title: Different patterns of visual processing for shapes in V1 of mouse and monkey

abstract: Recently, mice became a popular research model for the visual system including objects processing. However, while primates use their fovea to inspect objects at high resolution, mice have low visual acuity and lack a foveal region. Thus, how objects are encoded in the visual cortex of mice and whether monkeys and mice use similar neural mechanisms to discriminate between shapes, is unknown. Using voltage sensitive dye imaging, we investigated spatio-temporal patterns of population response in the primary visual cortex (V1) of mice and monkeys to shape contours. Neural responses in mice showed blurred spatial patterns, low correlation to the original stimuli and only subtle spatial differences between the shapes. In contrast, the neural responses in monkeys V1 fovea, showed prominent differences between the shapes at sub-degree resolution with high resemblance to the original shapes, in agreement with a topographic code. Furthermore, the spatial patterns of neural synchronization in monkeys V1 suggested contour binding by synchrony, but not in mice V1. Finally, monkeys V1 responses at larger eccentricities, resembled more to those in mice. These results may suggest that to discriminate between shapes, mice rely on neural processing of low-level cues, whereas monkeys rely more on a topographic code.


title: Retinal ganglion cell endowment is correlated with optic tract fiber cross section, not density

abstract: There is substantial variation between healthy individuals in the number of retinal ganglion cells (RGC) in the eye, with commensurate variation in the number of axons in the optic tracts. Fixel-based analysis of diffusion MR produces estimates of fiber density (FD) and cross section (FC). Using these fixel measurements along with retinal imaging, we asked if individual differences in RGC tissue volume are correlated with individual differences in FD and FC measurements obtained from the optic tracts, and subsequent structures along the cortical visual pathway. We find that RGC endowment is correlated with optic tract FC, but not with FD. RGC volume had a decreasing relationship with measurements from subsequent regions of the visual system (LGN volume, optic radiation FC/FD, and V1 surface area). However, we also found that the variations in each visual area were correlated with the variations in its immediately adjacent visual structure. We only observed these serial correlations when FC is used as the measure of interest for the optic tract and radiations, but no significant relationship was found when FD represented these white matter structures. From these results, we conclude that the variations in RGC endowment, LGN volume, and V1 surface area are better predicted by the overall cross section of the optic tract and optic radiations as compare to the intra-axonal restricted signal component of these white matter pathways. Additionally, the presence of significant correlations between adjacent, but not distant, anatomical structures suggests that there are multiple, local sources of anatomical variation along the visual pathway.

category: neuroscience


title: Responses of cortical neurons to intracortical microstimulation in awake primates

abstract:
Intracortical microstimulation (ICMS) is commonly used in many experimental and clinical paradigms; however, its effects on the activation of neurons are still not completely understood. To document the responses of cortical neurons in non-human primates to stimulation, we recorded single unit activity while delivering single-pulse stimulation via Utah arrays implanted in primary motor cortex of three macaque monkeys. Stimuli between 5-50 A delivered to single channels reliably evoked spikes in neurons recorded throughout the array with delays of up to 12 milliseconds. ICMS pulses also induced a period of inhibition lasting up to 150 ms that typically followed the initial excitatory response. Higher current amplitudes led to a greater probability of evoking a spike and extended the duration of inhibition. The likelihood of evoking a spike in a neuron was dependent on the spontaneous firing rate as well as the delay between its most recent spike time and stimulus onset. Tonic repetitive stimulation between 2 and 20 Hz often modulated both the probability of evoking spikes and the duration of inhibition, although high frequency stimulation in particular was more likely to change both responses. On a trial-by-trial basis, whether a stimulus evoked a spike did not affect the subsequent inhibitory response; however, their changes over time were frequently positively or negatively correlated. Our results document the complex dynamics of cortical neural responses to electrical stimulation that need to be considered when utilizing ICMS for scientific and clinical applications.

Significance statementIntracortical microstimulation (ICMS) is commonly used to probe the cortex, and previous studies have characterized the responses of single neurons to ICMS. However, these studies typically explored the averaged effects of ICMS throughout each experimental session, rather than by a trial-by-trial basis for each stimulation pulse. By shifting the approach, we explored the dependence of neural responses to ICMS on the spontaneous neural activity as well as the dynamics of responses over time due to repetitive stimulation. Our results highlight how the responses of neurons to ICMS are likely the result of interactions between local excitatory and inhibitory cortical circuits. These results will help inform the design of ICMS for both basic research and clinically relevant stimulation protocols.


title: CRISPR activation rescues abnormalities in SCN2A haploinsufficiency-associated autism spectrum disorder

abstract: The majority of autism spectrum disorder (ASD) risk genes are associated with ASD due to haploinsufficiency, where only one gene copy is functional. Here, using SCN2A haploinsufficiency, a major risk factor for ASD, we show that increasing the expression of the existing functional SCN2A allele with CRISPR activation (CRISPRa) can provide a viable therapeutic approach. We first demonstrate therapeutic potential by showing that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency. Next, using an rAAV-CRISPRa based treatment, we restore electrophysiological deficits in both Scn2a heterozygous mice and human stem-cell-derived neurons. Our results provide a novel therapeutic approach for numerous ASD-associated genes and demonstrate that rescue of Scn2a haploinsufficiency, even at adolescent stages, can ameliorate neurodevelopmental phenotypes.


title: Medial prefrontal-hippocampal concordance in child-parent dyads underlies children's psychological wellbeing

abstract: Negative family environments can be harmful to childrens psychological wellbeing via altered child-parent interactions. Single-brain paradigms have uncovered changes in brain systems related to negative family environments, but how neurobiological reciprocity between child and parent brains contribute to childrens psychological wellbeing remains unknown. Across two studies examining 395 child-parent dyads, we show that children from negative family environments experienced more psychopathological symptoms. Dyad-based analysis of movie-watching functional magnetic resonance imaging data revealed higher inter-subject correlations in dorsal- and ventral-MPFC, and greater concordance of activity with regions critical for socioemotional skills in child-parent than child-stranger dyads. Negative family environment reduced inter-subject correlation of ventral-MPFC with the hippocampus in child-parent dyads, which further mediated childrens internalizing symptoms. The ventral-MPFC and hippocampus exhibited reciprocal activity, which helped child-parent dyads form shared meaning during movie watching. Our findings suggest child-parent brain-to-brain concordance may be a neurobiological mechanism for how family environments affect psychological wellbeing.


title: Brain-to-brain concordance in child-parent dyads underlies children's psychological wellbeing


title: Autophagy slows the aging of Germline stem cells in Drosophila through modulation of E-cadherin

abstract: Autophagy is a conserved process that degrades cytoplasmic components and organelles in metazoan cells including germline stem cells. Although autophagy is implicated in the aging of stem cells, the precise mechanism are still unknown. Here we show that elevating autophagy by overexpressing (OE) Drosophila Autophagy-related gene 8a (Atg8a) in the female Germline stem cells (GSCs) delays their loss due to aging. However, sustained elevated autophagy levels in old flies promote GSC loss due to cell death. In contrast, knockdown of Atg8a (Atg8aRNAi) in GSCs accelerates their loss. Atg8aOE GSCs show elevated autophagy flux, and increased mitotic activity even at 8 weeks of age. Atg8aOE GSCs possess smaller-sized mitochondria and exhibit reduced mitochondrial oxidative stress in the GSCs. However, in contrast Atg8aRNAi GSCs have elevated mitochondrial ROS and possess larger mitochondria. Finally, our data show that Atg8aOE GSCs occupy the stem cell niche for longer duration with the aid of elevated E-cadherin at the GSC-cap cell contact sites. Our data suggests that elevated autophagy promotes GSC maintenance and activity, and delays their aging.

category: developmental biology


title: High predation risk decimates survival duringthe reproduction season

abstract: Predators attack conspicuous prey phenotypes that are abundant in the environment. Male display behaviour of conspicuous nuptial colouration becomes risky in the presence of a predator, and adult males face higher predation risk. High predation risk in one sex will lead to low survival and sex ratio bias in adult cohorts, unless the increased predation risk is compensated by higher escape rate. Here, we tested the hypothesis that sand lizards (Lacerta agilis) have sex-specific predation risk and escape rate. We found statistically stable sex ratio in age groups, equal frequency of tail regenerates between sexes, and similar survival rate. Predation risk is similar between sexes, and escape rate increases survival by about 5%. We found low survival rate and a low number of tail autotomy events during the months when sand lizard females mate and lay eggs, indicating high predator pressure throughout reproduction, when females fail to escape predation. The risky reproduction season in an ectotherm is a convolution of morphological changes (conspicuous colouration in males, body allometry changes in gravid females), behaviour (nuptial displays), and environmental conditions which challenge lizard thermal performance, favouring performance of endotherm predators in cold spring months.

