bluuebunny/crossref_metadata_2025 huggingface dataset:
Biomass formation and sugar release efficiency ofPopulusmodified by altered expression of a NAC transcription factor
Authors: Payyavula Raja S , Badmi Raghuram , Jawdy Sara S , Rodriguez Miguel , Gunter Lee , Sykes Robert W , Winkeler Kimberly A. , Collins Cassandra M. , Rottmann William H. , Chen Jin-Gui , Yang Xiaohan , Tuskan Gerald A , Kalluri Udaya C
Published: October 2021

Woody biomass is an important feedstock for biofuel production. Manipulation of wood properties that enable efficient conversion of biomass to biofuel reduces cost of biofuel production. Wood cell wall composition is regulated at several levels that involve expression of transcription factors such as wood-/secondary cell wall-associated NAC domains (WND or SND). InArabidopsis thaliana, SND1regulates cell wall composition through activation of its down-stream targets such as MYBs. The functional aspects ofSND1homologs in the woodyPopulushave been studied through transgenic manipulation. In this study, we investigated the role ofPdWND1B, Populus SND1sequence ortholog, in wood formation using transgenic manipulation through over-expression or silencing under the control of a vascular-specific4-coumarate-CoAligase (4CL) promoter. As compared to control plants,PdWND1B-RNAi plants were shorter in height, with significantly reduced stem diameter and dry biomass, whereas there were no significant differences in growth and productivity ofPdWND1Bover-expression plants. Conversely,PdWND1Bover-expression lines showed a significant reduction in cellulose and increase in lignin content, whereas there was no significant impact on lignin content of down-regulated lines. Stem carbohydrate composition analysis revealed a decrease in glucose, mannose, arabinose, and galactose, but an increase in xylose in the over-expression lines. Transcriptome analysis revealed upregulation of several downstream transcription factors and secondary cell wall related structural genes in thePdWND1Bover-expression lines that corresponded to significant phenotypic changes in cell wall chemistry observed inPdWND1Boverexpression lines. Relative to the control, glucose release and ethanol production from stem biomass was significantly reduced in over-expression lines but appeared enhanced in the RNAi lines. Our results show thatPdWND1Bis an important factor determining biomass productivity, cell wall chemistry and its conversion to biofuels inPopulus.
Using TrackMate to Analyze Drosophila Larval and Adult Locomotion
Authors: Omelchenko Alisa A. , Huda Ainul , Castaneda Allison N. , Vaden Thomas J. , Ni Lina
Published: October 2021

Drosophila adult and larvae exhibit sophisticated behaviors that are widely used in development, synaptic transmission, sensory physiology, and learning and memory research. Many of these behaviors depend on locomotion, the ability of an animal to move. However, the statistical analysis of locomotion is not trivial. Here we use an open-source Fiji plugin TrackMate to track the locomotion of Drosophila adults and larvae. We build optimal experimental setups to rapidly process recordings by Fiji and analyze by TrackMate. We also provide tips for analyzing non-optimal recordings. TrackMate extracts the X and Y positions of an animal on each frame of an image sequence or a video. This information allows for generating moving trajectories, calculating moving distances, and determining preference indices in two-choice assays. Notably, this free-cost analysis method does not require programming skills.

Summary statement

This study uses an open-source Fiji plugin TrackMate to computationally analyze Drosophila adult and larval behavioral assays, which does not require programming skills.
Sustained Released of Bioactive Mesenchymal Stromal Cell-Derived Extracellular Vesicles from 3D-Printed Gelatin Methacrylate Hydrogels
Authors: Born Louis J. , McLoughlin Shannon T. , Dutta Dipankar , Mahadik Bhushan , Jia Xiaofeng , Fisher John P. , Jay Steven M.
Published: October 2021

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate therapeutic potential of MSC EV-laden GelMA bioinks.
Ex VivoandIn VivoCD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26)
Authors: Hemsath Jack R. , Liaci A. Manuel , Rubin Jeffrey D. , Parrett Brian J. , Lu Shao-Chia , Nguyen Tien V. , Turner Mallory A. , Chen Christopher Y. , Cupelli Karolina , Reddy Vijay S. , Stehle Thilo , Liszewski M. Kathryn , Atkinson John P. , Barry Michael A.
Published: October 2021

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against SARS-CoV-2 and HIV-1. Yet, its primary receptor portfolio remains controversial, potentially including sialic acid, CAR, integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned by the inability to co-crystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infectionin vitroandin vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domains CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (IM) injection, but not after intranasal (IN) administration. Ad26 transduction was 10-fold lower than Ad5 after intratumoral (IT) injection of CD46-expressing tumors. Ad26 transduction of liver was 1000-fold lower than Ad5 after intravenous (IV) injection. These data demonstrate the use of CD46 by Ad26 under certain situations, but also show that the receptor has little consequence by other routes of administration. Finally, IV injection of high doses of Ad26 into CD46 mice induced release of liver enzymes in the bloodstream and reduced white blood cell counts, but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during COVID-19 vaccination with this serotype of adenovirus.

