subsamples

abstract: Counting the number of species, items, or genes that are shared between two sets is a simple calculation when sampling is complete. However, when only partial samples are available, quantifying the overlap between two sets becomes an estimation problem. Furthermore, to calculate normalized measures of {beta}-diversity, such as the Jaccard and Sorenson-Dice indices, one must also estimate the total sizes of the sets being compared. Previous efforts to address these problems have assumed knowledge of total population sizes and then used Bayesian methods to produce unbiased estimates with quantified uncertainty. Here, we address populations of unknown size and show that this produces systematically better estimates--both in terms of central estimates and quantification of uncertainty in those estimates. We further show how to use species count data to refine estimates of population size in a Bayesian joint model of populations and overlap.


title: Molecular adaptation to folivory and the conservation implications for Madagascar's lemurs

abstract: Folivory evolved independently at least three times over the last 40 million years among Madagascars lemurs. Many extant lemuriform folivores exist in sympatry in Madagascars remaining forests. These species avoid feeding competition by adopting different dietary strategies within folivory, reflected in behavioral, morphological, and microbiota diversity across species. These conditions make lemurs an ideal study system for understanding adaptation to leaf-eating. Most folivorous lemurs are also highly endangered. The significance of folivory for conservation outlook is complex. Though generalist folivores may be relatively well equipped to survive habitat disturbance, specialist folivores occupying narrow dietary niches may be less resilient. Characterizing the genetic bases of adaptation to folivory across species and lineages can provide insights into their differential physiology and potential to resist habitat change. We recently reported accelerated genetic change in RNASE1, a gene encoding an enzyme (RNase 1) involved in molecular adaptation in mammalian folivores, including various monkeys and sifakas (genus Propithecus; family Indriidae). Here, we sought to assess whether other lemurs, including phylogenetically and ecologically diverse folivores, might show parallel adaptive change in RNASE1 that could underlie a capacity for efficient folivory. We characterized RNASE1 in 21 lemur species representing all five families and members of the three extant folivorous lineages: 1) bamboo lemurs (family Lemuridae), 2) sportive lemurs (family Lepilemuridae), and 3) indriids (family Indriidae). We found pervasive sequence change in RNASE1 across all indriids, a dN/dS value > 3 in this clade, and evidence for shared change in isoelectric point, indicating altered enzymatic function. Sportive and bamboo lemurs, in contrast, showed more modest sequence change. The greater change in indriids may reflect a shared strategy emphasizing complex gut morphology and microbiota to facilitate folivory. This case study illustrates how genetic analysis may reveal differences in functional traits that could influence species ecology and, in turn, their resilience to habitat change. Moreover, our results support the contention that not all primate folivores are built the same and highlight the need to avoid generalizations about dietary guild in considering conservation outlook, particularly in lemurs where such diversity in folivory has probably led to extensive specialization via niche partitioning.


title: Biomechanical study of cellulose scaffolds for bone tissue engineering in vivo and in vitro.

abstract: Plant-derived cellulose biomaterials have recently been utilized in several tissue engineering applications. These naturally-derived cellulose scaffolds have been shown to be highly biocompatible in vivo, possess structural features of relevance to several tissues, and support mammalian cell invasion and proliferation. Recent work utilizing decellularized apple hypanthium tissue has shown that it possesses a pore size similar to trabecular bone and can successfully host osteogenic differentiation. In the present study, we further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) and analyzed their mechanical properties in vitro and in vivo. MC3T3-E1 pre-osteoblasts were seeded in cellulose scaffolds. Following chemically-induced osteogenic differentiation, scaffolds were evaluated for mineralization and for their mechanical properties. Alkaline phosphatase and Alizarin Red staining confirmed the osteogenic potential of the scaffolds. Histological analysis of the constructs revealed cell invasion and mineralization throughout the constructs. Furthermore, scanning electron microscopy demonstrated the presence of mineral aggregates on the scaffolds after culture in differentiation medium, and energy-dispersive spectroscopy confirmed the presence of phosphate and calcium. However, although the Youngs modulus significantly increased after cell differentiation, it remained lower than that of healthy bone tissue. Interestingly, mechanical assessment of acellular scaffolds implanted in rat calvaria defects for 8 weeks revealed that the force required to push out the scaffolds from the surrounding bone was similar to that of native calvarial bone. In addition, cell infiltration and extracellular matrix deposition were visible within the implanted scaffolds. Overall, our results confirm that plant-derived cellulose is a promising candidate for BTE applications. However, the discrepancy in mechanical properties between the mineralized scaffolds and healthy bone tissue may limit their use to low load-bearing applications. Further structural re-engineering and optimization to improve the mechanical properties may be required for load-bearing applications.