category: ecology


title: Predicting catchment suitability for biodiversity at national scales

abstract: Biomonitoring of water quality and catchment management are often disconnected, due to mismatching scales. Great effort and money is spent each year on routine reach-scale surveying across many sites, particularly in the UK, and typically with a focus on pre-defined indicators of organic pollution to compare observed vs expected subsets of common macroinvertebrate indicator species. Threatened species are often ignored due to their rarity as are many invasive species, which are seen as undesirable even though they are increasingly common in freshwaters, especially in urban ecosystems. However, these taxa are monitored separately for reasons related to biodiversity concerns rather than for gauging water quality. Repurposing such monitoring data could therefore provide important new biomonitoring tools that can help catchment managers to directly link the water quality that they aim to control with the biodiversity that they are trying to protect. Here we used the England Non-Native and Rare/Protected species records that track these two groups of species as a proof-of-concept for linking catchment scale management of freshwater ecosystems and biodiversity to a range of potential drivers across England. We used national land use (Centre for Ecology and Hydrology land cover map) and water quality indicator (Environment Agency water quality data archive) datasets to predict the presence or absence of 48 focal threatened or invasive species of concern routinely sampled by the English Environment Agency at catchment scale, with a median accuracy of 0.81 area under the receiver operating characteristic curve. A variety of water quality indicators and land-use types were useful in predictions, highlighting that future biomonitoring schemes could use such complementary measures to capture a wider spectrum of drivers and responses. In particular, the percentage of a catchment covered by freshwater was the single most important metric, reinforcing the need for space/habitat to support biodiversity. We show how our method could inform new catchment management approaches, by highlighting how key relationships can be identified and how to understand, visualise and prioritise catchments that are most suitable for restorations or water quality interventions. The scale of this work, in terms of number of species, drivers and locations, represents a step towards a new approach to catchment management that enables managers to link drivers they can control (water quality and land use) to the biota they are trying to protect (biodiversity).


title: The genetic architecture of multiple mutualisms and mating system in Turnera ulmifolia

abstract: Plants often associate with multiple arthropod mutualists. These partners provide important services to their hosts, but multiple interactions can constrain a plants ability to respond to complex, multivariate selection. Here, we quantified patterns of genetic variance and covariance among rewards for pollination, biotic defence, and seed dispersal mutualisms in multiple populations of Turnera ulmifolia to better understand how the genetic architecture of multiple mutualisms might influence their evolution. We phenotyped plants cultivated from 17 Jamaican populations for several mutualism and mating system-related traits. We then fit genetic variance-covariance (G) matrices for the island metapopulation and the 5 largest individual populations. At the metapopulation level, we observed significant positive genetic correlations among stigma-anther separation, floral nectar production, and extrafloral nectar production. These correlations have the potential to significantly constrain or facilitate the evolution of multiple mutualisms in T. ulmifolia and suggest that pollination, seed dispersal, and dispersal mutualisms do not evolve independently. In particular, we found that positive genetic correlations between floral and extrafloral nectar production may help explain their stable co-existence in the face of physiological trade-offs and negative interactions between pollinators and ant bodyguards. Locally, we found only small differences in G among our T. ulmifolia populations, suggesting that geographic variation in G may not shape the evolution of multiple mutualisms.

category: evolutionary biology


title: The genome sequence of the scarce swallowtail, Iphiclides podalirius

abstract: The scarce swallowtail, Iphiclides podalirius (Linnaeus, 1758), is a species of butterfly in the family Papilionidae. Here we present a chromosome-level genome assembly for I. podalirius as well as gene and transposable element annotations. We estimate heterozygosity within different partitions of the genome and find a negative correlation between chromosome length and heterozygosity at fourfold-degenerate sites. This high quality genome assembly, the first for any species in the tribe Leptocircini, will be a valuable resource for population genomics in the genus Iphiclides and comparative genomics more generally.


title: Gallbladder adenocarcinomas undergo subclonal diversification and selection from precancerous lesions to metastatic tumors

abstract: We aimed to elucidate the evolutionary trajectories of gallbladder adenocarcinoma (GBAC) using multi-regional and longitudinal tumor samples. Using whole-exome sequencing data, we constructed phylogenetic trees in each patient, and analyzed mutational signatures. A total of 11 patients including 2 rapid autopsy cases were enrolled. The most frequently altered gene in primary tumors was ERBB2 (54.5%), followed by TP53 (45.5%), and FBXW7 (27.3%). Most mutations in frequently altered genes in primary tumors were detectable in concurrent precancerous lesions (biliary intraepithelial neoplasia, BilIN), but some of them were subclonal. Subclonal diversity was common in BilIN (n=4). However, among subclones in BilIN, a certain subclone commonly shrank in concurrent primary tumors. In addition, selected subclones underwent linear and branching evolution, maintaining subclonal diversity. In combined analysis with metastatic tumors (n=11), branching evolution was identified in 9 (81.8%) patients. Of these, 8 patients (88.9%) had a total of 11 subclones expanded at least 7-fold during metastasis. These subclones harbored putative metastasis-driving mutations in tumor suppressor genes such as SMAD4, ROBO1, and DICER1. In mutational signature analysis, 6 mutational signatures were identified: 1, 3, 7, 13, 22, and 24 (cosine similarity >0.9). Signatures 1 (age) and 13 (APOBEC) decreased during metastasis while signatures 22 (aristolochic acid) and 24 (aflatoxin) were relatively highlighted. Subclonal diversity arose early in precancerous lesions and the clonal selection was a common event during malignant transformation in GBAC. However, selected cancer clones continued to evolve and thus maintained subclonal diversity in metastatic tumors.

category: genetics


title: Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping

abstract:
CYP2D6 is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants.

Author SummaryDuring the last decades, the usefulness of personalized medicine has become increasingly apparent. Directly linked to that is the need for accurate genotyping assays to determine the pharmacogenetic profile of patients. Continuing research has led to the development of genotyping assays that perform quite robustly. However, complex genes remain an issue when it comes to determining the complete sequence correctly. An example of such a complex but very important pharmacogene is CYP2D6. Therefore, we developed a genotyping assay in an attempt to generate complete allele-specific consensus sequences of CYP2D6, by optimizing a targeted amplification-free long-read sequencing method and developing a new analysis pipeline. In reference samples, we showed that our genotyping assay performed accurately and confirmed the presence of variants that go undetected by most current assays. However, the implementation of this assay in practice is still hampered as the selected enrichment strategies inherently lead to a low percentage of on-target reads, resulting in low on-target sequencing depths. Further optimization and validation of the assay is thus needed, but definitely worth considering for follow-up research as we already demonstrated the added value for generating more complete genotypes, which on its turn will result in more accurate gene function predictions.


title: OTTERS: A powerful TWAS framework leveraging summary-level reference data

abstract: Transcriptome-wide association studies (TWAS) identify genes that influence complex traits through genetic regulation of gene expression. Existing TWAS tools like FUSION require individual-level eQTL reference data from a source like GTEx (n=~100s) to impute gene expression in a test GWAS. Therefore, these TWAS tools are not applicable to enormous summary-level reference eQTL datasets like those generated by the eQTLGen consortium (n=~32K). Development of TWAS methods that can harness summary-level reference data like eQTLGen is valuable not only to enable TWAS in more general settings but also to permit more powerful analyses, since we expect expression prediction to improve with increasing reference sample size. To fill this important gap, we develop a TWAS framework called OTTERS (Omnibus Transcriptome Test using Expression Reference Summary data) that adapts multiple polygenic risk score (PRS) methods to estimate eQTL weights from summary-level eQTL reference data. For each PRS method, OTTERS estimates eQTL weights to impute gene expression per gene and then tests for association between imputed expression and outcome for a GWAS dataset. OTTERS then combines the PRS-based association tests together to create an optimal TWAS p-value. By simulation, we show that OTTERS is powerful across a wide range of models. We also applied OTTERS to conduct a TWAS of cardiovascular disease using GWAS summary data from UK Biobank and summary-level eQTLGen reference data. OTTERS identified 11 additional TWAS risk genes that were missed by FUSION using GTEx reference data. These findings suggest that OTTERS provides a practical and powerful tool for TWAS analysis.


title: Robust inference of expression state in bulk and single-cell RNA-Seq using curated intergenic regions

abstract: RNA-Seq is a powerful technique to provide quantitative information on gene expression. While many applications focus on estimated expression levels, it is also important to determine which genes are actively transcribed, and which are not. The problem can be viewed as simply setting a biologically meaningful threshold for calling a gene expressed. We propose to define this threshold per sample relative to the background level for non-expressed genomic features, inferred by the amount of reads mapped to intergenic regions of the genome. To this aim, we first define a stringent set of reference intergenic regions, based on available bulk RNA-Seq libraries for each species. We provide predefined regions selected for different animal species with varying genome annotation quality through the Bgee database. We then call genes expressed if their level of expression is significantly higher than the background noise. This approach can be applied to bulk as well as single-cell RNA-Seq, on a single library as well as on a combination of libraries over one condition. We show that the estimated proportion of expressed genes is biologically meaningful and stable between libraries originating from the same tissue, in both model and non-model organisms.