IMPORTANCE

Human species D Ad26 is being pursued as a low seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection, and its role in virus biology, vaccine efficacy, and importantly, in vaccine safety.
A whole-brain monosynaptic input connectome to neuron classes in mouse visual cortex
Authors: Yao Shenqin , Wang Quanxin , Hirokawa Karla E. , Ouellette Benjamin , Ahmed Ruweida , Bomben Jasmin , Brouner Krissy , Casal Linzy , Caldejon Shiella , Cho Andy , Dotson Nadezhda I. , Daigle Tanya L. , Egdorf Tom , Enstrom Rachel , Gary Amanda , Gelfand Emily , Gorham Melissa , Griffin Fiona , Gu Hong , Hancock Nicole , Howard Robert , Kuan Leonard , Lambert Sophie , Lee Eric Kenji , Luviano Jennifer , Mace Kyla , Maxwell Michelle , Mortrud Marty T. , Naeemi Maitham , Nayan Chelsea , Ngo Nhan-Kiet , Nguyen Thuyanh , North Kat , Ransford Shea , Ruiz Augustin , Seid Sam , Swapp Jackie , Taormina Michael J , Wakeman Wayne , Zhou Thomas , Nicovich Philip R. , Williford Ali , Potekhina Lydia , McGraw Medea , Ng Lydia , Groblewski Peter A. , Tasic Bosiljka , Mihalas Stefan , Harris Julie A , Cetin Ali , Zeng Hongkui
Published: October 2021

Identification of the structural connections between neurons is a prerequisite to understanding brain function. We developed a pipeline to systematically map brain-wide monosynaptic inputs to specific neuronal populations using Cre-driver mouse lines and the recombinant rabies tracing system. We first improved the rabies virus tracing strategy to accurately identify starter cells and to efficiently quantify presynaptic inputs. We then mapped brain-wide presynaptic inputs to different excitatory and inhibitory neuron subclasses in the primary visual cortex and seven higher visual areas. Our results reveal quantitative target-, layer- and cell-class-specific differences in the retrograde connectomes, despite similar global input patterns to different neuronal populations in the same anatomical area. The retrograde connectivity we define is consistent with the presence of the ventral and dorsal visual information processing streams and reveals further subnetworks within the dorsal stream. The hierarchical organization of the entire visual cortex can be derived from intracortical feedforward and feedback pathways mediated by upper- and lower-layer input neurons, respectively. This study expands our knowledge of the brain-wide inputs regulating visual areas and demonstrates that our improved rabies virus tracing strategy can be used to scale up the effort in dissecting connectivity of genetically defined cell populations in the whole mouse brain.
Ensemble–function relationships to evaluate catalysis in the ketosteroid isomerase oxyanion hole
Authors: Yabukarski Filip , Doukov Tzanko , Pinney Margaux , Biel Justin , Fraser James , Herschlag Daniel
Published: October 2021

Following decades of insights from structure–function studies, there is now a need to progress from a static to dynamic view of enzymes. Comparison of prior cryo X-ray structures suggested that deleterious effects from ketosteroid isomerase (KSI) mutants arise from misalignment of the oxyanion hole catalytic residue, Y16. However, multi-conformer models from room temperature X-ray diffraction revealed an ensemble of Y16 conformers indistinguishable from WT for Y32F/Y57F KSI and a distinct, non-native ensemble for Y16 in Y57F KSI. Functional analyses suggested rate effects arise from weakened hydrogen bonding, due to disruption of the Y16/Y57/Y32 hydrogen bond network, and repositioning of the general base. In general, catalytic changes can be deconvoluted into effects on the probability of occupying a state (P-effects) and the reactivity of each state (k-effects). Our results underscore the need for ensemble–function analysis to decipher enzyme function and ultimately manipulate their extraordinary capabilities.
Real-time Noise-suppressed Wide-Dynamic-Range Compression in Ultrahigh-Resolution Neuronal Imaging
Authors: Borah Bhaskar Jyoti , Sun Chi-Kuang
Published: October 2021

With a limited dynamic range of an imaging system, there are always regions with signal intensities comparable to the noise level, if the signal intensity distribution is close to or even wider than the available dynamic range. Optical brain/neuronal imaging is such a case where weak-intensity ultrafine structures, such as, nerve fibers, dendrites and dendritic spines, often coexist with ultrabright structures, such as, somas. A high fluorescence-protein concentration makes the soma order-of-magnitude brighter than the adjacent ultrafine structures resulting in an ultra-wide dynamic range. A straightforward enhancement of the weak-intensity structures often leads to saturation of the brighter ones, and might further result in amplification of high-frequency background noises. An adaptive illumination strategy to real-time-compress the dynamic range demands a dedicated hardware to operate and owing to electronic limitations, might encounter a poor effective bandwidth especially when each digitized pixel is required to be illumination optimized. Furthermore, such a method is often not immune to noise-amplification while locally enhancing a weak-intensity structure. We report a dedicated-hardware-free method for rapid noise-suppressed wide-dynamic-range compression so as to enhance visibility of such weak-intensity structures in terms of both contrast-ratio and signal-to-noise ratio while minimizing saturation of the brightest ones. With large-FOV aliasing-free two-photon fluorescence neuronal imaging, we validate its effectiveness by retrieving weak-intensity ultrafine structures amidst a strong noisy background. With compute-unified-device-architecture (CUDA)-acceleration, a time-complexity of <3 ms for a 1000×1000-sized 16-bit data-set is secured, enabling a real-time applicability of the same.
Deficiency of ZC3HC1 modulates vascular smooth muscle cell phenotype and increases neointima formation
Authors: Aherrahrou Redouane , Reinberger Tobias , Werner Julia , Otto Miriam , Al-Hasani Jaafar , Munoz-Venegas Loretto , Yin XiaoKe , Mayr Manuel , Civelek Mete , Schunkert Heribert , Kessler Thorsten , Erdmann Jeanette , Aherrahrou Zouhair
Published: October 2021