category: bioengineering


title: Multi-modal locomotor costs explain sexual size but not shape dimorphism in a leaf-mimicking insect

abstract:
In most arthropods, adult females are larger than males, and male competition is a race to quickly locate and mate with scattered females (scramble competition polygyny). In this context, smaller males may be favored due to more efficient locomotion leading to higher mobility during mate searching while larger males may benefit from increased speed and higher survivorship. Understanding how body size affects different aspects of the locomotor performance of males is therefore essential to shed light on the evolution of this widespread mating system. Using a combination of empirical measures of flight performance and substrate adhesion, and modelling of body aerodynamics, we show that large body size impairs both flight and landing (attachment) performance in male leaf insects (Phyllium philippinicum), a species where relatively small and skinny males fly through the canopy in search of large sedentary females. Smaller males were more agile in the air, ascended more rapidly during flight, and had a lower risk of detaching from the substrates on which they walk and land. Our models revealed variation in body shape affected body lift and drag, but tradeoffs with weight meant that effects were negligible, suggesting that flight costs do not explain the evolution of strong sexual dimorphism in body shape in this species.

Graphical abstract

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title: Comparative Transcriptomics of Tropical Woody Plants Supports Fast and Furious Strategy along the Leaf Economics Spectrum in Lianas.

abstract: Lianas, climbing woody plants, influence the structure and function of tropical forests. Climbing traits have evolved multiple times, including ancestral groups such as gymnosperms and pteridophytes, but the genetic basis of the liana strategy is largely unknown. Here, we use a comparative transcriptomic approach for 47 tropical plant species, including ten lianas of diverse taxonomic origins, to identify genes that are consistently expressed or downregulated only in lianas. Our comparative analysis of full-length transcripts enabled the identification of a core interactomic network common to lianas. Sets of transcripts identified from our analysis reveal features related to functional traits pertinent to leaf economics spectrum in lianas, include upregulation of genes controlling epidermal cuticular properties, cell wall remodeling, carbon concentrating mechanism, cell cycle progression, DNA repair and a large suit of downregulated transcription factors and enzymes involved in ABA-mediated stress response as well as lignin and suberin synthesis. All together, these genes are known to be significant in shaping plant morphologies through responses such as gravitropism, phyllotaxy and shade avoidance.

category: plant biology


title: Multivalent designed proteins protect against SARS-CoV-2 variants of concern

abstract:
Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness.

One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

category: synthetic biology


title: The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior

abstract:
We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying reduced methamphetamine behavioral sensitivity. Mice with a heterozygous frameshift deletion in the first coding exon of Hnrnph1 showed reduced methamphetamine-induced dopamine release and behaviors. To inform the mechanism linking hnRNP H dysfunction with reduced methamphetamine-induced dopamine release and behavior, we surveyed the RNA targetome of hnRNP H via cross-linking immunoprecipitation coupled with RNA-sequencing in striatal tissue at baseline and at 30 min post-methamphetamine (2 mg/kg, i.p.). Methamphetamine induced opposite changes in RNA-binding targets of hnRNP H in Hnrnph1 mutants versus wild-types, including 3UTR targets in mRNAs enriched for synaptic proteins involved in dopamine release and excitatory synaptic plasticity. Targetome, transcriptome, and spliceome analyses triangulated on a methamphetamine-induced upregulation of Cacna2d2 transcript and decreased 3UTR usage in hyposensitive Hnrnph1 mutants. Our study identifies a dynamic methamphetamine-induced RNA targetome of hnRNP H that has the potential to rapidly regulate gene expression, synaptic transmission, plasticity, and behavior.

HIGHLIGHTSO_LIThe hnRNP H RNA targetome contains an enrichment of G-rich binding motifs within introns
C_LIO_LIThe hnRNP H RNA targetome reveals gene networks enriched for synaptic function
C_LIO_LIHnrnph1 mutation and methamphetamine treatment induce changes in RNA-binding targets of hnRNP H
C_LIO_LITargetome, transcriptome, and spliceome analysis triangulated on Cacna2d2 as a methamphetamine-induced regulatory target with potential physiological relevance to synaptic transmission and behavior
C_LI


title: A single full-length VAR2CSA ectodomain variant purifies broadly neutralizing antibodies against placental malaria isolates

abstract:
Placental malaria (PM) is a deadly syndrome most frequent and severe in first pregnancies. PM results from accumulation of Plasmodium falciparum (Pf)-infected erythrocytes (IE) that express the surface antigen VAR2CSA and bind to chondroitin sulfate A (CSA) in the placenta. Women become PM-resistant over successive pregnancies as they develop anti-adhesion and anti-VAR2CSA antibodies, supporting VAR2CSA as the leading PM-vaccine candidate. However, the first VAR2CSA subunit vaccines failed to induce broadly neutralizing antibody and it is still unclear whether naturally acquired protective antibodies target variant or conserved epitopes. This is crucial to determine whether effective vaccines will require incorporation of many or only a single VAR2CSA allele.