category: genomics


title: dartR v2: an accessible genetic analysis platform for conservation, ecology, and agriculture

abstract:
O_LIInnumerable approaches to analyse genetic data are now available to guide conservation, ecological and agricultural projects. However, streamlined and accessible tools are needed to bring these approaches within reach of a broader user base. dartR was released in 2018 to lessen the intrinsic complexity of analysing single nucleotide polymorphisms (SNPs) and dominant markers (presence/absence of amplified sequence tags) by providing user-friendly data quality control and marker selection functions. dartR users have grown steadily since its release and provided valuable feedback on their interaction with the package allowing us to enhance dartR capabilities.
C_LIO_LIHere, we present Version 2 of dartR. In this iteration, we substantially increased the number of available functions from 45 to 144. In addition to improved functionality, we focused on enhancing the user experience by extending plot customisation, function standardisation, increasing user support and function speed. dartR provides functions for various stages in analysing genetic data, from data manipulation to reporting.
C_LIO_LIdartR provides many functions for importing, exporting and linking to other packages, to provide an easy-to-navigate conduit between data generation and analysis options already available via other packages. We also implemented simulation functions whose results can be analysed seamlessly with several other dartR functions.
C_LIO_LIAs more methods and approaches mature to inform conservation, we envision that accessible platforms to analyse genetic data will play a crucial role in translating science into practice.
C_LI


title: Improved Centromere Assemblies for RefGen_v4

abstract: Genome assemblies based on long read sequencing technology have revolutionized the assembly of repeat-rich centromere regions. However, because maize centromeres are highly enriched for the tandem repeat CentC and centromeric retrotransposons (CR), automated genome assembly left gaps even in the excellent B73 RefGen_v4 reference genome constructed from long-read data. Manual editing of >140 Mb spanning the ten centromeres of maize inbred B73 resulted in the closure of 127 sequence gaps and the addition of >8.4 Mb of previously unanchored sequence (unitigs and reads) containing 24 genes, 2 Mb of CR repeat and 887 kb of CentC without including any additional sequence data. The functional centromeres of five maize chromosomes were closed completely, including a 7 Mb region spanning the extremely CR2-rich CEN2. This improved assembly, B73 RefGen_v4CEN, was completed in February 2019 and has been available at https://doi.org/10.25739/7y1p-5169, both as pseudomolecules and as centromere assemblies alone. Thus, the manual editing of existing sequence data significantly improved the centromere regions of the B73 RefGen_v4 reference genome. These data were used for centromere analyses until the release of RefGen_v5.


title: A robust method for measuring social reward in adult mice

abstract: Social interactions can be and often are rewarding. The effect of social contact strongly depends on circumstances, and the reward may be driven by varied motivational processes, ranging from parental or affiliative behaviors to investigation or aggression. Reward associated with nonreproductive interactions in rodents is measured using the social conditioned place preference (sCPP) paradigm, where a change in preference for an initially neutral context confirms reinforcing effects of social contact. Here, we revised the sCPP method and reexamined social reward in adult female mice. Contrary to earlier studies, we found that robust rewarding effects of social contact could be detected in adult (14-week-old) female C57BL/6 mice when the sCPP task was refined to remove confounding factors. Strikingly, the rewarding effects of social interaction were only observed among female siblings who remained together from birth. Contact with same-age nonsiblings was not rewarding even after 8 weeks of cohousing. Other factors critical for the social reward effect in the sCPP paradigm included the number of conditioning sessions and the inherent preference for contextual cues. Thus, we show that social interaction is rewarding in adult female mice, but this effect strictly depends on the familiarity of the interaction partners. Furthermore, by identifying confounding factors, we provide a model to study the mechanisms underlying the rewarding effects of nonreproductive social interaction in adult mice.

category: animal behavior and cognition


title: Characterization of a miniaturized ultrasound transducer customized for oral health applications

abstract: Periodontitis affects nearly 50% of Americans. Conventional clinical assessment based on periodontal probing remains the gold standard despite being operator-dependent, time-consuming, and often painful. In contrast, ultrasound imaging is non-invasive, portable, low-cost, and free of ionizing radiation. Unfortunately, most conventional ultrasound transducers can only access anterior teeth due to the size of the imaging equipment. Here, we characterized a novel and customized periodontal ultrasound transducer SS-19-128 that can examine the entire human mouth. The transducer is 1.8 cm width by 1 cm height, including 128 channels with a center frequency of 19 MHz. The axial and lateral spatial resolution at 5.5 mm depth is 102.1 m and 142.9 m, respectively. The penetration depth in tissue mimicking phantom is over 31 mm. Imaging was performed on 3 human subjects, and statistical analysis on swine jaws indicated a high agreement with the gold standard.

category: bioengineering


title: Human iPSC-Vascular Smooth Muscle Cell Spheroids Demonstrate Size-dependent Alterations in Cellular Viability and Secretory Function

abstract: Human induced pluripotent stem cells and their differentiated vascular cells have been revolutionizing the field of regenerative wound healing. These cells are shown to be rejuvenated with immense potentials in secreting paracrine factors. Recently, hiPSC-derived vascular smooth muscle cells (hiPSC-VSMC) have shown regenerative wound healing ability via their paracrine secretion. The quest to modulate the secretory function of these hiPSC-VSMC is an ongoing effort and involves the use of both biochemical and biophysical stimuli. This study explores the development and optimization of a reproducible, inexpensive protocol to form hiPSC-VSMC derived spheroids to investigate the implications of spheroid size on viability and paracrine secretion. Our data shows the successful formation of different sizes of spheroids using various amount of hiPSC-VSMC. The hiPSC-VSMC spheroids formed with 10000 cells strike an ideal balance between overall cell health and maximal paracrine secretion. The conditioned medium from these spheroids was found to be bioactive in enhancing human dermal fibroblast cell proliferation and migration. This research will inform future studies on the optimal spheroid size for regenerative wound healing applications.


title: Mesenchymal Stem Cells Sense the Toughness of Nanomaterials and Interfaces

abstract: Stem cells are known to sense and respond to a broad range of physical stimuli arising from their extra-cellular environment. In particular, the role of the mechanical properties (Youngs or shear modulus, viscoelasticity) of biomaterials has extensively been shown to have a significant impact on the adhesion, spreading, expansion and differentiation of stem cells. In turn, cells exert forces on their environment that can lead to striking changes in shape, size and contraction of associated tissues, and may result in mechanical disruption and functional failure. However, no study has so far correlated stem cell phenotype and biomaterials toughness. Indeed, disentangling toughness-mediated cell response from other mechanosensing processes has remained elusive as it is particularly challenging to uncouple Youngs or shear moduli from toughness, within a range relevant to cell-generated forces. In this report, we show how the design of macromolecular architecture of polymer nanosheets regulates interfacial toughness, independently to interfacial shear storage modulus, and how this, in turn, controls the expansion of mesenchymal stem cells at liquid interfaces.


title: RNAinsecta: A tool for prediction of pre-microRNA in insects using machine learning algorithms.

abstract: Pre-MicroRNAs are the hairpin loops which produces microRNAs that negatively regulate gene expression in several organisms. In insects, microRNAs participate in several biological processes including metamorphosis, reproduction, immune response, etc. Numerous tools have been designed in recent years to predict pre-microRNA using binary machine learning classifiers where predictive models are trained with true and pseudo pre-microRNA hairpin loops. Currently however, there are no existing tool that is exclusively designed for insect pre-microRNA detection. In this experiment we trained machine learning classifiers such as Random Forest, Support Vector Machine, Logistic Regression and k-Nearest Neighbours to predict pre-microRNA hairpin loops in insects while using Synthetic Minority Over-sampling Technique and Near-Miss to handle the class imbalance. The trained model on Support Vector Machine achieved accuracy of 92.19% while the Random Forest attained an accuracy of 80.28% on our validation dataset. These models are hosted online as web application called RNAinsecta. Further, searching target for the predicted pre-microRNA in insect model organism Drosophila melanogaster has been provided in RNAinsecta using miRanda at the backend where experimentally validated genes regulated by microRNA are collected from miRTarBase as target sites. RNAinsecta is freely available at https://rnainsecta.in

category: bioinformatics


title: Designing microplate layouts using artificial intelligence

abstract: Microplates are indispensable in large-scale biological experiments but the layout of samples and controls can have a large effect on results. Here we introduce an artificial intelligence based method for designing microplate layouts that reduces unwanted bias and limits the impact of batch effects, leading to more accurate and reliable experimental results. The method relies on constraint programming, and produces effective multiplate layouts for different experimental settings, while at the same time remaining flexible and modifiable to take into account particular laboratory settings. First we discuss the desired properties of effective microplate layouts, which we then implement as a constraint model. We show that our method produces layouts that lead to smaller errors in dose response experiments when estimating EC50/IC50 values, to the point of frequently obtaining smaller errors even when using fewer doses or replicates. We also show how effective layouts lead to more robust results in high-throughput screening experiments. Finally, we make our method easily accessible by providing a suite of tools, an online service (PLAID), a data format to enable automated construction of designs programmatically, and notebooks to evaluate and compare designs aiding decisions on the number of doses and replicates when planning microplate experiments.


title: A Novel Sequence Labeling Framework for Extracting Drug-Protein Relations from Biomedical Literature

abstract:
Automatic extracting interactions between chemical compound/drug and gene/protein is significantly beneficial to drug discovery, drug repurposing, drug design, and biomedical knowledge graph construction. To promote the development and evaluation of systems that are able to automatically detect in relations between drug and protein, the DrugProt track was organized during the BioCreative VII challenge. This paper describes our approach to this challenging task. In addition to the conventional text classification framework that has been widely used in relation extraction tasks, we propose a novel sequence labeling framework to drug-protein relation extraction. We first comprehensively compared the cutting-edge biomedical pre-trained language models for both frameworks. Then, we explored several ensemble methods to further improve the final performance. In the challenge, our best submission (i.e., the ensemble of models in two frameworks via major voting) achieved the F1-score of 0.795 on the official test set. Further, we realized the sequence labeling framework is more efficient and achieves better performance than does the text classification framework. Finally, our ensemble of the sequence labeling models with majority voting achieves the best F1-score of 0.800 on the test set.