The ZC3HC1 gene has been linked to various cardiovascular traits. One variant, rs11556924-T, has been found to lower the risk of coronary artery disease (CAD) and blood pressure, but increases carotid intima-media thickness (IMT). This study aimed to determine how ZC3HC1 affects IMT using in vitro and in vivo models. In this study, we analyzed the effect of the rs11556924-T allele on ZC3HC1 expression in vascular smooth muscle cells (SMCs) from 151 multi-ethnic heart transplant donors. The results showed that rs11556924-T was associated with lower ZC3HC1 expression and faster SMC migration. ZC3HC1 knockdown (KD) experiments supported these findings, showing increase migration and proliferation. Mechanistically ZC3HC1 KD led to decreased expression of contractile marker genes and the accumulation of cyclin B1, a key cell cycle protein. Pathway analysis of differentially expressed genes between ZC3HC1 KD and controls SMCs showed decreased expression of genes in the cell division and cytoskeleton organization pathways, as well as higher expression of genes involved in extracellular matrix organization and cytokine-mediated signaling. To validate these findings in vivo, we generated and characterized knockout (Zc3hc1-/-) mice. These mice had enhanced neointima formation in response to arterial injury and faster SMCs migration ability. However, complete loss of Zc3hc1 led to a significant reduction in SMC proliferation and lower cyclin B1 protein level. In addition, immunostaining and confocal microscopy demonstrated, for the first time, that ZC3HC1 and Cyclin B1 were located at the cleavage furrow during mitotic progression of SMCs. Taken together, our study suggests that lower ZC3HC1/NIPA level leads to increased SMC migration and neointima formation. Moreover, we proposed a biphasic role of NIPA in proliferation. Lower levels of NIPA promote SMC proliferation, while complete loss of NIPA hampers cell division and abrogates proliferation.
The use of barcodedAsaiabacteria in mosquitoin vivoscreens for identification of systemic insecticides and inhibitors of malaria transmission
Authors: Sturm Angelika , Vos Martijn W. , Henderson Rob , Eldering Maarten , Koolen Karin M.J. , Sheshachalam Avinash , Favia Guido , Samby Kirandeep , Herreros Esperanza , Dechering Koen J.
Published: October 2021

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the bloodmeals was achieved through recombinant DNA-taggedAsaiabacteria that successfully colonizedAedesandAnophelesmosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles and neonicotinoids were enriched for compounds with systemic adulticide activity againstAnopheles. Using a luminescentPlasmodium falciparumreporter strain, barcoded screens identified 48 drug-like transmission blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects, and identifies novel candidate small molecules for disease control.
The evolutionary maintenance of Lévy flight foraging
Authors: Campeau Winston , Simons Andrew M. , Stevens Brett
Published: October 2021

Lévy flight is a type of random walk that models the behaviour of many phenomena across a multiplicity of academic disciplines; within biology specifically, the behaviour of fish, birds, insects, mollusks, bacteria, plants, slime molds, t-cells, and human populations. The Lévy flight foraging hypothesis states that because Lévy flights can maximize an organism’s search efficiency, natural selection should result in Lévy-like behaviour. Empirical and theoretical research has provided ample evidence of Lévy walks in both extinct and extant species, and its efficiency across models with a diversity of resource distributions. However, no model has addressed the maintenance of Lévy flight foraging through evolutionary processes, and existing models lack ecological breadth. We use numerical simulations, including lineage-based models of evolution with a distribution of move lengths as a variable and heritable trait, to test the Lévy flight foraging hypothesis. We include biological and ecological contexts such as population size, searching costs, lifespan, resource distribution, speed, and consider both energy accumulated at the end of a lifespan and averaged over a lifespan. We demonstrate that selection often results in Lévy-like behaviour, although conditional; smaller populations, longer searches, and low searching costs increase the fitness of Lévy-like behaviour relative to Brownian behaviour. Interestingly, our results also evidence a bet-hedging strategy; Lévy-like behaviour reduces fitness variance, thus maximizing geometric mean fitness over multiple generations.