Here, IgG from multigravidae was sequentially purified on five full-length VAR2CSA ectodomain variants, thereby depleting IgG reactivity to each. The five VAR2CSA variants purified [~]0.7% of total IgG and yielded both strain-transcending and strain-specific reactivity to VAR2CSA and IE-surface antigen. IgG purified on the first VAR2CSA antigen displayed broad cross-reactivity to both recombinant and native VAR2CSA variants, and inhibited binding of all isolates to CSA. IgG remaining after depletion on all variants showed significantly reduced binding-inhibition activity compared to initial total IgG.

These findings demonstrate that a single VAR2CSA ectodomain variant displays neutralizing epitopes shared by multiple parasites, including maternal isolates, and suggest that a broadly effective PM-vaccine can be achieved with a limited number of VAR2CSA variants.

category: immunology


title: Defining the Immune Responses for SARS-CoV-2-Human Macrophage Interactions

abstract: Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end organ malfunctions. All follow an abortive viral infection. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding consequent end-organ tissue damage.


title: Protease activities of vaginal Porphyromonas species disrupt coagulation and extracellular matrix in the cervicovaginal niche

abstract:
Porphyromonas asaccahrolytica and Porphyromonas uenonis are frequently isolated from the human vagina and are linked to bacterial vaginosis and preterm labour. However, little is known about the pathogenesis mechanisms of these bacteria. The related oral opportunistic pathogen, Porphyromonas gingivalis, is comparatively well-studied and known to secrete numerous extracellular matrix-targeting proteases. Among these are the gingipain family of cysteine proteases that drive periodontal disease progression and hematogenic transmission to the placenta. Given their phylogenetic relatedness, we hypothesized that vaginal Porphyromonas species possess gingipain-like protease activity targeting host extracellular matrix in the female reproductive tract. In this study, we demonstrate that vaginal Porphyromonas species degrade type I collagen (cervix), type IV collagen (chorioamnion/placenta), and fibrinogen, but not through the activity of gingipain orthologs. Bioinformatic queries identified 5 candidate collagenases in each species, including serine, cysteine and metalloproteases, with signal peptides directing them to the extracellular environment. Inhibition assays revealed both species secrete metalloproteases that degrade collagen and casein, while P. asaccharolytica also secretes a metalloprotease that degrades fibrinogen. Phylogenetic analysis of the predicted collagen-degrading metalloprotease revealed an orthologous relationship with the P. gingivalis endopeptidase PepO. Cloning and expression of P. asaccharolytica PepO confirmed this proteins collagenase and caseinase activities, which have not previously been attributed to PepO homologs in other bacteria. Altogether, this description of the first known virulence factor in Porphyromonas species colonizing the human vagina sheds light on their potential to alter the structural integrity and homeostasis of reproductive tissues.

ImportancePorphyromonas species are common inhabitants of the vaginal microbiome, but their presence has been liked to adverse health outcomes for women, including bacterial vaginosis and preterm birth. We determined that P. asaccharolytica and P. uenonis secrete broad-acting proteases capable of freely diffusing within the cervicovaginal niche and degrading important components of host tissues, namely the extracellular matrix. We show that secreted Porphyromonas proteases degrade collagens that are enriched within the cervix (type I) and chorioamniotic membranes (type IV). Furthermore, these Porphyromonas proteases can also degrade fibrinogen and inhibit clot formation. These activities can be partially attributed to a metalloprotease that exhibits broad-acting protease activity and is distantly related to the P. gingivalis endopeptidase PepO. This initial characterization of virulence activities in vaginal Porphyromonas species highlights their potential to harm human pregnancy through clotting disruption, fetal membrane weakening, and premature cervical remodeling.