Database URLhttps://github.com/lingluodlut/BioCreativeVII_DrugProt


title: Small molecules from Bacopa monneiri as potent inhibitors against Neurodegenerative disorders

abstract: Alzheimers is characterized by the formation of senile plaques and fibril tangles. Several methodologies have been employed to treat the disease. Albeit engineered medications which are accessible for the treatment of Alzheimers, due to their numerous side-effects, it becomes imperative to formulate and synthesize novel drug candidates. Plants could be utilized as an alternative for these manufactured medications because of their low incidental effects in contrast with the engineered drugs. Bacopa monneiri (BM) is a therapeutic plant which is accounted for to be utilized to treat NDs. Therefore, in current study an in-silico approach was carried out to evaluate the pharmacological effect of BM. Molecular Docking was carried out to screen the active phytochemicals of BM which can act as potential drug candidates against amyloid-{beta} plaques. A total of 8 biologically active phytochemicals from BM were docked against p75NTR receptor. Based on molecular docking study it was observed that the phytocompounds Bacopasaponin D and Bacopasaponin G of BM significantly fits to the active site of p75NTR. Further Molecular Dynamics simulation study was performed to examine the stability of the binding of these phytochemicals with the selected targets. Our findings suggested that the phytocompounds Bacopasaponin D and Bacopasaponin G significantly binds with p75NTR and thus might have a potential to inhibit the natural binding activity of amyloid-b plaques and act as a potential anti-neurodegenerative drug.


title: Does Tesla valve work for microscale active swimmers? - a computational study

abstract: Although the Tesla valve is well-known for its diodicity for fluid flows and pressure drops, it is not clear whether and how the resistances experienced by active swimmers in the forward and reverse directions are different. Here we carried out a computational study on the diodicity of the Tesla valve for active swimmers in the absence of fluid flows. We simulated the active Brownian motion of the swimmers in a Tesla valve, followed by examining their trajectories and quantifying the fraction of active swimmers reaching the left or right end of the Tesla valve (in the forward or reverse direction, respectively). We also estimated the first passage time of the swimmers reaching the valve ends. We confirmed that, in the absence of fluid flows, the Tesla valve shows much higher resistance to active swimmers in the reverse direction than the forward direction. The current study provides a better understanding of the interaction of the Tesla valve with active swimmers and gives insight into potential applications of the Tesla valve in the filtering and sorting of motile microbes.

category: biophysics


title: Bladder cancer organoids as a functional system to model different disease stages and therapy response

abstract: Bladder Cancer (BLCa) inter-patient heterogeneity is considered the primary cause of tumor reoccurrence and treatment failure, suggesting that BLCa patients could benefit from a more personalized treatment approach. Patient-derived organoids (PDOs) have been successfully used as a functional model for predicting drug response in different cancer types. In our study, we established BLCa PDO cultures from different BLCa stages. BLCa PDOs preserve the histological and molecular heterogeneity of the parental tumors, including their multiclonal genetic landscapes. BLCa PDOs consistently share key genetic alterations detected in parental tumors, mirroring tumor evolution in longitudinal sampling. Our drug screening pipeline was implemented using BLCa PDOs, testing both standard-of-care and additional FDA-approved compounds for other solid tumors. Integrative analysis of drug response profiles with matched PDO genomic analysis was used to determine enrichment thresholds for candidate markers of therapy resistance and sensitivity. By assessing the clinical history of longitudinally sampled cases, the clonal evolution of the disease could be determined and matched with drug response profiles. In conclusion, we have developed a clinically relevant pipeline for drug response profile assessment and discovery of candidate markers of therapy resistance.

category: cancer biology


title: Aberrant MYCN expression drives oncogenic hijacking of EZH2 as a transcriptional activator in peripheral T cell lymphoma

abstract:
Peripheral T cell lymphoma (PTCL) is a heterogeneous group of hematological cancers arising from the malignant transformation of mature T cells. In a cohort of 28 PTCL cases, we identified recurrent overexpression of MYCN, a member of the MYC family of oncogenic transcription factors. Approximately half of all PTCL cases was characterized by a MYC expression signature. Inducible expression of MYCN in lymphoid cells in a mouse model caused T cell lymphoma that recapitulated human PTCL with a MYC expression signature. Integration of mouse and human expression data identified EZH2 as a key downstream target of MYCN. Remarkably, EZH2 was found to be an essential co-factor for the transcriptional activation of the MYCN-driven gene expression program, which was independent of methyltransferase activity, but dependent on phosphorylation by CDK1. MYCN-driven T cell lymphoma was sensitive to EZH2 degradation or CDK1 inhibition, which displayed synergy with FDA-approved HDAC inhibitors.

Key points- Transcriptomic analysis of PTCL tumors reveals recurrent MYCN overexpression and the presence of a MYC signature in 50% of PTCL cases
- EZH2 is a transcriptional cofactor for the MYCN-driven gene expression program, which confers sensitivity to HDAC inhibition


title: Transcriptional landscape in BRAF wild type metastatic melanoma

abstract: Melanoma is a relative rare disease worldwide, nevertheless it has a great relevance in some countries such as in Europe. In order to shed some light upon the transcriptional profile of skin melanoma, we compared the gene expression of 6 independent tumours (all progressed towards metastatic disease and with wild type BRAF) to the expression profile of healthy melanocytes. Paraffin-embedded samples were manually microdissected to obtain enriched samples and then RNA was extracted and analysed through a microarray-based approach. An exhaustive bioinformatics analysis was performed to identify differentially expressed transcripts between the two groups as well as enriched functional terms. Overall, 50 up- and 19 down-regulated transcripts were found to be significantly changed in tumour compared to control tissue. Among the upregulated transcripts, the majority belonged to the immune response group and to proteasome, while most of the downregulated genes were related to cytosolic ribosomes. Interestingly, Gene Set Enrichment Analysis (GSEA), along with the analysis of the expression data retrieved from TCGA/GTEx databases, confirmed the general trend of downregulation affecting cytoribosome proteins. In contrast, transcripts coding for mitori-bosome proteins showed the opposite trend.


title: Pulmonary dust foci as rat pneumoconiosis lesion induced by titanium dioxide nanoparticles in 13-week inhalation study

abstract:
BackgroundMost toxicological studies on titanium dioxide (TiO2) particles to date have concentrated on carcinogenicity and acute toxicity, with few studies focusing of pneumoconiosis, which is a variety of airspace and interstitial lung diseases caused by particle-laden macrophages. The present study examined rat pulmonary lesions associated with pneumoconiosis after inhalation exposure to TiO2 nanoparticles (NPs).

MethodsMale and female F344 rats were exposed to 6.3, 12.5, 25, or 50 mg/m3 anatase type TiO2 NPs for 6 hours/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. After the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses.

ResultsNumerous milky white spots were present in the lungs after exposure 25 and 50 mg/m3 TiO2 NPs. Histopathological analysis revealed that the spots were alveolar lesions, characterized predominantly by the agglomeration of particle-laden macrophages and the presence of reactive alveolar epithelial type 2 cell (AEC2) hyperplasia. We defined this characteristic lesion as pulmonary dust foci (PDF). The PDF is an inflammatory niche, with decreased vascular endothelial cells in the interstitium, and proliferating AEC2 transformed into alveolar epithelial progenitor cells. The AEC2 in the PDF had acquired DNA damage. Based on PDF induction, the lowest observed adverse effect concentration for pulmonary disorders in male and female rats in this study was 12.5 mg/m3 and 6.3 mg/m3, respectively. The no observed adverse effect concentration for male rats was 6.3 mg/m3. There was a sex difference in lung lesion development, with females showing more pronounced lesion parameters than males.

ConclusionsInhalation exposure to TiO2 NPs caused PDF, an air-space lesion which is an alveolar inflammatory niche containing particle-laden macrophages and proliferating AEC2. This PDF histopathologically resembles some pneumoconiosis lesions (pulmonary siderosis and hard metal pneumoconiosis) in workers and lung disease in smokers, suggesting that it is an early pneumoconiosis lesion caused by exposure to TiO2 NPs in rats and a common alveolar reaction in mammals.

category: pathology


title: Frontal Cortex Gates Distractor Stimulus Encoding in Target Sensory Cortex

abstract:
Suppressing behavioral responses to distractor stimuli is a fundamental cognitive process, essential for performing goal-directed tasks. A common framework for the neuronal implementation of distractor suppression is the attenuation of distractor stimuli from early sensory to higher-order processing. However, details of the localization and mechanisms of attenuation are poorly understood. In this study, we trained mice to selectively respond (lick) to whisker deflections from one whisker field and ignore identical deflections from the opposite whisker field. We found that suppression of frontal cortex, aligned to the target stimulus or the distractor stimulus, increases responses to distractors (false alarms). By simultaneously recording spiking activity in the mouse whisker region of primary somatosensory cortex (S1), we found that frontal cortex selectively suppresses the neuronal encoding of distractor stimuli in target-aligned S1. Frontal cortex decorrelates the encoding of target and distractor stimuli in single units, thereby increasing stimulus selectivity across the population of target-aligned S1 neurons. Even before stimulus onset, effects of frontal cortex on S1 were robust and context-dependent, indicating proactive modulation. Overall, our study provides important mechanistic details about the roles of frontal cortex in gating sensory responses in sensory cortex.