Author summary

In heterotrophs, incuding animals, survival depends on the net energy gained through foraging. The expectation, then, is that natural selection results in adaptations for efficient foraging that optimize the balance of searching costs and rewards. Lévy flight foraging has been proposed as an optimal foraging solution. The hypothesis states, if no information about resource locations are available, and the locations are re-visitable, then selection will result in adaptations for Lévy flight foraging, a type of random walk. It has been argued that Levy-like foraging behaviour may simply reflect how resources are distributed, but empirical and theoretical research suggests that this behaviour is intrinsic or innate. However, this research does not address evolutionary mechanisms, and lacks ecological breadth. We extend the current theoretical framework by including evolutionary ecological contexts. We treat an organism’s random walk as a heritable trait, and explore ecological contexts such as population size, lifespan, carrying capacity, searching costs, reproductive strategies, and different distributions of food. Our evolutionary simulations overwhelmingly resulted in selection for Lévy-like foraging, regardless of the distribution of food, and evidences Lévy flight foraging as a bet-hedging strategy. Thus, here we provide some of the first evidence for the evolutionary maintenance of Lévy flight foraging.
Towards integration of time-resolved confocal microscopy of a 3D in vitro microfluidic platform with a hybrid multiscale model of tumor angiogenesis
Authors: Phillips Caleb M. , Lima Ernesto A. B. F. , Gadde Manasa , Jarrett Angela M. , Rylander Marissa Nichole , Yankeelov Thomas E.
Published: October 2021

The goal of this study is to calibrate a multiscale model of tumor angiogenesis with time-resolved data to allow for systematic testing of mathematical predictions of vascular sprouting. The multi-scale model consists of an agent-based description of tumor and endothelial cell dynamics coupled to a continuum model of vascular endothelial growth factor concentration. First, we calibrate ordinary differential equation models to time-resolved protein expression data to estimate the rates of secretion and consumption of vascular endothelial growth factor by endothelial and tumor cells, respectively. These parameters are then input into the multiscale tumor angiogenesis model, and the remaining model parameters are then calibrated to time resolved confocal microscopy images obtained within a 3D vascularized microfluidic platform. The microfluidic platform mimics a functional blood vessel with a surrounding collagen matrix seeded with inflammatory breast cancer cells, which induce tumor angiogenesis. Once the multi-scale model is fully parameterized, we forecast the spatiotemporal distribution of vascular sprouts at future time points and directly compare the predictions to experimentally measured data. We assess the ability of our model to globally recapitulate angiogenic vasculature density, resulting in an average relative calibration error of 17.7% ± 6.3% and an average prediction error of 20.2% ± 4% and 21.7% ± 3.6% using one and four calibrated parameters, respectively. We then assess the model’s ability to predict local vessel morphology (individualized vessel structure as opposed to global vascular density), initialized with the first time point and calibrated with two intermediate time points. To the best of our knowledge, this represents the first study to integrate well-controlled, experimental data into a mechanism-based, multiscale, mathematical model of angiogenic sprouting to make specific, testable predictions.
tiRNA-Val promotes angiogenesis via Sirt1–Hif-1α axis in mice with diabetic retinopathy
Authors: Xu Yan , Zou Haidong , Ding Qi , Zou Yuelan , Tang Chun , Lu Yuyu , Xu Xun
Published: October 2021

Diabetic retinopathy (DR) is a specific microvascular complication arising from diabetes, and its pathogenesis is notcompletely understood. tRNA-derived stress-induced RNAs (tiRNAs), a new type of small noncoding RNA generated by specific cleavage of tRNAs, has become a promising target forseveral diseases. However, the regulatory function of tiRNAs in DR and its detailed mechanism remain unknown. Here, we analyzed the tiRNA profiles of normal and DR retinal tissues. The expression level of tiRNA-Val was significantly upregulated in DR retinal tissues. Consistently, tiRNA-Val was upregulated in human retinal microvascular endothelial cells (HRMECs) under high glucose conditions. The overexpression of tiRNA-Val enhanced cell proliferation and inhibited cell apoptosis in HRMECs, but the knockdown of tiRNA-Val decreased cell proliferation and promoted cell apoptosis. Mechanistically, tiRNA-Val, derived from mature tRNA-Val with Ang cleavage, decreased Sirt1 expression level by interacting with sirt1 3’UTR, leading to the accumulation of Hif-1α, a key target for DR. In addition, subretinal injection of adeno-associated virus to knock down tiRNA-Val in DR mice ameliorated the symptoms of DR. Therefore, these data suggest that tiRNA-Val is a potential target in treating diabetic retinopathy.
Disrupted dynamic network reconfiguration of the executive and reward networks in internet gaming disorder: Why IGD subjects cannot control their gaming cravings
Authors: Wang Min , Zheng Hui , Zhou Weiran , Yang Bo , Wang Lingxiao , Chen Shuaiyu , Dong Guang-Heng
Published: October 2021

Background

Studies have shown that people with internet gaming disorder (IGD) show impaired executive control over their gaming cravings; however, the neural mechanisms underlying this process remain unknown. In addition, these conclusions were based on the hypothesis that brain networks were temporally stationary and neglected changes in cognitive processes.

Methods

Resting-state fMRI data were collected from 402 subjects (162 subjects with IGD and 240 recreational game users (RGUs)). The community structure (recruitment and integration) of the executive control network and the basal ganglia network (BGN, representing the reward network) of patients with IGD and healthy controls were analyzed and compared. Mediation effects were analyzed among the different networks.

Results

When compared to RGUs, subjects with IGD had a lower recruitment coefficient within the right executive control network (ECN). Further analysis showed that only male subjects had a lower recruitment coefficient. Mediation analysis showed that the integration coefficient of the right ECN mediated the relationship between the recruitment coefficients of both the right ECN and the BGN in RGUs.

