title: The inositol-requiring enzyme 1α/X-box protein 1 (IRE1α/XBP1) pathway of the unfolded protein response is impaired in pediatric cholestatic liver disease explants

abstract:
Background and AimsCholestatic liver diseases (CLD) are the leading indication for pediatric liver transplantation. Bile acids are known to trigger endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is a coordinated response which functions to decrease ER stress and maintain cellular homeostasis. Dysregulation of the UPR, specifically the inositol-requiring enzyme 1/X-box protein 1 (IRE1/XBP1) pathway, has been associated with several adult liver diseases. In this study, we sought to evaluate the UPR in pediatric livers with end-stage CLD and hypothesized there would be altered IRE1/XBP1 pathway activation compared to normal control livers.

MethodsWe evaluated 34 human liver ex-plants which included pediatric CLD, pediatric normal, pediatric non-cholestatic liver disease (autoimmune hepatitis, AIH), adult CLD and adult normal cohorts. We performed RNA sequencing (RNAseq), qPCR and western blotting to identify significantly different pathway activation and assess for changes within the UPR.

ResultsPathway analysis of RNAseq data demonstrated that protein processing in the ER was a significantly differentially expressed pathway in all liver disease cohorts compared to appropriate controls. Pediatric CLD samples had evidence of ongoing ER stress with activation of the PKR-like ER kinase (PERK) pathway but had significantly decreased expression of the IRE1/XBP1 pathway of the UPR compared to normal controls. Pediatric AIH samples had similar activation of the PERK pathway but notably did not have decreased activation of the IRE1/XBP1 pathway compared to normal controls.

ConclusionPediatric CLD ex-plants have evidence of ongoing ER stress but decreased activation of the protective IRE1/XBP1 pathway of the UPR. Dysregulation of this pathway may contribute to disease progression and could be a therapeutic target for treating pediatric CLD.


title: Structure of the human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp bound to a dsDNA-ssDNA junction

abstract:
The RAD9-RAD1-HUS1 (9-1-1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5-recessed end of a dsDNA-ssDNA junction by the RAD17-RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9-1-1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA.

Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17-RFC clamp loader bound to human 9-1-1, engaged with a dsDNA-ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9-1-1 over PCNA, and how the clamp loader specifically recognises the recessed 5 DNA end and fixes the orientation of 9-1-1 on the ssDNA.

category: biochemistry


title: Oligomer-to-Monomer Transition Underlies the Chaperone Function of AAGAB in AP1/AP2 Assembly

abstract: Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homotetramer. AAGAB tetramerization is mediated by its C-terminal domain, which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the tetramerization domain (TD), revealing a dimer of dimer assembly. Interestingly, AAGAB uses the same TD to recognize and stabilize the {gamma} subunit in the AP1 complex and the  subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of TD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.


title: State-of-the-Art Estimation of Protein Model Accuracy using AlphaFold

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe problem of predicting a proteins 3D structure from its primary amino acid sequence is a longstanding challenge in structural biology. Recently, approaches like AlphaFold have achieved remarkable performance on this task by combining deep learning techniques with coevolutionary data from multiple sequence alignments of related protein sequences. The use of coevolutionary information is critical to these models accuracy, and without it their predictive performance drops considerably. In living cells, however, the 3D structure of a protein is fully determined by its primary sequence and the biophysical laws that cause it to fold into a low-energy configuration. Thus, it should be possible to predict a proteins structure from only its primary sequence by learning a highly-accurate biophysical energy function. We provide evidence that AlphaFold has learned such an energy function, and uses coevolution data to solve the global search problem of finding a low-energy conformation. We demonstrate that AlphaFolds learned potential function can be used to rank the quality of candidate protein structures with state-of-the-art accuracy, without using any coevolution data. Finally, we propose a method for utilizing this potential function to predict protein structures without the need for MSAs.