category: microbiology


title: Genome-wide association study reveals putative bacterial risk factors for cavitary Mycobacterium avium complex lung disease

abstract:
Mycobacterium avium complex (MAC) lung disease is a slowly progressive disease, and its increasing incidence has garnered increased research interests. Cavitary MAC lung disease is associated with a higher mortality rate. Though genetic studies have unraveled the human risk factors, the role of microbial factors on pathogenesis behind the disease remains elusive. In this study, M. avium isolates were collected from sputum specimens of 109 distinct Japanese patients with or without a cavity (60 with a cavity and 49 without cavity) in a hospital located in Osaka prefecture. M. avium genomes were sequenced and searched for DNA motifs associated with cavity formation using a bacterial GWAS. Excluding known macrolide resistance mutations; cavity formation was found to be primarily associated with variants of cytochrome P450 of the CYP139 family, type I polyketide synthase Pks13, and the promoter region of an operon encoding membrane-anchored protease FtsH and folate synthesis pathway enzymes. Cavity risk variants at these three loci were frequent in the MahEastAsia2 lineage among the six lineages detected in M. avium global populations. Furthermore, the study demonstrated a correlation between the cavity risk promoter variant and increased sulfamethoxazole/trimethoprim resistance. Together, these findings suggest that natural variation in the biosynthesis and maintenance processes of M. avium membrane components influences the disease type of MAC lung disease. Although further validation is needed, the bacterial genetic markers listed in the present study could contribute to prognosis prediction based on bacterial genotyping and help develop treatment strategies in the future.

IMPORTANCENontuberculous mycobacterial lung disease is of great concern in countries with an increasingly aging population. The disease types can largely be classified into non-cavitary nodular bronchiectasis and cavitary diseases (fibrocavitary, nodular bronchiectasis with cavity) that require different treatment strategies depending on the causal agents. Several studies have reported human risk factors for the disease; however, little efforts were made to investigate the risk factors in nontuberculous mycobacteria. Moreover, molecular genetics experiments have been difficult to search for virulence factors in M. avium, which the population genomics approaches could overcome. Here, the GWAS results suggested variants in three chromosomal loci associated with mycobacterial membrane components as risk factors for cavitary MAC lung disease. These findings could help develop treatment strategies for MAC lung disease in the future.


title: Immunogenicity of low dose prime-boost vaccination of mRNA vaccine CV07050101 in non-human primates

abstract: Many different vaccine candidates against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of COVID-19, are currently approved and under development. Vaccine platforms vary from mRNA vaccines to viral-vectored vaccines, and several candidates have been shown to produce humoral and cellular responses in small animal models, non-human primates and human volunteers. In this study, six non-human primates received a prime-boost intramuscular vaccination with 4 {micro}g of mRNA vaccine candidate CV07050101, which encodes a pre-fusion stabilized spike (S) protein of SARS-CoV-2. Boost vaccination was performed 28 days post prime vaccination. As a control, six animals were similarly injected with PBS. Humoral and cellular immune responses were investigated at time of vaccination, and two weeks afterwards. No antibodies could be detected two and four weeks after prime vaccination. Two weeks after boost vaccination, binding but no neutralizing antibodies were detected in 4 out of 6 non-human primates. SARS-CoV-2 S protein specific T cell responses were detected in these 4 animals. In conclusion, prime-boost vaccination with 4 {micro}g of vaccine candidate CV07050101 resulted in limited immune responses in 4 out of 6 non-human primates.


title: Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition

abstract: The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryo-EM structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT PAM sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing.


title: Receptor-binding domain recombinant protein RBD219-N1C1 on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

abstract: We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B. 1.351 (Beta), B. 1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.


title: Receptor-binding domain recombinant protein on alum-CpG induces broad protection against SARS-CoV-2 variants of concern


title: Microchromosomes are building blocks of bird, reptile and mammal chromosomes

abstract:
Microchromosomes, once considered unimportant shreds of the chicken genome, are gene rich elements with a high GC content and few transposable elements. Their origin has been debated for decades. We used cytological and whole genome sequence comparisons, and chromosome conformation capture, to trace their origin and fate in genomes of reptiles, birds and mammals. We find that microchromosomes as well as macrochromosomes are highly conserved across birds, and share synteny with single small chromosomes of the chordate amphioxus, attesting to their origin as elements of an ancient animal genome. Turtles and squamates (snakes and lizards) share different subsets of ancestral microchromosomes, having independently lost microchromosomes by fusion with other microchromosomes or macrochromosomes. Patterns of fusions were quite different in different lineages.

Cytological observations show that microchromosomes in all lineages are spatially separated into a central compartment at interphase and during mitosis and meiosis. This reflects higher interaction between microchromosomes than with macrochromosomes, as observed by chromosome conformation capture, and suggests some functional coherence. In highly rearranged genomes fused microchromosomes retain most ancestral characteristics, but these may erode over evolutionary time; surprisingly de novo microchromosomes have rapidly adopted high interaction.