Conclusions

Subjects with IGD had a lower recruitment coefficient than RGUs, and this feature was only observed in male subjects, making them less efficient at impulse control. The meditation results suggest a top-down control mechanism of the ECN is missing in subjects with IGD. All of these findings could explain why subjects with IGD have impaired executive control over their gaming cravings.
Sound disrupts sleep-associated brain oscillations in rodents according to its meaning
Authors: van Kronenberg Philipp , Milinski Linus , Kruschke Zoë , de Hoz Livia
Published: October 2021

Sleep is essential but poses a risk to the animal. Filtering acoustic information according to its relevance, a process generally known as sensory gating, is crucial during sleep to ensure a balance between rest and danger detection. The mechanisms of this sensory gating and its specificity are not understood. Here, we tested the effect that sounds of different meaning had on sleep-associated ongoing oscillations. We recorded EEG and EMG from mice during rapid-eye movement (REM) and non-REM (NREM) sleep while presenting sounds with or without behavioural relevance. We found that sound presentation per se, in the form of an unfamiliar neutral sound, elicited a weak or no change in the sleep-dependent EEG power during NREM and REM sleep. In contrast, the presentation of a sound previously conditioned in an aversive task, elicited a clear and fast decrease in the sleep-dependent EEG power during both sleep phases, suggesting a transition to lighter sleep without awakening. The observed changes generally weakened over training days and were not present in animals that failed to learn. Interestingly, the effect could be generalized to unfamiliar neutral sounds if presented following conditioned training, an effect that depended on sleep phase and sound type. The data demonstrate that sounds are differentially gated during sleep depending on their meaning and that this process is reflected in disruption of sleep-associated brain oscillations without an effect on behavioural arousal.
Identification of fetal liver stroma in spectral cytometry using the parameter autofluorescence
Authors: Peixoto Marcia Mesquita , Soares-da-Silva Francisca , Schmutz Sandrine , Mailhe Marie-Pierre , Novault Sophie , Cumano Ana , Ait-Mansour Cedric
Published: October 2021

The fetal liver is the main hematopoietic organ during embryonic development. The fetal liver is also the unique anatomical site where hematopoietic stem cells expand before colonizing the bone marrow, where they ensure life-long blood cell production and become mostly resting. The identification of the different cell types that comprise the hematopoietic stroma in the fetal liver is essential to understand the signals required for the expansion and differentiation of the hematopoietic stem cells. We used a panel of monoclonal antibodies to identify fetal liver stromal cells in a 5-laser equipped spectral flow cytometry analyzer. The “Autofluorescence Finder” of SONY ID7000 software identified two distinct autofluorescence emission spectra. Using autofluorescence as a fluorescence parameter we could assign the two autofluorescent signals to three distinct cell types and identified surface markers that characterize these populations. We found that one autofluorescent population corresponds to hepatoblasts and cholangiocytes whereas the other expresses mesenchymal transcripts and was identified as stellate cells. Importantly, after birth, autofluorescence becomes the unique identifying property of hepatoblasts because mature cholangiocytes are no longer autofluorescent.

These results show that autofluorescence used as a parameter in spectral flow cytometry is a useful tool to identify new cell subsets that are difficult to analyze in conventional flow cytometry.
A novel approach to develop wheat chromosome-specific KASP markers for detecting Amblyopyrum muticum segments in doubled haploid introgression lines
Authors: Grewal Surbhi , Coombes Benedict , Joynson Ryan , Hall Anthony , Fellers John , Yang Cai-yun , Scholefield Duncan , Ashling Stephen , Isaac Peter , King Ian P. , King Julie
Published: October 2021

Many wild relative species are being used in pre-breeding programmes to increase the genetic diversity of wheat. Genotyping tools such as single nucleotide polymorphism (SNP)-based arrays and molecular markers have been widely used to characterise wheat-wild relative introgression lines. However, due to the polyploid nature of the recipient wheat genome, it is difficult to develop SNP-based KASP markers that are codominant to track the introgressions from the wild species. Previous attempts to develop KASP markers have involved both exome- and PCR-amplicon-based sequencing of the wild species. But chromosome-specific KASPs assays have been hindered by homoeologous SNPs within the wheat genome. This study involved whole genome sequencing of the diploid wheat wild relative Amblyopyrum muticum and development of a SNP discovery pipeline that generated ∼38,000 SNPs in single-copy wheat genome sequences. New assays were designed to increase the density of Am. muticum polymorphic KASP markers. With a goal of one marker per 60 Mbp, 335 new KASP assays were validated as functional. Together with assays validated in previous studies, 498 well distributed chromosome-specific markers were used to recharacterize previously genotyped wheat-Am. muticum doubled haploid (DH) introgression lines. The chromosome specific nature of the KASP markers allowed clarification of which wheat chromosomes were involved with recombination events or substituted with Am. muticum chromosomes and the higher density of markers allowed detection of new small introgressions in these DH lines.