category: biophysics


title: Cryo-EM structure of amyloid fibril formed by α-synuclein familial A53E mutation

abstract: Synucleinopathies, including Parkinsons disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA) have the same hallmark pathologic feature of misfolded -synuclein protein accumulation in the brain. PD patients who carry -syn hereditary mutations tend to have an earlier onset and more severe clinical symptoms and pathology than sporadic PD patients who carry wild-type (WT) -syn. Therefore, revealing the structural effect of -syn hereditary mutations on the wild-type fibril structure can help us understand synucleinopathies structural basis. Here, we present a 3.38 [A] cryo-electron microscopy structure of -synuclein fibrils containing the hereditary A53E mutation. The A53E fibril is symmetrically composed of two protofilaments, as are many other synucleopathic structures - including WT. Interestingly, the interface between the protofilaments in A53E has significantly less buried surface area than all other documented fibril structures of -syn and its other mutants. The A53E fibril also exhibits slower formation/growth in in vitro fibrillation experiment compared to other mutants. This implies that the structural differences - both in the protofilament and between each protofilament of A53E - change the aggregation mechanism, or in the least, its kinetics of formation. These differences influence the molecular characteristics of each fibril mutant and likely plays a macro-scale role in progressing one clinical pathology over another.


title: Loss of tumor suppressor WWOX enhances RAS activity in pancreatic cancer

abstract: Pancreatic cancer is one of the most lethal cancers, due to late diagnoses and chemotherapy resistance. The tumor suppressor WWOX, spanning one of the most active common fragile sites in the human genome (FRA16D), is commonly altered in pancreatic cancer. However, the direct contribution of WWOX loss to pancreatic cancer development and progression is largely unknown. Here, we report that combined conditional deletion of Wwox and activation of KRasG12D in Ptf1a-CreER-expressing mice resulted in accelerated formation of precursor lesions and pancreatic carcinoma. In addition, these mice displayed enhanced MAPK and IL6/Jak/Stat3 signalling, combined with higher rates of macrophage infiltration and inflammation signature at sites of acinar to ductal metaplasia (ADM). Moreover, accumulated DNA double strand breaks were observed at ADM lesions. Finally, overexpression of WWOX in patient-derived xenografts led to reduction in KRAS MAPK activity and IL6/Jak/Stat3 signalling so diminishing their aggressiveness and in vivo tumor growth. Our data underscore the important role of WWOX in regulating RAS activity in pancreatic carcinogenesis.

category: cancer biology


title: The KASH5 protein involved in meiotic chromosomal movements is a novel dynein activating adaptor

abstract: Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.

category: cell biology


title: Visualization of mRNA Expression in Pseudomonas aeruginosa Aggregates Reveals Spatial Patterns of Fermentative and Denitrifying Metabolism

abstract:
Gaining insight into the behavior of bacteria at the single cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels both in liquid culture and in biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression was much stronger in anoxic environments compared to other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and demonstrate the utility of HCR in unveiling cellular activities at the microscale in heterogeneous populations.

ImportanceTo understand bacteria in diverse contexts we must understand the variations in behaviors and metabolisms they express spatiotemporally. Populations of bacteria are known to be heterogeneous, but the ways this variation manifests can be challenging to characterize due to technical limitations. By focusing on energy conservation, we demonstrate that HCR v3.0 can visualize nuances in gene expression, allowing us to understand how metabolism in Pseudomonas aeruginosa biofilms responds to microenvironmental variation at high spatial resolution. We validated probes for four catabolic genes: a constitutively expressed oxidase, acetate kinase, nitrite reductase, and nitrous oxide reductase. We showed that the genes for different modes of metabolism are expressed in overlapping but distinct subpopulations according to oxygen concentrations in a predictable fashion. The spatial transcriptomic technique described here has the potential to be used to map microbial activities across diverse environments.