Some chromosomes of early branching monotreme mammals align to several bird microchromosomes, suggesting multiple microchromosome fusions in a mammalian ancestor. Subsequently multiple rearrangements fueled the extraordinary karyotypic diversity of therian mammals.

Thus microchromosomes, far from being aberrant genetic elements, represent fundamental building blocks of amniote chromosomes, and it is mammals, rather than reptiles, that are atypical.

Significance StatementGenomes of birds and reptiles, but not mammals, consist of a few large chromosomes and many tiny microchromosomes. Once considered unimportant shreds of the genome, microchromosomes are gene rich and highly conserved among bird and reptiles, and share homology with one or more of the tiny chromosomes of an invertebrate that diverged from the vertebrate lineage 684 million years ago. Microchromosomes interact strongly and crowd together at the centre of cells, suggesting functional coherence. Many microchromosomes have been lost independently in turtles, snakes and lizards as they have fused with each other, or with larger chromosomes. In mammals they have completely disappeared, yet some chromosomes of the basal platypus line up with several microchromosomes, suggesting that they are the building blocks of the atypically variable chromosomes of mammals.

category: genomics


title: A single-cell transcriptomic atlas characterizes liver non-parenchymal cells in healthy and diseased mice

abstract: The heterogeneity of liver non-parenchymal cells (NPCs) is essential for liver structure and function. However, the current understanding of liver NPCs, especially in different liver diseases, remains incompletely elucidated. Here, a single-cell transcriptome atlas of 171,814 NPCs from healthy and 5 typical liver disease mouse models, including alcoholic liver disease, nonalcoholic steatohepatitis (NASH), drug-induced liver injury, cholestatic, and ischemia-reperfusion liver injury is constructed. The inter- and intra-group heterogeneity of 12 types (and numerous subtypes) of NPCs involving endothelial cells, hepatic stellate cells (HSCs), neutrophils, T cells, and mononuclear phagocytes (MPs) are summarized. A protective subtype of neutrophils characterized by Chil3high is validated and found significantly increasing only in drug-induced and cholestatic liver injury models. Transcriptional regulatory network analysis reveals disease-specific transcriptional reprogramming. Metabolic activity analysis indicates that fibrosis is accompanied by increases in glycolysis and retinol metabolism in activated HSCs and MPs. Moreover, we found that cell-cell interactions between cholangiocytes and immune cells contribute more to cholestatic liver fibrosis compared with NASH, while HSCs are more important for NASH fibrosis. Our atlas, together with an interactive website provides a systematic view of highly heterogeneous NPCs and a valuable resource to better understand pathological mechanisms underlying liver diseases.


title: The E. coli helicase does not use ATP during replication.

abstract:
The replisome is responsible for replication of DNA in all domains of life, with several of its individual enzyme components relying on hydrolysis of nucleoside triphosphates to provide energy for replisome function. Half a century of biochemical studies have demonstrated a dependence on ATP as an energy source for helicases to unwind duplex DNA during replication. Through single-molecule visualization of DNA replication by the Escherichia coli replisome, we demonstrate that the DnaB helicase does not rely on hydrolysis of ATP (or any ribo-NTPs) in the context of the elongating replisome. We establish that nucleotide incorporation by the leading-strand polymerase is the main motor driving the replication process.

One Sentence SummaryPolymerases provide the energy for helicase-mediated DNA unwinding during E. coli DNA replication.

category: biochemistry


title: Rapid extraction and kinetic analysis of protein complexes from single cells

abstract: Proteins contribute to cell biology by forming dynamic, regulated interactions, and measuring these interactions is a foundational approach in biochemistry. We present a rapid, quantitative in vivo assay for protein-protein interactions, based on optical cell lysis followed by time-resolved single-molecule analysis of protein complex binding to an antibody-coated substrate. We show that our approach has better reproducibility, higher dynamic range, and lower background than previous single-molecule pull-down assays. Furthermore, we demonstrate that by monitoring cellular protein complexes over time after cell lysis, we can measure the dissociation rate constant of a cellular protein complex, providing information about binding affinity and kinetics. Our dynamic single-cell, single-molecule pull-down method thus approaches the biochemical precision that is often sought from in vitro assays, while being applicable to native protein complexes isolated from single cells in vivo.