Key Message

A novel methodology to generate chromosome-specific SNPs between wheat and its wild relative Amblyopyrum muticum and their use in the development of KASP markers to genotype wheat-Am. muticum introgression lines.
Modeling spatial, developmental, physiological, and topological constraints on human brain connectivity
Authors: Oldham S. , Fulcher B. D. , Aquino K. , Arnatkevičiūtė A. , Paquola C. , Shishegar R. , Fornito A.
Published: October 2021

The complex connectivity of nervous systems is thought to have been shaped by competitive selection pressures to minimize wiring costs and support adaptive function. Accordingly, recent modeling work indicates that stochastic processes, shaped by putative trade-offs between the cost and value of each connection, can successfully reproduce many topological properties of macroscale human connectomes measured with diffusion magnetic resonance imaging. Here, we derive a new formalism with the aim to more accurately capture the competing pressures of wiring cost minimization and topological complexity. We further show that model performance can be improved by accounting for developmental changes in brain geometry and associated wiring costs, and by using inter-regional transcriptional or microstructural similarity rather than topological wiring-rules. However, all models struggled to capture topologies spatial embedding. Our findings highlight an important role for genetics in shaping macroscale brain connectivity and indicate that stochastic models offer an incomplete account of connectome organization.
Transmembrane Signaling Regulates the Remodeling of the Actin Cytoskeleton: Roles of PKC and Adducin
Authors: Shieh Bih-Hwa , Sun Wesley , Ferng Darwin
Published: October 2021

We tested the hypothesis that Pkc53E regulates adducin to orchestrate the remodeling of the membrane skeleton following the transmembrane GPCR-Gq signaling. Adducin is a known substrate of PKC and is critical for the assembly of the membrane skeleton by cross-linking actin filaments with the spectrin network. In Drosophila photoreceptors, loss of function in pkc53E leads to retinal degeneration while Pkc53E-RNAi negatively impacts the actin cytoskeleton of the visual organelle rhabdomeres. Unexpectedly, Pkc53E-RNAi enhances the degeneration caused by the loss of PLCβ4 (norpAP24). We show that when PLCβ4 is absent Plc21C may be activated instead for activating Pkc53E. We investigate whether Pkc53E phosphorylates adducin in vivo and observed that levels of phosphorylated adducin at the conserved PKC site were greatly reduced in a null allele of pkc53E. We show Pkc53E-RNAi did not modify adducin-RNAi, which exerts a more severe effect on the actin cytoskeleton. Moreover, overexpression of the mCherry-tagged adducin that appears to act in a dominant-negative manner interferes with the spectrin interaction leading to the apical expansion of rhabdomeres similar to that of β-spectrin-RNAi. We performed epistasis analysis and show that double mutants of the tagged adducin and Pkc53E-RNAi display the expansion phenotype at the eclosion, but progress to severe degeneration in adult photoreceptors. Together, most of our findings support that adducin is likely regulated by Pkc53E in Drosophila photoreceptors.
In vivo production of pederin by labrenzin pathway expansion
Authors: Kačar Dina , Schleissner Carmen , Cañedo Librada M. , Rodríguez Pilar , de la Calle Fernando , Cuevas Carmen , Galán Beatriz , García José Luis
Published: October 2021

Pederin is a potent polyketide toxin that causes severe skin lesions in humans after contact with insects of genus Paederus. Due to its potent anticancer activities, pederin family compounds have raised the interest of pharmaceutical industry. Despite extensive studies on the cluster of biosynthetic genes responsible for the production of pederin, it has not yet been possible to isolate and cultivate its bacterial endosymbiont producer. However, the marine bacterium Labrenzia sp. PHM005 was recently reported to produce labrenzin, the closest pederin analog. By cloning a synthetic pedO gene encoding one of the three O-methyltraferase of the pederin cluster into Labrenzia sp. PHM005 we have been able to produce pederin for the first time by fermentation in the new recombinant strain.
Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse
Authors: Fernández-Hermira Sara , Sanz-Fernández Irene , Botas Marta , Calvo Víctor , Izquierdo Manuel
Published: October 2021

T cell receptor (TCR) and B cell receptor (BCR) stimulation of T and B lymphocytes, by antigen presented on an antigen-presenting cell (APC) induces the formation of the immunological synapse (IS). IS formation is associated with an initial increase in cortical filamentous actin (F-actin) at the IS, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. This is followed by the convergence of secretion vesicles towards the centrosome, and the polarization of the centrosome to the IS. These reversible, cortical actin cytoskeleton reorganization processes occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, proteolytic granules secretion in B lymphocytes and during cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. In addition, several findings obtained in T and B lymphocytes forming IS show that actin cytoskeleton reorganization also occurs at the centrosomal area. F-actin reduction at the centrosomal area appears to be associated with centrosome polarization. In this chapter we deal with the analysis of centrosomal area F-actin reorganization, as well as the centrosome polarization analysis towards the IS.
PTPN14, a modifier of HHT, protects SMAD4 from ubiquitination and turnover to potentiate BMP9 signaling in endothelial cells
Authors: Mamai Ons , Beleford Daniah T. , Taylor Mark , Basu Sugandha , Cen Xinjian , Trilok Suprita , Zhang Jiamin , Balmain Allan , Akhurst Rosemary J.
Published: October 2021