category: microbiology


title: IRONMAN interacts with OsHRZ1 and OsHRZ2 to maintain Fe homeostasis

abstract: IRONMAN is a family of small peptides which positively regulate the Fe deficiency response. However, the molecular mechanism by which OsIMA1 and OsIMA2 regulate Fe homeostasis was unclear. Here, we reveal that OsIMA1 and OsIMA2 interact with the potential Fe sensors, OsHRZ1 and OsHRZ2. OsIMA1 and OsIMA2 contain a conserved 17-amino acid C-terminal region which is responsible for the interactions with OsHRZ1 and OsHRZ2. The OsIMA1 overexpressing plants have the increased seed Fe concentration and the reduced fertility, as observed in the hrz1-2 loss-of-function mutant plants. Moreover, the expression trends of Fe deficiency inducible genes in the OsIMA1 overexpressing plants are the same to those in the hrz1-2. Co-expression assays suggest that OsHRZ1 and OsHRZ2 promote the degradation of OsIMA1 proteins. As the interaction partners of OsHRZ1, the OsPRI proteins also interact with OsHRZ2. The conserved C-terminal region of four OsPRIs contributes to the interactions with OsHRZ1 and OsHRZ2. An artificial IMA (aIMA) derived from the C-terminal of OsPRI1 can be also degraded by OsHRZ1. Moreover, the aIMA overexpressing rice plants accumulate more Fe without reduction of fertility. This work establishes the link between OsIMAs and OsHRZs, and develops a new strategy for Fe fortification in rice.

category: molecular biology


title: Accurate detection of structural variation is hard

abstract:
The accurate characterization of structural variation is crucial for our understanding of how large chromosomal alterations affect phenotypic differences and contribute to genome evolution. Whole-genome sequencing is a popular approach for identifying structural variants, but the accuracy of popular tools remains unclear due to the limitations of existing benchmarks. Moreover, the performance of these tools for predicting variants in non-human genomes is less certain, as most tools were developed and benchmarked using data from the human genome.

To address this problem, multiple short- and long-read tools were benchmarked using real and simulated Caenorhabditis elegans whole-genome sequence data. To evaluate the use of long-read data for the validation of short-read predictions, the agreement between predictions from a short-read ensemble learning method and long-read tools were compared. The results obtained indicate that the best performing tool is contingent on the type and size of the variant, as well as the sequencing depth of coverage. These results also highlight the need for reference datasets generated from real data that can be used as  ground truth in benchmarks.

category: genomics


title: Impact of dominance rank specification in dyadic interaction models

abstract: Dominance rank is a vital descriptor of social dynamics in animal societies and regularly used in studies to explain observed interaction patterns. However, researchers can choose between different indices and standardizations, and can specify dyadic rank relations differently when studying interaction distributions. These researcher degrees of freedom potentially introduce biases into studies and reduce replicability. Here, I demonstrate the impact of researcher choices by comparing the performance of different combinations of rank index, standardization, and model specification when explaining dyadic interaction patterns in sooty mangabeys (Cercocebus atys atys). I show that while no combination consistently performed best across interaction types (aggression, grooming, proximity, supplants), model specifications allowing for non-linear patterns performed better than other models on average. Choices made in pre-processing and model building impacted model performance and subsequent interpretation of results. Researchers could end up describing social systems differently based on the same data. These results highlight the impact of researcher choices in the processing of behavioural data and potential limitations when using indirect species comparisons in animal behaviour research. To increase repeatability, researchers could make the impact of their processing choices more transparent and report results using a variety of indices and model specifications.