title: Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling

abstract: Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto full-length proteins with precise control over chain length and modification site. A series of semi-synthetic ADP-ribosylated histone proteins are prepared which demonstrate that ADPr at H2BS6 or H3S10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. Importantly, we found ALC1 selectively remodels  activated substrates within heterogeneous nucleosome populations and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a method that is broadly applicable to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.


title: Wireless endovascular nerve stimulation with a millimeter-sized magnetoelectric implant

abstract: Implanted bioelectronic devices have the potential to treat disorders that are resistant to traditional pharmacological therapies; however, reaching many therapeutic nerve targets requires invasive surgeries and implantation of centimeter-sized devices. Here we show that it is possible to stimulate peripheral nerves from within blood vessels using a millimeter-sized wireless implant. By directing the stimulating leads through the blood vessels we can target specific nerves that are difficult to reach with traditional surgeries. Furthermore, we demonstrate this endovascular nerve stimulation (EVNS) with a millimeter sized wireless stimulator that can be delivered minimally invasively through a percutaneous catheter which would significantly lower the barrier to entry for neuromodulatory treatment approaches because of the reduced risk. This miniaturization is achieved by using magnetoelectric materials to efficiently deliver data and power through tissue to a digitally-programmable 0.8 mm2 CMOS system-on-a-chip. As a proof-of-principle we show wireless stimulation of peripheral nerve targets both directly and from within the blood vessels in rodent and porcine models. The wireless EVNS concept described here provides a path toward minimally invasive bioelectronics where mm-sized implants combined with endovascular stimulation enable access to a number of nerve targets without open surgery or implantation of battery-powered pulse generators.


title: CAT, AGTR2, L-SIGN and DC-SIGN are potential receptors for the entry of SARS-CoV-2 into human cells

abstract: Since December 2019, the COVID-19 caused by SARS-CoV-2 has been widely spread all over the world. It is reported that SARS-CoV-2 infection affects a series of human tissues, including lung, gastrointestinal tract, kidney, etc. ACE2 has been identified as the primary receptor of the SARS-CoV-2 Spike (S) protein. The relatively low expression level of this known receptor in the lungs, which is the predominantly infected organ in COVID-19, indicates that there may be some other co-receptors or alternative receptors of SARS-CoV-2 to work in coordination with ACE2. Here, we identified twenty-one candidate receptors of SARS-CoV-2, including ACE2-interactor proteins and SARS-CoV receptors. Then we investigated the protein expression levels of these twenty-one candidate receptors in different human tissues and found that five of which CAT, MME, L-SIGN, DC-SIGN, and AGTR2 were specifically expressed in SARS-CoV-2 affected tissues. Next, we performed molecular simulations of the above five candidate receptors with SARS-CoV-2 S protein, and found that the binding affinities of CAT, AGTR2, L-SIGN and DC-SIGN to S protein were even higher than ACE2. Interestingly, we also observed that CAT and AGTR2 bound to S protein in different regions with ACE2 conformationally, suggesting that these two proteins are likely capable of the co-receptors of ACE2. Conclusively, we considered that CAT, AGTR2, L-SIGN and DC-SIGN were the potential receptors of SARS-CoV-2. Moreover, AGTR2 and DC-SIGN tend to be highly expressed in the lungs of smokers, which is consistent with clinical phenomena of COVID-19, and further confirmed our conclusion. Besides, we also predicted the binding hot spots for these putative protein-protein interactions, which would help develop drugs against SARS-CoV-2.


title: Structural snapshots of human PepT1 and PepT2 reveal mechanistic insights into substrate and drug transport across epithelial membranes

abstract: The uptake of peptides in mammals plays a crucial role in nutrition and inflammatory diseases. This process is mediated by promiscuous transporters of the Solute Carrier Family 15, which form part of the Major Facilitator superfamily. Besides the uptake of short peptides, Peptide transporter 1 (PepT1) is a highly abundant drug transporter in the intestine and represents a major route for oral drug delivery. Peptide transporter 2 (PepT2) allows in addition renal drug reabsorption from ultrafiltration and brain-to-blood efflux of neurotoxic compounds. Here we present cryo-EM structures of human PepT1 in an outward open state and of human PepT2 in an inward facing partially occluded state with a bound substrate. The structures reveal the architecture of human peptide transporters and provide mechanistic insights into substrate recognition and conformational transitions during transport. Importantly, this may support future drug design efforts to increase the bioavailability of different drugs in the human body.