Major findingsO_LIBehavior: Suppressing either target-aligned or distractor-aligned frontal cortex increases behavioral responses to distractor stimuli.
C_LIO_LIPopulation neuronal encoding: Suppressing target-aligned frontal cortex increases distractor stimulus encoding in target-aligned S1.
C_LIO_LISingle unit neuronal encoding: Frontal cortex decorrelates encoding of target and distractor stimuli in target-aligned sensory cortex.
C_LIO_LIProactive modulation: Frontal cortex modulation of sensory cortex pre-stimulus is robust and context-dependent.
C_LI


title: Frontal Cortex Gates Distractor Stimulus Encoding in Sensory Cortex


title: Affective enhancement of episodic memory is associated with widespread patterns of intrinsic functional connectivity across the adult lifespan

abstract: Subjectively arousing experiences tend to be better remembered than neutral ones. While numerous task-related neuroimaging studies have revealed the neural mechanisms associated with this phenomenon, it remains unclear how variability in the extent to which individuals show superior memory for subjectively arousing stimuli is associated with the intrinsic functional organization of their brains. Here, we addressed this issue using functional magnetic resonance imaging data collected at rest from a sample drawn from the Cambridge Centre for Ageing Neuroscience cohort (N = 269, 18-86 years). Specifically, we performed multi-voxel pattern analysis of intrinsic functional connectivity, an unbiased, data-driven approach to examine whole-brain voxel-wise connectivity patterns. This technique allowed us to reveal the most important features from the high-dimensional, whole-brain connectivity structure without a priori hypotheses about the topography and direction of functional connectivity differences. Behaviorally, both item and associative memory accuracy were enhanced for trials with affectively arousing (positive or negative) stimuli than those with neutral ones. Whole-brain multi-voxel pattern analysis of functional connectivity revealed that the affective enhancement of memory was associated with intrinsic connectivity patterns of spatially distributed brain regions belonging to several functional networks in the cerebral cortex. Post hoc seed-based brain-behavior regression analysis and principal component analysis of the resulting correlation maps showed that these connectivity patterns were in turn primarily characterized by the involvement of heteromodal association (dorsal attention, salience, and default mode) networks as well as select subcortical structures (striatum, thalamus, and cerebellum). Collectively, these findings suggest that the affective enhancement of episodic memory may be characterized as a whole-brain phenomenon, possibly supported by intrinsic functional interactions across several networks and structures in the brain.


title: Unraveling the functional attributes of the language connectome: crucial subnetworks, flexibility and variability.

abstract: Language processing is a highly integrative function, intertwining linguistic operations (processing the language code intentionally used for communication) and extra-linguistic processes (e.g., attention monitoring, predictive inference, long-term memory). This synergetic cognitive architecture requires a distributed and specialized neural substrate. Brain systems have mostly been examined at rest. However, task-related functional connectivity provides additional and valuable information about how information is processed when various cognitive states are involved. We gathered thirteen language fMRI tasks in a unique database of one hundred and fifty neurotypical adults (InLang database). The tasks were designed to assess a wide range of linguistic processes and subprocesses. From this database, we applied network theory as a computational tool to model the task-related functional connectome of language (LANG). The organization of this data-driven neurocognitive atlas of language is examined at multiple levels, uncovering its major components (or crucial subnetworks) and its anatomical and functional correlates. Furthermore, we estimate its reconfiguration as a function of linguistic demand (flexibility), or several factors such as age or gender (variability). By accounting for the multifaceted nature of language and modulating factors, this study can contribute to enrich and refine existing neurocognitive models of language. The LANG atlas can also be considered as a reference for comparative or clinical studies, involving a variety of patients and conditions.


title: Performance comparison of systemic activity correction in fNIRS for methods with and without short distance channels

abstract:
SignificanceFunctional Near Infrared Spectroscopy (fNIRS) is a promising tool for neurofeedback (NFB) or brain computer interfaces (BCIs). However, fNIRS signals are typically highly contaminated by systemic activity (SA) artifacts and, if not properly corrected, NFB or BCIs run the risk of being based on noise instead of brain activity. This risk can likely be reduced by correcting for SA, in particular when short distance channels (SDCs) are available. Literature comparing correction methods with and without SDCs is still sparse, specifically comparisons considering single trials are lacking.

AimThis study aimed at comparing the performance of SA correction methods with and without SDCs.

ApproachSemi-simulated and real motor task data of healthy elderly individuals were used. Correction methods without SDCs included a simple and a more advanced spatial filter. Correction methods with SDCs included a regression approach considering only the closest SDC and two GLM-based methods, one including all eight SDCs and one using only two a priori selected SDCs as regressors. All methods were compared to data uncorrected for SA and correction performance was assessed with quality measures quantifying signal improvement and spatial specificity at single trial level.

ResultsAll correction methods were found to improve signal quality and to enhance spatial specificity as compared to the uncorrected data. Methods with SDCs usually outperformed methods without SDCs. Correction methods with-out SDCs tended to overcorrect the data. However, the exact pattern of results and the degree of differences observable between correction methods varied between semi-simulated and real data, and also between quality measures.

ConclusionsOverall, results confirmed that both {Delta}[HbO] and {Delta}[HbR] are affected by SA and that correction meth-ods with SDCs outperform methods without SDCs. Nonetheless, improvements in signal quality can also be achieved without SDCs and should therefore be given priority over not correcting for SA.


title: EEG-representational geometries and psychometric distortions in approximate numerical judgment

abstract: When judging the average value of sample stimuli (e.g., numbers) people tend to either over- or underweight extreme sample values, depending on task context. In a context of overweighting, recent work has shown that extreme sample values were overly represented also in neural signals, in terms of an anti-compressed geometry of number samples in multivariate electroencephalography (EEG) patterns. Here, we asked whether neural representational geometries may also reflect underweighting of extreme values (i.e., compression) which has been observed behaviorally in a great variety of tasks. We used a simple experimental manipulation (instructions to average a single-stream or to compare dual-streams of samples) to induce compression or anti-compression in behavior when participants judged rapid number sequences. Model-based representational similarity analysis (RSA) replicated the previous finding of neural anti-compression in the dual-stream task, but failed to provide evidence for neural compression in the single-stream task, despite the evidence for compression in behavior. Instead, the results suggested enhanced neural processing of extreme values in either task, regardless of whether extremes were over- or underweighted in subsequent behavioral choice. We further observed more general differences in the neural representation of the sample information between the two tasks. The results suggest enhanced processing of extreme values as the brains default. Such a default raises new questions about the origin of common psychometric distortions, such as diminishing sensitivity for larger values.


title: The role of capacity constraints in Convolutional Neural Networks for learning random versus natural data

abstract: Convolutional neural networks (CNNs) are often described as promising models of human vision, yet they show many differences from human abilities. We focus on a superhuman capacity of top-performing CNNs, namely, their ability to learn very large datasets of random patterns. We verify that human learning on such tasks is extremely limited, even with few stimuli. We argue that the performance difference is due to CNNs overcapacity and introduce biologically inspired mechanisms to constrain it, while retaining the good test set generalisation to structured images as characteristic of CNNs. We investigate the efficacy of adding noise to hidden units activations, restricting early convolutional layers with an information bottleneck, and using a bounded activation function. Internal noise was the most potent intervention and the only one which, by itself, could reduce random data performance in the tested models to chance levels. We also investigated whether networks with biologically inspired capacity constraints show improved generalisation to out-of-distribution stimuli, however little benefit was observed. Our results suggest that constraining networks with biologically motivated mechanisms paves the way for closer correspondence between network and human performance, but the few manipulations we have tested are only a small step towards that goal.


title: Normative sleep spindle database and findings from 772 healthy children from birth through 18 years

abstract: Work in the last two decades has identified sleep spindles, discrete "sigma band" oscillations during stage 2 sleep, as a key oscillatory mechanism required for off-line memory consolidation. Although, sleep spindles are known to evolve concomitant with brain maturation and reflect cognitive function across the lifespan, the details of this developmental trajectory are unknown. To address this, we curated a database of sleep electroencephalograms from 772 developmentally normal children to characterize spindles from birth through 18 years. After validating an automated spindle detector against ~20,000 hand-marked spindles across ages, we demonstrate that sleep spindle features follow distinct age-specific patterns in distribution, rate, duration, frequency, estimated refractory period, and inter-hemispheric spindle lag. These data expand our current knowledge of normal physiological brain development and provide a large normative database to detect deviations in sleep spindles to aid discovery, biomarker development, and diagnosis in pediatric neurodevelopmental disorders.


title: Endo-lysosomal assembly variations among Human Leukocyte Antigen class I (HLA-I) allotypes

abstract: The extreme polymorphisms of HLA-I proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes (CTL). The canonical endoplasmic reticulum (ER) HLA-I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA-I molecules. We hypothesized variable influences of HLA-I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA-I are variable. For example, in moDCs, compared with HLA-B*08:01, HLA-B*35:01 displays reduced cell-surface stability and greater receptivity to exogenous peptide. Perturbations of endo-lysosomal pH negatively affect the surface expression of moDC HLA-B*35:01 but not HLA-B*08:01, causing HLA-B*35:01 accumulation in LAMP1+ compartments. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for HLA-B*35:01. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA-I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA-I expression and increase the efficiency of cross-presentation.

category: immunology


title: Identification of large offspring syndrome during pregnancy through ultrasonography and maternal blood transcriptome analyses

abstract: The use of assisted reproductive technologies (ART) in cattle can result in large/abnormal offspring syndrome (LOS/AOS) which is characterized by macrosomia. LOS can cause dystocia and lead to the death of dam and calf. Currently, no test exists to identify LOS pregnancies. We hypothesized that fetal ultrasonography and/or maternal blood markers are useful to identify LOS. Bovine fetuses were generated by artificial insemination (control) or ART. Fetal ultrasonographies were taken on gestation day 55 (D55) and fetal collections performed on D56 or D105 (gestation in cattle {approx}280 days). ART fetuses weighing [≥]97 percentile of the control weight were considered LOS. Ultrasonography results show that the product of six D55 measurements can be used to identify extreme cases of LOS. To determine whether maternal blood can be used to identify LOS, leukocyte mRNA from 23 females was sequenced. Unsupervised hierarchical clustering grouped the transcriptomes of the two females carrying the two largest LOS fetuses. Comparison of the leukocyte transcriptomes of these two females to the transcriptome of all other females identified several misregulated transcripts on gestation D55 and D105 with LOC783838 and PCDH1 being misregulated at both time-points. Together our data suggest that LOS is identifiable during pregnancy in cattle.


title: String-Pulling as a Behavioral Assessment of Skilled Forelimb Motor Function in a Middle Cerebral Artery Occlusion Rat Model

abstract:
Stroke is a leading cause of long-term disability in humans and frequently results in bilateral impairments in fine motor control. Many behavioral tasks used to assess rodent models of stroke evaluate a single limb; however, recent work has demonstrated that bilateral hand-over-hand movements used to pull in a string assess skilled movement of both hands.