category: animal behavior and cognition


title: Interspecies isobaric labeling-based quantitative proteomics reveals protein changes in the ovary of Aedes aegypti co-infected with ZIKV and Wolbachia

abstract:
Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by Ae. aegypti. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barre syndrome. One of the actual most promising strategies to mitigate arbovirus transmission involves releasing Ae. aegypti mosquitoes carrying the maternally inherited endosymbiont bacteria Wolbachia pipientis. The presence of Wolbachia is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits as fecundity and fertility. However, the mechanisms by which Wolbachia influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of co-infections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of Wolbachia wMel and ZIKV infection in Ae. aegypti ovaries. To the best of our knowledge, this is the most complete proteome of Ae. aegypti ovaries reported so far, with a total of 3,913 proteins identified, also,were able to quantify a total of 1,044 Wolbachia proteins in complex sample tissue of Ae. aegypti ovary.. Furthermore, we discuss proteins and pathways altered in Ae. aegypti during ZIKV infections, Wolbachia infections, co-infection Wolbachia/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of Ae. aegypti. The modified aspects were mostly related to the immune priming enhancement by Wolbachia presence and the modulation of the Juvenile Hormone pathway caused by both microorganisms infection.

HighlightsO_LIProteome changes in Ae. aegypti, Wolbachia, and ZIKV interactions
C_LIO_LIA great diversity of Wolbachia proteins were quantified in Ae. aegypti ovary
C_LIO_LIJuvenile Hormone pathway is modulated by both infections
C_LIO_LIWolbachia enhances Ae. aegypti immune priming mechanism
C_LIO_LIZIKV unsettles host immune response by reducing antimicrobial peptides production
C_LIO_LICoinfection triggers oxidative stress and a lack of vitellogenin precursors
C_LI


title: Single-cell microfluidic analysis unravels individual cellular fates during Double-Strand Break Repair

abstract: Trinucleotide repeat expansions are responsible for two dozen human disorders. Contracting expanded repeats by Double-Strand Break Repair (DSBR) might be a therapeutic approach. Given the complexity of manipulating human cells, recent assays were made to quantify DSBR efficacy in yeast, using a fluorescent reporter. In this study DSBR is characterized with an interdisciplinary approach, linking large population dynamics and individual cells. Time-resolved molecular measurements of changes in the population are first confronted to a coupled differential equation model to obtain repair processes rates. Comparisons with measurements in microfluidic devices, where the progeny of 80-150 individual cells are followed, show good agreement between individual trajectories and mathematical and molecular results. Further analysis of individual progenies shows the heterogeneity of individual cell contributions to global repair efficacy. Three different categories of repair are identified: high-efficacy error-free, low-efficacy error-free and low-efficacy error-prone. These categories depend on the type of endonuclease used and on the target sequence.

category: bioengineering


title: Continuous extraction and concentration of secreted metabolites from engineered microbes using membrane technology

abstract: Microalgal cultivation in photobioreactors and membrane separations are both considered sustainable processes. Here we explore their synergistic combination to extract and concentrate a heterologous sesquiterpenoid produced by engineered green algal cells. A hydrophobic hollow-fiber membrane contactor was used to allow interaction of culture broth and cells with a dodecane solvent phase to accumulate algal produced patchoulol. Subsequent continuous membrane extraction of patchoulol from dodecane enabled product concentration in a methanol stream as well as dodecane recovery for its reuse. A structure-based prediction using machine learning was used to model a process whereby 100% patchoulol recovery from dodecane could be achieved with solvent-resistant nanofiltration membranes. Solvent consumption, E-factor, and economic sustainability were assessed and compared with existing patchoulol production processes. Our extraction and product purification process offers six- and two-orders of magnitude lower solvent consumption compared to synthetic production and thermal-based separation, respectively. Our proposed methodology is transferable to other microbial systems for the isolation of high-value isoprenoid and hydrocarbon products.


title: Combining two Genetic Sexing Strains allows sorting of non-transgenic males for Aedes genetic control

abstract:
Chemical control of the mosquito vectors Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is an autocidal control tactic that represents a valuable alternative but is limited by the slow, error-prone, and wasteful sex-separation stage.