title: m6A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small cell lung cancer

abstract: The modification of N6-methyladenosine (m6A) in RNA and its eraser ALKBH5, an m6A demethylase, play important roles across various steps of human carcinogenesis. However, the involvement of ALKBH5 in non-small cell lung cancer (NSCLC) development remains to be completely elucidated. The current study revealed that the expression of ALKBH5 were increased in NSCLC and increased expression of ALKBH5 worsened the prognosis of patients with NSCLC. In vitro study revealed that ALKBH5 knockdown suppressed cell proliferation ability of PC9 and A549 cells as well as promoted G1 arrest and increased the number of apoptotic cells. Furthermore, ALKBH5 overexpression increased the cell proliferation ability of the immortalized cell lines. Microarray analysis and western blotting revealed that the expression of CDKN1A or TIMP3 were increased by ALKBH5 knockdown. These alterations were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs. The decline of mRNAs was, at least partly, owing to the destabilization of these mRNAs by one of the IGF2BPs. In conclusions, the ALKBH5-IGF2BPs axis promotes cell proliferation and tumorigenicity, which in turn causes the unfavorable prognosis of NSCLC.

category: cancer biology


title: Re-convolving the compositional landscape of primary and recurrent glioblastoma using single nucleus RNA sequencing

abstract: Glioblastoma is an aggressive diffusely infiltrating neoplasm that spreads beyond surgical resection margins, where it intermingles with non-neoplastic brain cells. This complex microenvironment harbouring infiltrating glioma and non-neoplastic brain cells is the origin of tumor recurrence. Thus, understanding the cellular and molecular features of the glioma microenvironment is therapeutically and prognostically important. Here, we used single-nucleus RNA sequencing (snRNAseq) of primary and recurrent glioma to define three compositional  tissue-states rooted in cohabitation of cell-types and transcriptional states. These comprise a state featuring A) abundant normal brain microenvironment cells, B) reactive/inflamed brain microenvironment, and C) cellular/proliferative tumor. All these states exhibited variable degrees of infiltration by glioma cells. Spatial transcriptomics confirmed that the cell-types and transcriptomics states which compositionally cohabitate tissue states also colocalize in space. Tissue states are clinically significant because they correlate with radiographic, histopathologic, and prognostic features. We found that enrichment of tissue state B signature correlated with shorter survival. Importantly, we found that our compositionally defined tissue states are enriched in distinct metabolic pathways. One such pathway is fatty acid biosynthesis, which was enriched in tissue state B - a state enriched in recurrent glioblastoma and composed of astrocyte-like/mesenchymal glioma cells, reactive astrocytes resembling those seen in neurodegeneration, and monocyte-like myeloid cells. We showed that treating acute slices of GBM with a fatty acid synthesis inhibitor is sufficient to deplete the transcriptional signature of tissue state B. Our findings define a novel compositional approach to the glioma-infiltrated tissue which allows us to discover prognostic and targetable features, paving the way to new mechanistic and therapeutic discoveries.


title: Re-convolving the compositional landscape of primary and recurrent glioblastoma reveals prognostic andtargetable tissue states


title: Optimization of the TeraTox assay for preclinical teratogenicity assessment

abstract: Current animal-free methods to assess teratogenicity of drugs under development still deliver high numbers of false negatives, and more sensitive approaches of toxicity prediction are required. To address this issue, we characterized the TeraTox test, a newly developed multi-lineage differentiation assay for human teratogenicity prediction using 3D human induced pluripotent stem cells. TeraTox produces as primary output concentration-dependent data sets for each test compound on cytotoxicity and altered gene expression. These data are then fed into a prediction model based on an interpretable machine-learning approach. The final information obtained relates to the concentration-dependent human teratogenicity potential of drug candidates. We applied TeraTox to profile 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. This way, it was possible to relate the test predictions to known human or animal toxicity. The TeraTox had an accuracy of 69% (specificity: 53%, sensitivity: 79%). It clearly performed better than two quantitative structure-activity relationship (QSAR) models and it had a higher sensitivity than the murine embryonic stem cell test (mEST) run in the same laboratory. By combining TeraTox and mEST data, the overall prediction accuracy was further improved. The knowledge on the pattern of altered gene expression may provide additional value in grouping toxicologically similar compounds and possibly deducing common modes of action. The assay will thus be a helpful additional tool in drug discovery, and the dataset provided here will be a valuable resource for the field of stem cell-based drug profiling.


title: Identification of the functions of 4-coumarate-CoA ligase/ acyl-CoA synthetase paralogs in potato

abstract:
BackgroundThe 4CL/ ACS protein family is well known for its 4-coumarate-CoA ligase (4CL) enzymes but there are many aspects of this family that are still unclear or generally known. Cytosolic class I and class II 4CL enzymes control the biosynthesis of lignin/ suberin and flavonoids, respectively. Many 4CL homologs have broad substrate permissiveness in vitro and have no clear cut function. However, it has been demonstrated unequivocally that a peroxisomal 4CL-like homolog from Arabidopsis efficiently uses p-coumarate for ubiquinone biosynthesis. Another homolog has been shown to act as a fatty acyl-CoA synthetase and yet another as OPDA-CoA ligase. Hence, despite this knowledge, most homologs remain annotated as "4CL-like" whereas other researches study the ACS protein family.