Devascularization focused on the forelimb portion of sensorimotor cortex has been observed to produce persistent disruptions in the topographical organization of string-pulling behavior. The current study examined changes in string-pulling after a more clinically relevant rodent model of stroke via middle cerebral artery occlusion (MCAO). Detailed movement analyses revealed disruptions in the bilateral organization of string-pulling and fine motor control of both hands. Rats missed the string more often with both hands, and when the string was missed on the impaired side, rats continued to cycle through subcomponents of string-pulling behavior as if the string were grasped in the hand. Rats also failed to make a grasping motion with the impaired hand when the string was missed and instead, demonstrated an open-handed raking-like motion. No differences were found in time to approach or to complete the string-pulling task to obtain a reward, demonstrating the importance of using a detailed functional analysis of movement to detect changes in performance. String-pulling behavior is sensitive at detecting changes in bilateral rhythmical hand control following MCAO providing a foundation for future work to investigate other models of stroke and to evaluate the efficacy of therapeutic interventions that enhance neuroplasticity.


title: Quantitative crosslinking and mass spectrometry determine binding interfaces and affinities mediating kinetochore stabilization

abstract: Crosslinking and mass spectrometry (XLMS) are used in integrative structural biology to acquire spatial restraints. We found a dependency between crosslink distances and intensities and developed a quantitative workflow to simultaneously estimate apparent dissociation constants (KD) of contacts within multi-subunit complexes and to aid interface prediction. Quantitative XLMS was applied to study the assembly of the macromolecular kinetochore complex, which is built on centromeric chromatin and establishes a stable link to spindle microtubules in order to segregate chromosomes during cell division. Inter-protein crosslink intensities facilitated determination of phosphorylation-induced binding interfaces and affinity changes. Phosphorylation of outer and inner kinetochore proteins mediated cooperative kinetochore stabilization and decreased the KD values of its interactions to the centromeric nucleosome by ~200-fold, which was essential for cell viability. This work demonstrates the potential of quantitative XLMS for characterizing mechanistic effects on protein assemblies upon post-translational modifications or cofactor interaction and for biological modeling.

category: biochemistry


title: Imaging optically thick tissues simply and reproducibly: a practical guide to Lightsheet Macroscopy

abstract: Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting glitchy and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardized 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes should be non-aberrating with objective numerical apertures less than 0.65, and present an inexpensive tool for alignment and calibration of standard lightsheet parameters. Mouse embryo, liver and heart imaging are demonstrated as examples with practical recommendations for acquisition and post-processing.


title: Full-mouth photoacoustic/ultrasound imaging of the periodontal pocket with a compact intraoral transducer

abstract: Periodontitis is a public issue and imaging periodontal pocket is important to evaluate periodontitis. Regular linear transducers have limitations in imaging the posterior teeth due to their geometry restrictions. Here we characterized a transducer that can image the entire human mouth including assessment of periodontal pockets via a combination of photoacoustic and ultrasound imaging. Unlike conventional transducer design, this device has a toothbrush-shaped form factor with a side-view transducer to image molars (total size: 1 x 1.9 cm). A laser diode was integrated as the light source to reduce the cost and size and facilitates clinical transition. The in vivo imaging of a molar of a periodontal patient demonstrated that the transducer could image in the posterior area of gum in vivo; the value determined by imaging was within 7% of the value measured clinically.


title: StORF-Reporter: Finding Genes between Genes

abstract:
Large regions of prokaryotic genomes are currently without any annotation, in part due to well-established limitations of annotation tools. We present StORF-Reporter, a tool that takes as input an annotated genome and returns missed CDS genes from the unannotated regions. StORF-Reporter consists of two parts. The first part begins with the extraction of unannotated regions from an annotated genome. Next, Stop-ORFs (StORFs) are identified in these unannotated regions. StORFs are Open Reading Frames that are delimited by stop codons.

We show that this methodology recovers complete coding sequences (with/without similarity to known genes) which were missing from both canonical and novel genome annotations. StORF-Reporter recovered sequences that exhibited high levels of sequence identity to proteins in the SwissProt database and the proteomes of the genome they were identified from (gene-duplicates). We inspected in detail the results from the genomes of six model organisms, the pangenome of Escherichia coli, and a further 6,223 annotated prokaryotic genomes of 179 genera from the Ensembl Bacteria database. StORF-Reporter was able to extend the core, soft-core and accessory gene-collections, and identify novel gene families and families which were extended into additional genera, not previously identified in the canonical annotations. Many of the gene families these sequences form are routinely misreported or completely omitted by state-of-the-art annotation methods.


title: ShrinkCRISPR : A flexible method for differential fitness analysis of CRISPR-Cas9 screen data.

abstract:
CRISPR screens provide large-scale assessment of cellular gene functions. Pooled libraries typically consist of several single guide RNAs (sgRNAs) per gene, for a large number of genes, which are transduced in such a way that every cell receives at most one sgRNA, resulting in the disruption of a single gene in that cell. This approach is often used to investigate effects on cellular fitness, by measuring sgRNA abundance at different time points. Comparing gene knockout effects between different cell populations is challenging due to variable cell-type specific parameters and between replicates variation. Failure to take those into account can lead to inflated or false discoveries.

We propose a new, flexible approach called ShrinkCRISPR that can take into account multiple sources of variation. Impact on cellular fitness between conditions is inferred by using a mixed-effects model, which allows to test for gene-knockout effects while taking into account sgRNA-specific variation. Estimates are obtained using an empirical Bayesian approach. ShrinkCRISPR can be applied to a variety of experimental designs, including multiple factors. In simulation studies, we compared ShrinkCRISPR results with those of drugZ and MAGeCK, common methods used to detect differential effect on cell fitness. ShrinkCRISPR yielded as many true discoveries as drugZ using a paired screen design, and outperformed both drugZ and MAGeCK for an independent screen design. Although conservative, ShrinkCRISPR was the only approach that kept false discoveries under control at the desired level, for both designs. Using data from several publicly available screens, we showed that ShrinkCRISPR can take data for several time points into account simultaneously, helping to detect early and late differential effects.

ShrinkCRISPR is a robust and flexible approach, able to incorporate different sources of variations and to test for differential effect on cell fitness at the gene level. These improve power to find effects on cell fitness, while keeping multiple testing under the correct control level and helping to improve reproducibility. ShrinkCrispr can be applied to different study designs and incorporate multiple time points, making it a complete and reliable tool to analyze CRISPR screen data.


title: Phasor-Based Multi-Harmonic Unmixing for in-vivo Hyperspectral Imaging

abstract: Hyperspectral imaging (HSI) is paramount technique in biomedical science, however, dealing with the data it produces is challenging. Traditional unmixing relies on algorithms that need spectroscopic parameters from the fluorescent species in the sample. The phasor-based multi-harmonic unmixing method is a model-free and intuitive approach, that requires only the empirical measurement of the pure species and the phasor approach in multi-harmonic. Using simulations, we demonstrate the feasibility of the approach for up to 5 components and adventure in the use of adding an 6th unknown component as autofluorescence. The method computes the pixel-wise photon fraction of every spectral component, and the simulations show that it can be successfully used in the habitual photon count regimes dealt with in every day imaging experiments (between 101 and 103). We additionally test the method in living cells, using 5 common commercial dyes for organelle labeling and we easily and accurately separate them. Finally, we challenge the method by introducing a solvatochromic probe, 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (LAURDAN), intended to measure membrane dynamics on specific subcellular membrane-bound organelles by taking advantage of the linear combination between the organelle probes and LAURDAN. We succeed in monitoring the membrane order at the Golgi apparatus, Mitochondria, and plasma membrane, and quantitatively compared them. The phasor-based multi-harmonic unmixing method can help to expand the outreach of HSI and democratize its use by the community for it does not require of special know-how.


title: RNA helicase DDX5 mediates adaptive response to multi-kinase inhibitors in liver cancer

abstract:
ObjectiveTo determine the role of RNA helicase DDX5 in sorafenib/multi-tyrosine kinase inhibitor (mTKI) response. Sorafenib and mTKIs downregulate DDX5 in vitro and preclinical hepatocellular carcinoma (HCC) models. In turn, sorafenib-mediated DDX5 downregulation activates Wnt/{beta}-catenin and non-canonical NF-{kappa}B signaling, resulting in ferroptosis escape and mTKI resistance.