Here, we present four genetic sexing strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing the isolation of transgenic males. Furthermore, we show how combining these sexing strains allows the production of non-transgenic males. Scaling-up would allow the sorting of 100,000 neonate male larvae in under 1.5 hour with 0.01-0.1% female contamination. The resulting males present similar survival and flight ability to laboratory-reared wild-type males.

By facilitating the sorting of transgenic or non-transgenic males, these Genetic Sexing Strains should enable a major upscaling in control programmes against these major vectors.


title: Uncertainty quantification in the cerebral circulation simulation focusing on the collateral flow: Surrogate model approach with machine learning

abstract:
Collateral circulation in the circle of Willis (CoW), closely associated with disease mechanisms and treatment outcomes, can be effectively investigated using one-dimensional-zero-dimensional hemodynamic simulations. As the entire cardiovascular system is considered in the simulation, it captures the systemic effects of local arterial changes, thus reproducing collateral circulation that reflects biological phenomena. The simulation facilitates rapid assessment of clinically relevant hemodynamic quantities under patient-specific conditions by incorporating clinical data. During patient-specific simulations, the impact of clinical data uncertainty on the simulated quantities should be quantified to obtain reliable results. However, as uncertainty quantification (UQ) is time-consuming and computationally expensive, its implementation in time-sensitive clinical applications is considered impractical. Therefore, we constructed a surrogate model based on machine learning using simulation data. The model accurately predicts the flow rate and pressure in the CoW in a few milliseconds. This reduced computation time enables the UQ execution with 100 000 predictions in a few minutes on a single CPU core and in less than a minute on a GPU. We performed UQ to predict the risk of cerebral hyperperfusion (CH), a life-threatening condition that can occur after carotid artery stenosis surgery if collateral circulation fails to function appropriately. We predicted the statistics of the postoperative flow rate increase in the CoW, which is a measure of CH, considering the uncertainties of arterial diameters, stenosis parameters, and flow rates measured using the patients clinical data. A sensitivity analysis was performed to clarify the impact of each uncertain parameter on the flow rate increase. Results indicated that CH occurred when two conditions were satisfied simultaneously: severe stenosis and when arteries of small diameter serve as the collateral pathway to the cerebral artery on the stenosis side. These findings elucidate the biological aspects of cerebral circulation in terms of the relationship between collateral flow and CH.

Author summaryCerebral arteries generate a ring-like network that provides alternative routes for blood supply in the case of carotid artery stenosis. This collateral circulation is closely associated with the potential risk of stroke and treatment outcomes in patients with stenosis. In this study, we propose a method to elucidate the cerebral circulation of individual patients using a blood flow simulation that incorporates the patients clinical data. A key feature of our approach is its capability to obtain the probability of the different outputs using simulation, considering the uncertainty of patient conditions. Although this capability is essential for obtaining reliable results, the process is time-consuming and requires numerous computer resources. We solved this problem by combining the blood flow simulation with machine learning to perform predictions 43 000 times faster than conventional simulations. We applied the proposed method to predict cerebral circulation following surgery in three patients with stenosis and verified that the method can assess the surgical risk almost in real-time, even on a desktop computer. Additionally, extensive prediction results (100 000 cases for each patient) obtained using this method clarify the relationship between collateral circulation and life-threatening surgical outcomes.


title: Towards unsupervised classification of macromolecular complexes in cryo electron tomography: challenges and opportunities

abstract:
Background and ObjectivesCryo electron tomography visualizes native cells at nanometer resolution, but analysis is challenged by noise and artifacts. Recently, supervised deep learning methods have been applied to decipher the 3D spatial distribution of macromolecules. However, in order to discover unknown objects, unsupervised classification techniques are necessary. In this paper, we provide an overview of unsupervised deep learning techniques, discuss the challenges to analyze cryo-ET data, and provide a proof-of-concept on real data.

MethodsWe propose an unsupervised sub-tomogram classification method based on transfer learning. We use a deep neural network to learn a clustering friendly representation able to capture 3D shapes in the presence of noise and artifacts. This representation is learned here from a synthetic data set.