ResultsWe set out identify the specific functions of 4CL/ ACS homologs, specifically in order to study the 4CL family in Solanum tuberosum. An in depth phylogenetic analysis was done. Using clustering techniques, functional annotation and taxonomic signals, three major clades were depicted. Clade 1 is composed of class I from monocotyledons, class I from dicotyledons and class II canonical 4CL enzymes subclades. Specificity determining positions and 3D structure analysis shows that clade 2 cytosolic 4CL-like enzymes show a rather different binding cleft and presumably use medium- to long-chain fatty acids. Clade 3 is composed of five subclades, four of which have a broad taxonomic contribution and a similar binding cleft as 4CLs whereas a fifth, specific for dicotyledons shows a significantly different binding pocket. The potato 4CL family comprises four class I (St4CL-I(A-D)) and one class II (St4CL-II) members. Transcript levels of St4CLs and of marker genes of the flavonoid (chalcone synthase, CHS) and suberin (feruloyl-CoA transferase, FHT) pathways were determined by qRT-PCR in flesh and skin from Andean varieties. St4CL-IA was barely detected in the skin of some varieties whereas St4CL-IB did not show a clear pattern. St4CL-IC and St4CL-ID could not be detected. St4CL-II expression pattern was similar to CHS. St4CL-IA and St4CL-IB were induced by wounding as did FHT whereas St4CL-II and CHS expression was repressed. Constitutive and wound-induced expression suggests that St4CL-IA and St4CL-IB isoforms are likely involved in soluble and/ or suberin-bound phenolic compounds while St4CL-II appears to be involved in flavonoid biosynthesis.


title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

abstract:
Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modes controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce  synthetic regulatory reconstitution, a novel conceptual framework for studying gene regulation and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130-170 kilobases each) to an ectopic location in the mouse genome. We find that a HoxA cluster lacking distal enhancers recapitulates correct patterns of chromatin remodeling and transcription in response to patterning signals, while distal enhancers are required for full transcriptional output. Synthetic regulatory reconstitution is a generalizable strategy to decipher the regulatory logic of gene expression in complex genomes.

One-Sentence SummaryReconstitution of gene regulation using large DNA constructs unravels the regulatory logic of a developmental gene locus.


title: Chronic CNS Pathology is Associated with Abnormal Collagen Deposition and Fibrotic-like Changes

abstract: Multiple sclerosis is a chronic debilitating disease of the CNS. The relapsing remitting form of the disease is driven by CNS directed inflammation. However, in the progressive forms of the disease, inflammation has abated and the underlying pathology is less well understood. In this paper, we show that chronic lesions in progressive MS are associated with fibrotic changes, a type of pathology that has previously not thought to occur in the CNS. In an animal model of chronic MS, late stage disease contains no inflammatory infiltrates and is instead characterized by collagen deposition that is histologically similar to fibrosis. In human MS samples, chronic, but not acute lesions, are devoid of inflammatory infiltrates and instead contain significant collagen deposition. Furthermore, we demonstrate that both mouse and human astrocytes are the cellular source of collagen. These results suggest that anti-fibrotic therapy may be beneficial in the treatment of progressive MS.


title: Adult medial habenula neurons require GDNF receptor GFRalpha1 for synaptic stability and function

abstract: The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatio-temporally controlled Cre-mediated recombination in adult mice, we found that the GDNF receptor alpha 1 (GFR1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFR1 in the mouse brain, and acute ablation of GFR1 results in loss of septo-habenular and habenulo-interpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and opto-genetic studies in mice lacking GFR1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index of AMPARs, suggesting reduced Ca2+-permeability. Further biochemical and proximity ligation assay studies defined the presence of GluA1/GluA2 (Ca2+-impermeable) as well as GluA1/GluA4 (Ca2+-permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits in mHb neurons lacking GFR1. Finally, acute loss of GFR1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFR1 in the maintenance and function of adult glutamatergic synapses, and reveal a potential new mechanism for regulating synaptic plasticity in the septo-habenulo-interpeduncular pathway and attuning of anxiety and fear behaviors.