Design and ResultsMolecular, pharmacologic and bioinformatic approaches were employed in human HCC cell lines, preclinical HCC models, and HCCs from TCGA. Earlier studies linked sorafenib effectiveness to ferroptosis. Herein we demonstrate sorafenib/mTKIs downregulate DDX5 in vitro and in vivo. To understand the effect of DDX5 downregulation, we compared TCGA-derived HCCs expressing low vs. high DDX5 focusing on ferroptosis-related genes. Glutathione Peroxidase 4 (GPX4), a key ferroptosis regulator, was significantly overexpressed in DDX5LOW HCCs. Importantly, DDX5-knockdown (DDX5KD) HCC cell lines lacked lipid peroxidation by GPX4 inhibition, indicating DDX5 downregulation suppresses ferroptosis. RNAseq of wild type vs. DDX5KD cells untreated or treated with sorafenib, identified a unique set of genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps genes from Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways, including NF-{kappa}B inducing Kinase required for non-canonical NF-{kappa}B activation. Pharmacologic inhibition of these pathways in combination with sorafenib reduced DDX5KD cell viability. Mechanistically, sorafenib-mediated NIK expression induced NRF2 transcription, while DDX5KD extended NRF2 half/life by stabilizing p62/SQSTM1, enhancing GPX4 expression and ferroptosis escape.

ConclusionSorafenib/mTKI-mediated DDX5 downregulation results in adaptive mTKI resistance by enhancing NRF2 expression, leading to ferroptosis escape. We propose inhibition of the pathways leading to NRF2 expression will enhance the therapeutic effectiveness of sorafenib/mTKIs.

O_LIWhat is already known on this topic -
In advanced HCC, mTKIs/sorafenib, offer limited survival benefits due to resistance. Combination of VEGF (bevacizumab) and PD-L1 antibody (atezolizumab) has led to its adoption as first line treatment; however, mTKIs are still widely used in patients with contra-indications to bevacizumab; also, in atezolizumab/bevacizumab failure, mTKIs are the gold standard in second or later-line systemic therapy, emphasizing the importance of delineating the mechanism of mTKI resistance.
C_LIO_LIWhat this study adds -
We show mTKIs and sorafenib downregulate the RNA helicase DDX5. This downregulation of DDX5 by sorafenib enables activation of Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways, leading to expression of genes that enable escape from ferroptotic cell death.
C_LIO_LIHow this study might affect research, practice or policy
The Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways activated by sorafenib/DDX5 downregulation can serve as druggable targets to enhance the anti-cancer effect of mTKIs by inducing ferroptosis, thereby opening-up new therapeutic directions against mTKI resistance.
C_LI


title: Shedding Light on the Black Box of a Neural Network Used to Detect Prostate Cancer in Whole Slide Images by Occlusion-Based Explainability

abstract:
Diagnostic histopathology is facing increasing demands due to aging populations and expanding healthcare programs. Semi-automated diagnostic systems employing deep learning methods are one approach to alleviate this pressure, with promising results for many routine diagnostic procedures. However, one major issue with deep learning approaches is their lack of interpretability--after adequate training they perform their assigned tasks admirably, but do not explain how they reach their conclusions. Knowledge of how a given method performs its task with high sensitivity and specificity would be advantageous to understand the key features responsible for diagnosis, and should in turn allow fine-tuning of deep learning approaches.

This paper presents a deep learning-based system for carcinoma detection in whole slide images of prostate core biopsies, achieving state-of-the-art performance; 100% area under curve and sensitivity of 0.978 for 8 detected false positives on average per slide.

Furthermore, we investigated various methods to extract the key features used by the neural network for classification. Of these, the technique called occlusion, adapted to whole slide images, analyzes the sensitivity of the detection system to changes in the input images. This technique produces heatmaps indicating which parts of the image have the strongest impact on the systems output that a histopathologist can examine to identify the networks reasoning behind a given classification. Reassuringly, the heatmaps identified several prevailing histomorphological features characterizing carcinoma, e.g. single-layered epithelium, presence of small lumina, and hyperchromatic nuclei with halos.

A convincing finding was the recognition of their mimickers in non-neoplastic tissue. The results show that the neural network approach to recognize prostatic cancer is similar to that taken by a human pathologist at medium optical resolution. The use of explain-ability heatmaps provides added value for automated digital pathology to analyze and fine-tune deep learning systems, and improves trust in computer-based decisions.


title: An untargeted metabolomics analysis of exogenous chemicals in human milk and transfer to the infant

abstract: Human milk is the optimal infant nutrition. However, while human-derived metabolites such as lipids and oligosaccharides in human milk are regularly reported, the presence of exogenous chemicals (such as drugs, food, and synthetic compounds) are often not addressed. To understand the types of exogenous compounds that might be present, human milk (n=996) was analyzed by untargeted metabolomics. This analysis revealed that lifestyle molecules such as medications and their metabolites, food, industrial sources such as plasticizers, cosmetics, microbial molecules, and other personal care products are found in human milk. We provide further evidence that some of these lifestyle molecules are also detectable in the newborns stool. Thus, this study gives important insight into the types of exposures infants receiving human milk might ingest due to the lifestyle choices, exposure, or medical status of the lactating parent.

category: pharmacology and toxicology


title: Mass red blood cell extravasation and tubular uptake results in toxic injury to the tubules during kidney ischemia from venous clamping

abstract:
Vascular congestion represents densely packed red blood cells (RBC) in the kidney circulation. In this study we tested the hypothesis that  vascular congestion directly promotes tubular injury.

All studies were performed in male and female Wistar-Kyoto rats. Vascular congestion and tubular injury were histologically examined between renal venous clamping, renal arterial clamping and venous clamping of blood free kidneys. Vessels were occluded for either 15 or 45 minutes without reperfusion.

We found that venous clamping resulted in greater vascular congestion than arterial clamping, particularly in the outer-medullary region (P<0.001). Venous clamping resulted in significant tubular injury, including cell swelling, tubular necrosis and luminal cast formation following 45-minutes of occlusion. Tubular injury was significantly less following 45 minutes of arterial clamping (P<0.001). Numerous red droplets were observed within tubular cells. These were most prominent in injured cells and following venous clamping. Electron microscopy identified these droplets as RBCs and indicated that RBCs from congested renal capillaries were extravasated and taken up by tubular cells. Labelling RBCs with a cell permanent dye confirmed tubular uptake of RBCs. Demonstrating that tubular injury was due to RBC uptake, tubular injury was markedly reduced in blood free kidneys following venous clamping (P<0.001).

Our data demonstrate that congestion of the kidney results in the rapid, mass extravasation and uptake of RBCs by tubular cells causing toxic injury to the tubules. Tubular toxicity from extravasation of RBCs appears to be a major component of tubular injury in ischemic AKI which has not previously been recognized.

category: physiology


title: Influence of ambient day light variations and age on the Iris-pupillary area ratio in beef cattle

abstract:
IntroductionIris-pupillary area ratio (IPR) is an objective and non-invasive index of autonomic nervous system activity and stress, which can be confounded by light intensity or age of an individual. Evaluating the influence of ambient light intensity variations or age on IPR is necessary to improve the validity of IPR for its clinical application in objective assessment of welfare and stress.

Materials and MethodsIn this study we evaluated the influence of ambient light intensity variations and age on IPR in beef cattle breeds (Limousin, Belgian Blue and Charolais) and analysed the data using correlation statistics.

ResultsThe correlation between the light intensity (20 to 500 lux; r = 0.22, p = 0.08) or age (10 to 145 months, r = 0.20, p = 0.12) and IPR was weak and statistically not significant. A sub-group analysis assessing the influence of gender on correlation between the light intensity and IPR also was not statistically significant.

ConclusionOur results suggest that within the ambient light intensity (20 to 500 lux) and age (10 to 145 months) the variation in IPR is minimal. Hence our results validate the merit of IPR in objectively measuring autonomic activity/stress and demonstrate the practicality of using IPR for welfare assessment under ambient light conditions in wider age cohorts of beef cattle.


title: Effect of tempol on post-thaw semen parameters and fertility during chicken semen cryopreservation

abstract: The present study evaluated addition of tempol during chicken semen cryopreservation on post-thaw semen parameters and fertility. Adult PD-1 line semen was cryopreserved using 4% dimethyl sulfoxide (DMSO) in Sasaki diluent (SD). In the semen cryomixture tempol (1 and 5 mM) was added at final concentrations. The semen with additives were filled in 0.5 ml French straws and exposed to liquid nitrogen vapours for 30 min and then stored in liquid nitrogen. The semen straw was thawed at 5{degrees}C for 100 sec and evaluated for sperm motility, live, abnormal and acrosome intact sperm. The seminal plasma was evaluated for lipid peroxidation. Fertilizing potential of the cryopreserved sperm was evaluated after insemination in the PD-1 line hens. The post-thaw sperm parameters were significantly (P<0.05) lower in the cryopreserved groups. The lipid peroxidation was significantly (P<0.05) higher in cryopreserved groups. The fertility was significantly (P<0.05) lower in all the cryopreserved groups. In conclusion, addition of tempol to cryopreservation mixture did not improve the post-thaw semen parameters or fertility.