ResultsWe show that when applying k-means clustering given a learning-based representation, it becomes possible to satisfyingly classify real sub-tomograms according to structural similarity. It is worth noting that no manual annotation is used for performing classification.

ConclusionsWe describe the advantages and limitations of our proof-of-concept and raise several perspectives for improving classification performance.

category: bioinformatics


title: Predicting the structure of large protein complexes using AlphaFold and sequential assembly

abstract: AlphaFold and AlphaFold-multimer can predict the structure of single- and multiple chain proteins with very high accuracy. However, predicting protein complexes with more than a handful of chains is still unfeasible, as the accuracy rapidly decreases with the number of chains and the protein size is limited by the memory on a GPU. Nevertheless, it might be possible to predict the structure of large complexes starting from predictions of subcomponents. Here, we take a graph traversal approach to assemble 175 protein complexes with 10-30 chains using predictions of subcomponents. We compute paths through a complex graph constructed of subcomponents using Monte Carlo Tree Search and assemble these in a stepwise fashion. Using subcomponents predicted from all possible trimeric interactions, 88 complexes (50%) are assembled to completion. We create a scoring function, mpDockQ, that can distinguish if assemblies are complete and predict their accuracy. Selecting complete complexes with TM-score [≥]0.9 at FPR 10% using mpDockQ results in 23 complexes with a median TM-score of 0.92. The complete assembly protocol, starting from the sequences, is freely available at: https://gitlab.com/patrickbryant1/molpc


title: Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

abstract: Summary/AbstractPARP1 contributes to genome architecture and DNA damage repair through its dynamic association with chromatin. PARP1 and PARP2 (PARP1/2) recognize damaged DNA and recruit the DNA repair machinery. Using single molecule microscopy in live cells, we monitored the movement of PARP1/2 on undamaged and damaged chromatin. We identify two classes of freely diffusing PARP1/2 and two classes of bound PARP1/2. The majority (> 60%) of PARP1/2 diffuse freely in both undamaged and damaged nuclei and in the presence of inhibitors of PARP1/2 used for cancer therapy (PARPi). Laser induced DNA damage results in a small fraction of slowly diffusing PARP1 and PARP2 to become transiently bound. Treatment of cells with PARPi in the presence of DNA damage causes subtle changes in the dynamics of bound PARP1/2, in contrast to bulk studies that suggest PARP trapping. Our results imply that next-generation PARPi could specifically target the small fraction of DNA-bound PARP1/2.


title: Dissecting β-Cardiac Myosin and Cardiac Myosin-Binding Protein C Interactions using a Nanosurf Assay

abstract:
Cardiac myosin-binding protein C (cMyBP-C) regulates cardiac contractility by slowing shortening velocity and sensitizing the thin filament to calcium. cMyBP-C has been shown to interact with the proximal myosin S2 tail and the thin filament. However, the relative contribution of these interactions to the collective modulation of actomyosin ensemble function remains unclear. Hence, we developed a "nanosurf" assay as a model system to interrogate cMyBP-C interactions with actin and/or myosin. Synthetic thick filaments were generated using recombinant human {beta}-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14 or 28 nm spacing, corresponding to the 14.3 nm myosin spacing found in native thick filaments. In vitro motility assays with myosin bound to the surrounding surface, exhibit enhanced thin filament interactions with synthetic thick filaments. No significant differences were observed in mean thin filament velocities between 14 and 28 nm spacing, consistent with our previous results for myosin V, VI, and {beta}-cardiac myosin S1. Our nanosurf assay demonstrates the slowing of actomyosin motility by cMyBP-C. Alternating {beta}-cardiac myosin HMM and cMyBP-C N-terminal fragments, C0-C2 or C1-C2, every 14 nm on the nanotube, reduced the mean thin filament velocity 4-6 fold relative to myosin alone. Interestingly, similar inhibition was observed using a {beta}-cardiac myosin S1 construct, which lacks the S2 region proposed to interact with cMyBP-C, suggesting the actin-cMyBP-C interactions may dominate the inhibitory mechanism. No significant inhibition of thin filament velocity was observed with a C0-C1f fragment, lacking the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A phosphomimetic C0-C2 fragment showed a 3-fold higher velocity compared to its phosphonull counterpart, further highlighting phosphorylation-dependent regulation via the M-domain. Together, we have established the nanosurf assay as a tool to precisely manipulate spatially dependent cMyBP-C binding partner interactions, shedding light on the molecular regulation of {beta}-cardiac myosin contractility.