Hereditary Hemorrhagic Telangiectasia (HHT) results from germline loss-of-function mutations of ENG, ACVRL1, or SMAD4, encoding TGFβ/BMP signaling components. Telangiectasias occur in most patients, and pulmonary, visceral, or cerebral arteriovenous malformations (AVMs) in 20-50% of these. How HHT mutations cause these clinical manifestations and why some patients suffer more serious sequelae than others is unknown. PTPN14 is a genetic modifier of pulmonary AVM incidence, and here we show by gene expression network analysis of a large panel of genetically diverse mouse lung RNA samples, that Ptpn14 is ontologically associated with markers of angiogenesis, vascular remodeling, and BMP/TGFβ and Rho kinase signaling. We demonstrate physical interaction between protein tyrosine phosphatase non-receptor, type 14 (PTPN14) and SMAD4 in nucleus and cytoplasm of primary human endothelial cells. PTPN14 suppresses ubiquitination and turnover of SMAD4 to augment tonic SMAD-mediated transcriptional readouts. This is the first report that PTPN14 binds and stabilizes SMAD4, a key component of the HHT signaling pathway. Through this mechanism, and its inhibition of YAP/TAZ signaling, PTPN14 levels may protect against development of AVMs in HHT. We discuss potential druggable targets for HHT within the ENG-ALK1-SMAD4-PTPN14 network.

One Sentence Summary

PTPN14 binds and stabilizes SMAD4 to potentiate BMP9 signaling in endothelial cells and components of the PTPN14 network may be drug targets for HHT.
TheDaphniacarapace and the origin of novel structures
Authors: Bruce Heather S. , Patel Nipam H.
Published: October 2021

Understanding how novel structures arise is a central question in evolution. Novel structures are often defined as structures that are not derived from (homologous to) any structure in the ancestor1. The carapace of the water fleaDaphnia magnais a bivalved “cape” of exoskeleton that has been proposed to be one of many novel arthropod structures that arose through repeated co-option of genes that also pattern insect wings2–4. To determine whether theDaphniacarapace is a novel structure, we compare the expression ofpannier, araucan, andvestigialbetweenDaphnia, Parhyale, andTribolium. Our results suggest that theDaphniacarapace did not arise by co-option, but instead derives from an exite (lateral lobe) that emerges from an ancestral proximal leg segment that was incorporated into theDaphniabody wall. TheDaphniacarapace therefore appears to be homologous to theParhyaletergal plate and the insect wing5. Remarkably, thevestigial-positive region that gives rise to theDaphniacarapace appears to be present inParhyale6andTriboliumas a small, inconspicuous protrusion. Similarly, thevestigial-positive regions that form thoracic tergal plates inParhyaleappear to be present inDaphnia, even thoughDaphniadoes not form thoracic tergal plates. Thus, rather than a novel structure resulting from gene co-option, theDaphniacarapace appears to have arisen from a shared, ancestral tissue (morphogenetic field) that persists in a cryptic state in other arthropod lineages. Cryptic persistence of unrecognized serial homologs may thus be a general solution for the origin of novel structures.
Moderate prenatal alcohol exposure modifies sex-specific CRFR1 activity in the central amygdala and anxiety-like behavior in adolescent offspring
Authors: Rouzer Siara Kate , Diaz Marvin R.
Published: October 2021

Anxiety disorders are highly prevalent among individuals with a history of prenatal alcohol exposure (PAE), and adolescent rodents demonstrate anxiety-like behavior following moderate PAE on Gestational Day (G) 12. A likely systemic target of PAE is the stress peptide corticotrophin-releasing factor (CRF), as activation of CRF receptor 1 (CRFR1) in the medial nucleus of the central amygdala (CeM) is known to increase anxiety-like behavior in adults. To determine if CRF-CRFR1 interactions underly PAE-induced anxiety, functional changes in CRF system activity were investigated in adolescent male and female Sprague Dawley rats following G12 PAE. Compared to air-exposed controls, PAE increased basal spontaneous (s) inhibitory post-synaptic current (IPSC) frequency in the CeM of males, but not females. Furthermore, PAE blunted CRFR1-regulated miniature (m) IPSCs in a sex- and dose-specific manner, and only PAE males demonstrated tonic CRFR1 activity in the CeM. It was further determined that G12 PAE decreased CRFR1 mRNA in the CeM of males while increasing regional expression in females. Finally, infusion of a CRFR1 agonist into the CeM of adolescents produced a blunted expression of CRFR1-induced anxiety-like behavior exclusively in PAE males, mirroring the blunted physiology demonstrated by PAE males. Cumulatively, these data suggest that CRFR1 function within the CeM is age- and sex-specific, and PAE not only increases the expression of anxiety-like behavior, but may reduce the efficacy of treatment for PAE-induced anxiety through CRFR1-associated mechanisms. Therefore, future research will be necessary to develop targeted treatment of anxiety disorders in individuals with a history of PAE.
Evaluation of calcium-sensitive adenylyl cyclase AC1 and AC8 mRNA expression in the anterior cingulate cortex of mice with neuropathic pain
Authors: Shiers Stephanie , Elahi Hajira , Hennen Stephanie , Price Theodore J
Published: October 2021