title: Characterization of EDS1-independent plant defense responses against bacterial pathogens using Duckweed/Pseudomonas pathosystems

abstract: ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) mediates the induction of defense responses against pathogens in most land plants. However, it has recently been shown that a few species have lost EDS1. It is unknown how defense against disease unfolds and evolves in the absence of EDS1. Here we utilize duckweeds; a collection of aquatic species that lack EDS1, to investigate this question. We successfully established duckweed-Pseudomonas pathosystems and were able to characterize pathogen-induced responses in an immune system that lacks the EDS1 signaling pathway. We show that the copy number of infection-associated genes and the infection-induced transcriptional responses of duckweeds differ from that of other model species. Moreover, we show that the conservation of canonical Microbe Triggered Immunity and Effector Triggered Immunity pathways varies between duckweed species. This work shows that pathogen defense has evolved along different trajectories and uncovers alternative genomic and transcriptional reprogramming. Specifically, the miAMP1 domain containing proteins, which are absent in Arabidopsis, show pathogen responsive upregulation in duckweeds. Despite such divergence between Arabidopsis and duckweed species, we find evidence for the conservation of upregulation of certain genes and the role of hormones in response to disease. Our work highlights the importance of expanding the pool of model species to study defense responses that have evolved in the plant kingdom, including those independent of EDS1.

category: plant biology


title: Relative stability of mRNA and protein severely limits inference of gene networks from single-cell mRNA measurements

abstract: Inference of gene regulatory networks from single-cell expression data, such as single-cell RNA sequencing, is a popular problem in computational biology. Despite diverse methods spanning information theory, machine learning, and statistics, it is unsolved. This shortcoming can be attributed to measurement errors, lack of perturbation data, or difficulty in causal inference. Yet, it is not known if kinetic properties of gene expression also cause an issue. We show how the relative stability of mRNA and protein hampers inference. Available inference methods perform benchmarking on synthetic data lacking protein species, which is biologically incorrect. We use a simple model of gene expression, incorporating both mRNA and protein, to show that a more stable protein than mRNA can cause loss in correlation between the mRNA of a transcription factor and its target gene. This can also happen when mRNA and protein are on the same timescale. The relative difference in timescales affects true interactions more strongly than false positives, which may not be suppressed. Besides correlation, we find that information-theoretic nonlinear measures are also prone to this problem. Finally, we demonstrate these principles in real single-cell RNA sequencing data for over 1700 yeast genes.

category: systems biology


title: Long-range inhibition synchronizes and updates prefrontal task activity

abstract: Changes in patterns of activity within the medial prefrontal cortex enable rodents, non-human primates, and humans to update their behavior to adapt to changes in the environment, e.g., during cognitive tasks1-5. Within medial prefrontal cortex, inhibitory neurons expressing parvalbumin are important for updating strategies in a rule shift task6-8. Nevertheless, causal mechanisms through which parvalbumin neurons recruit specific circuits to produce prefrontal network dynamics that switch from maintaining to updating task-related patterns of activity remain unknown. Here we identify a new long-range inhibitory projection from prefrontal parvalbumin neurons to the contralateral cortex that mediates this process. Whereas nonspecifically inhibiting all callosal projections does not prevent mice from learning rule shifts, selectively inhibiting these callosal parvalbumin projections disrupts rule shift learning, desynchronizes gamma-frequency activity that is necessary for learning8, and suppresses changes in prefrontal activity patterns that normally accompany rule shifts. Thus, callosal parvalbumin projections switch prefrontal circuits from maintaining to updating behavioral strategies by synchronizing callosal communication and preventing it from inappropriately maintaining outdated neural representations. These findings may explain how deficits in prefrontal parvalbumin neurons and gamma synchrony cause impaired behavioral flexibility in schizophrenia9-10, and identify long-range projections from prefrontal parvalbumin neurons as a key circuit locus for understanding and treating these deficits.

category: neuroscience


title: Impact of 98 LRRK2 variants linked to Parkinson's Disease on kinase activity and microtubule binding

abstract: Mutations enhancing the kinase activity of LRRK2 cause Parkinsons disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 98 LRRK2 variants linked to PD, including previously described pathogenic mutations, and identify 22 variants that robustly stimulate LRRK2 kinase activity. These include variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q)] as well as variants residing within the GTPase domain, predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORB (S1761R, L1795F)] and CORB:CORB dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer910/pSer935), consistent with the notion that the active kinase conformation blocks their phosphorylation. We find that the R1628P variant that has been linked to PD does not stimulate LRRK2 activity. We observe that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation at Thr73 compared to an in vitro kinase assay, as only a minority of activating variants enhance the activity of immunoprecipitated LRRK2, comprising the CORB (Y1699C, R1728H, S1761R) and kinase (G2019S, I2020T, T2031S) variants. Twelve variants including some that activated LRRK2, suppressed microtubule association in the presence of a Type 1 kinase inhibitor [ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA (A1589S), CORB (Y1699C, R1728H/L) and WD40 (R2143M, S2350I, G2385R)]. Our findings will aid in understanding the impact of these variants on LRRK2 kinase activation, stimulate work to reveal mechanisms by which variants impact biology, and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 clinical trials.


title: Downregulating α-synuclein in iPSC-derived dopaminergic neurons mimics electrophysiological phenotype of the A53T mutation

abstract: Parkinsons disease (PD) is a common debilitating neurodegenerative disorder, characterized by a progressive loss of dopaminergic (DA) neurons. Mutations, gene dosage increase, and single nucleotide polymorphisms in the -synuclein-encoding gene SNCA either cause or increase the risk for PD. However, neither the function of -synuclein in health and disease, nor its role throughout development is fully understood. Here, we introduce DeePhys, a new tool that allows for data-driven functional phenotyping of neuronal cell lines by combining electrophysiological features inferred from high-density microelectrode array (HD-MEA) recordings with a robust machine learning workflow. We apply DeePhys to human induced pluripotent stem cell (iPSC)-derived DA neuron-astrocyte co-cultures harboring the prominent SNCA mutation A53T and an isogenic control line. Moreover, we demonstrate how DeePhys can facilitate the assessment of cellular and network-level electrophysiological features to build functional phenotypes and to evaluate potential treatment interventions. We find that electrophysiological features across all scales proved to be highly specific for the A53T phenotype, enabled to predict the genotype and age of individual cultures with high accuracy, and revealed a mutant-like phenotype after downregulation of -synuclein.


title: A neural decision signal during internal sampling from working memory

abstract: How humans transform sensory information into decisions that steer purposeful behaviour is a central question in psychology and neuroscience that is traditionally investigated during the sampling of external sensations. The decision-making framework of gradual information sampling toward a decision has been proposed also to apply when sampling internal sensory evidence from memory. However, neural evidence for this proposal remains lacking. Here we report that sampling internal visual representations from working memory elicits a scalp-EEG potential associated with gradual evidence accumulation - the Central Parietal Positivity (CPP). Consistent with an evolving decision process, we show how this signal (i) builds up to, and scales with, the time participants require to reach a decision about the cued memory content and (ii) is amplified when having to select (decide) among multiple contents in working memory. These results bring the electrophysiology of decision making into the domain of working memory and suggest that variability in memory-guided behaviour may be driven (at least in part) by variations in the sampling of our inner mental contents.


title: Munc13 supports vesicle fusogenicity after disrupting active zone scaffolds and synaptic vesicle docking

abstract: Active zones consist of protein scaffolds that are tightly attached to the presynaptic plasma membrane. They dock and prime synaptic vesicles, couple them to Ca2+ entry, and target neurotransmitter release to postsynaptic receptor domains. Simultaneous RIM+ELKS ablation disrupts these scaffolds, abolishes vesicle docking and removes active zone-targeted Munc13, but some vesicles remain releasable. This enduring vesicular fusogenicity may be Munc13-independent or be mediated by non-active zone-anchored Munc13. We tested its Munc13-dependence by ablating Munc13-1 and Munc13-2 on top of RIM+ELKS in cultured hippocampal neurons. The hextuple knockout synapses lacked docked vesicles, but other ultrastructural features were near-normal despite the strong genetic manipulation. Removing Munc13 in addition to RIM+ELKS further impaired action potential-evoked release by decreasing the remaining pool of releasable vesicles. We conclude that Munc13 can support some fusogenicity without RIM and ELKS, and that presynaptic recruitment of Munc13, even without active zone-anchoring, suffices to generate some fusion-competent vesicles.


title: Spatially resolved transcriptomics reveals innervation-responsive functional clusters in skeletal muscle

abstract:
Striated muscle is a highly organized structure composed by well-defined anatomical domains with integrated but distinct assignments. So far, the lack of a direct correlation between tissue architecture and gene expression has limited our understanding of how each unit responds to physio-pathologic contexts.

Here, we show how the combined use of spatially resolved transcriptomics and immunofluorescence can bridge this gap by enabling the unbiased identification of such domains and the characterization of their response to external perturbations. Using a spatiotemporal analysis, we followed the changes in the transcriptomics profile of specific domains in muscle in a model of denervation. Furthermore, our approach allowed us to identify the spatial distribution and nerve dependence of atrophic signalling pathway and polyamine metabolism to glycolytic fibers. Indeed, we demonstrate a pronounced alteration of polyamine homeostasis upon denervation. Our dataset will serve as a resource for future studies of the mechanisms underlying skeletal muscle homeostasis and innervation.

Graphical Abstract

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category: molecular biology


title: Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

abstract: The enzyme soybean lipoxygenase provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen deuterium exchange experiments to detect a radiating cone of aliphatic side chains that extends from the iron active site of SLO to the protein-solvent interface. Employing eight variants of SLO, nanosecond fluorescence Stokes shifts have been measured using a probe appended to the identified surface loop. We report a remarkable identity of the enthalpies of activation for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within the identified thermal network. While the role of dynamics in enzyme function has been predominantly attributed to a rapidly equilibrating protein conformational landscape, these new data implicate a rapid and cooperative protein quake as the origin of the thermal activation of SLO. A mechanism of catalysis is presented that combines a distributed conformational landscape with a long range and site-specific thermal quake.