STATEMENT OF SIGNIFICANCECardiac myosin-binding protein C (cMyBP-C) is the most frequently mutated protein associated with hypertrophic cardiomyopathy (HCM), a common cause of sudden cardiac death. Despite the importance of cMyBP-C in cardiac contractility, the mechanisms underlying this regulation are unclear due to experimental challenges in studying the complex, transient, weak interactions of cMyBP-C with the contractile proteins of the sarcomere. In this study, we created a nanosurf synthetic DNA thick filament assay to dissect the cMyBP-C interactions with actin and human {beta}-cardiac myosin. We demonstrate actomyosin inhibition by cMyBP-C fragments regardless of recombinant human {beta}-cardiac myosin subfragment (HMM or S1) and highlight the importance of the cMyBP-C M-domain using cMyBP-C fragments and phosphomimetics.


title: Systemic circulating microRNA landscape in Lynch syndrome

abstract:
MicroRNAs (miRs) are non-coding RNA-molecules that regulate gene expression. Global circulating miR (c-miR) expression patterns (c-miRnome) change with carcinogenesis in various sporadic cancers. Therefore, aberrantly expressed c-miRs could have diagnostic, predictive and prognostic potential in molecular profiling of cancers. c-miR functions in carriers of inherited pathogenic mismatch-repair gene variants (path_MMR), also known as Lynch syndrome (LS), have remained understudied. LS cohort provides an ideal population for biomarker mining due to increased lifelong cancer risk and excessive cancer occurrence. Using high-throughput sequencing and bioinformatic approaches, we conducted an exploratory analysis to characterize systemic c-miRnomes of path_MMR carriers. Our discovery cohort included 81 healthy path_MMR carriers and 37 non-LS controls. Our analysis also included cancer cohort comprised of 13 path_MMR carriers with varying cancers and 24 sporadic rectal cancer patients. We showed for the first time that c-miRnome can discern healthy path_MMR carriers from non-LS controls but does not distinguish healthy path_MMR carriers from cancer patients with or without path_MMR. Our c-miR expression analysis combined with in silico tools suggest ongoing alterations of biological pathways shared in LS and sporadic carcinogenesis. We observed that these alterations can produce a c-miR signature which can be used to track oncogenic stress in cancer-free path_MMR carriers. Thus, c-miRs hold potential in monitoring which cancer patients would require more intensive surveillance or clinical management.

SignificanceC-miRnome can discern between healthy persons with or without path_MMR but does not distinguish healthy path_MMR carriers from cancer patients with or without path_MMR, indicating an ongoing alteration of biological pathways that can be used to track oncogenic stress at cancer-free state.


title: Predicting systemic and pulmonary tissue barrier concentration of orally inhaled drug products