The anterior cingulate cortex (ACC) is a critical region of the brain for the emotional and affective components of pain in rodents and humans. Hyperactivity in this region has been observed in neuropathic pain states in both patients and animal models and ablation of this region from cingulotomy, or inhibition with genetics or pharmacology can diminish pain and anxiety. Two adenylyl cyclases (AC), AC1 and AC8 play an important role in regulating nociception and anxiety-like behaviors through an action in the ACC, as genetic and pharmacological targeting of these enzymes reduces mechanical hypersensitivity and anxietylike behavior, respectively. However, the distribution of these ACs in the ACC has not been studied in the context of neuropathic pain. To address this gap in knowledge, we conducted RNAscope in situ hybridization to assess AC1 and AC8 mRNA distribution in mice with spared nerve injury (SNI). Given the key role of AC1 in nociception in neuropathic, inflammatory and visceral pain animal models, we hypothesized that AC1 would be upregulated in the ACC of mice following nerve injury. This hypothesis was also founded on data showing increased AC1 expression in the ACC of mice with zymosan-induced visceral inflammation. We found that AC1 and AC8 are widely expressed in many regions of the mouse brain including the hippocampus, ACC, medial prefrontal cortex and midbrain regions, but AC1 is more highly expressed. Contrary to our hypothesis, SNI causes an increase in AC8 mRNA expression in NMDAR-2B (Nr2b) positive neurons in the contralateral ACC but does not affect AC1 mRNA expression. Our findings show that changes in Adcy1 mRNA expression in the ACC are insufficient to explain the important role of this AC in mechanical hypersensitivity in mice following nerve injury and suggest a potential unappreciated role of AC8 in regulation of ACC synaptic changes after nerve injury.
Subcortical Segmentation of the Fetal Brain in 3D Ultrasound using Deep Learning
Authors: Hesse Linde S. , Aliasi Moska , Moser Felipe ,   the INTERGROWTH-21st Consortium, Haak Monique C. , Xie Weidi , Jenkinson Mark , Namburete Ana I.L.
Published: October 2021

The quantification of subcortical volume development from 3D fetal ultrasound can provide important diagnostic information during pregnancy monitoring. However, manual segmentation of subcortical structures in ultra-sound volumes is time-consuming and challenging due to low soft tissue contrast, speckle and shadowing artifacts. For this reason, we developed a convolutional neural network (CNN) for the automated segmentation of the choroid plexus (CP), lateral posterior ventricle horns (LPVH), cavum septum pellucidum et vergae (CSPV), and cerebellum (CB) from 3D ultrasound. As ground-truth labels are scarce and expensive to obtain, we applied few-shot learning, in which only a small number of manual annotations (n = 9) are used to train a CNN. We compared training a CNN with only a few individually annotated volumes versus many weakly labelled volumes obtained from atlas-based segmentations. This showed that segmentation performance close to intra-observer variability can be obtained with only a handful of manual annotations. Finally, the trained models were applied to a large number (n = 278) of ultrasound image volumes of a diverse, healthy population, obtaining novel US-specific growth curves of the respective structures during the second trimester of gestation.
The proSAAS chaperone provides neuroprotection and attenuates transsynaptic α–synuclein spread in rodent models of Parkinson’s disease
Authors: Lindberg Iris , Shu Zhan , Lam Hoa , Helwig Michael , Yucer Nur , Laperle Alexander , Svendsen Clive , Di Monte Donato A. , Maidment Nigel T.
Published: October 2021

Parkinson’s disease is a devastating motor disorder involving the aberrant aggregation of the synaptic protein synuclein (aSyn) and degeneration of the nigrostriatal dopaminergic tract. We previously showed that proSAAS, a small secreted chaperone protein widely expressed in neurons within the brain, is able to block aSyn-induced dopaminergic cytotoxicity in primary nigral neuron cultures. We show here that coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This positive functional outcome was accompanied by significant amelioration of the human aSyn-induced loss of both nigral tyrosine hydroxylase-positive cells and striatal tyrosine hydroxylase-positive terminals, demonstrating clear proSAAS-mediated protection of the nigro-striatal tract. ProSAAS overexpression also reduced the content of human aSyn protein in both the nigra and striatum and reduced the loss of tyrosine hydroxylase protein in both regions. Since proSAAS is a secreted protein, we tested the possibility that proSAAS is able to block the transsynaptic spread of aSyn from the periphery to the central nervous system, increasingly recognized as a potentially significant pathological mechanism. The number of human aSyn-positive neurites in the pons and caudal midbrain of mice following administration of human aSyn-encoding AAV into the vagus nerve was considerably reduced in mice coinjected with proSAAS-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. We suggest that proSAAS may represent a promising target for therapeutic development in Parkinson’s disease.

Significance

This paper describes two independent avenues of research that both provide support for the in vivo neuroprotective function of this small chaperone protein. In the first approach, we show that proSAAS overexpression provides remarkably effective protection against dopaminergic neurotoxicity in a rat model of Parkinson’s disease. This conclusion is supported both by three independent assays of motor function as well as by quantitative analysis of surviving dopaminergic neurons in brain areas involved in the control of motor function. In the second line of research, we show that in mice, the spread of human synuclein across synapses can be blunted by proSAAS overexpression.