abstract:
The complex physiology and anatomy of the lungs and the range of processes involved in pulmonary drug transport and disposition make it challenging to predict the fate of orally inhaled drugs. This study aimed to develop an integrated computational pharmacology approach to mechanistically describe the spatio-temporal dynamics of inhaled drugs in both systemic circulation and site-specific lung tissue. The model included all the physiologically relevant pulmonary processes, such as deposition, dissolution, transport across lung barriers, and mucociliary clearance, to predict the inhaled drug pharmacokinetics. For validation test cases, the model predicted the fate of orally inhaled budesonide (highly soluble, mildly lipophilic) and fluticasone propionate (practically insoluble, highly lipophilic) in healthy subjects for: i) systemic and site-specific lung retention profiles, ii) aerodynamic particle size-dependent deposition profiles, and iii) identified the most impactful drug-specific, formulation-specific, and system-specific property factors that impact the fate of both the pulmonary and systemic concentration of the drugs. In summary, the presented multiscale computational model can guide the design of orally inhaled drug products to target specific lung areas, identify the effects of product differences on lung and systemic pharmacokinetics, and be used to better understand bioequivalence of generic orally inhaled drug products.

Author summaryDespite widespread use of available orally inhaled drug products (OIDPs), much is unknown regarding their optimal lung deposition, targeted delivery to specific lung regions, and the effects of various device, formulation, and physiological factors on deposition, absorption, transport, and clearance. In this study, we have presented a multiscale computational framework that integrates a full-scale 24 generation 3D lung model with distinct barrier regions spanning trachea, tracheobronchial, alveolar, and the terminal alveolar sacs with multiple other modules to track the OIDP levels (concentration) in both blood and pulmonary tissue regions. Along with validating the framework on two different inhaled drug types, we have also presented a sensitivity analysis to highlight the most impactful drug and formulation parameters, and therefore, potential optimization parameters to modulate lung selectivity and to better understand the pulmonary retention of drugs in distinct lung regions.

category: pharmacology and toxicology


title: The pho1;2a'-m1.1 allele of Phosphate1 conditions mis-regulation of the phosphorus starvation response in maize (Zea mays ssp. mays L.)

abstract: PHO1 proteins play a central role in plant inorganic phosphorus translocation and sensing. The maize (Zea mays ssp. mays) genome encodes two co-orthologs of the Arabidopsis PHO1 gene, designated ZmPho1;2a and ZmPho1;2b. Here, we report the characterization of the transposon-footprint allele Zmpho1;2a-m1.1, which we refer to hereafter as pho1;2a. The pho1;2a allele is a stable derivative formed by excision of an Activator element from the ZmPho1;2a gene. The pho1;2a allele contains an 8 bp insertion at the point of excision that disrupts the reading frame and is predicted to generate a premature translational stop. We show that the pho1;2a allele is linked to a dosage-dependent reduction in transcript accumulation and a mild reduction in seedling growth that is enhanced under nutrient deficient conditions. Characterization of the shoot and root transcriptomes of seedlings segregating the pho1;2a mutation under different nutrient conditions revealed pho1;2a to have a dominant effect on patterns of transcript accumulation. Gene set enrichment analysis of the transcripts mis-regulated in pho1;2a mutants suggests that Pho1;2a functions in the fine-tuning of the transcriptional phosphate starvation response. We discuss our results with reference to possible genetic redundancy among maize Pho1 genes and in the context of reports linking functional variation in Pho1;2a to agronomically important traits.

category: plant biology


title: Cold-induced cyt elevations function to support osmoregulation in marine diatoms

abstract: Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater and intertidal environments. The latter can experience significant long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We have examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We find that the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations correlate with the rate of temperature decrease. We do not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance, but show that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As inter-tidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signalling interacts with osmotic signalling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signalling pathways may play an important role in their cellular responses to multiple simultaneous stressors.


title: Pulvinar Slits: Cellulose-deficient and De-esterified Homogalacturonan-rich Structures in a Legume Motor Cell

abstract: The cortical motor cells (CMCs) in a legume pulvinus execute the reversible deformation in leaf movement that is driven by changes in turgor pressure. In contrast to the underlying osmotic regulation, the cell wall structure of CMCs that contributes to the movement has yet to be characterized in detail. Here, we report that the cell wall of CMCs has tangential slits with low levels of cellulose deposition. This structure is unique and distinct from any other primary cell wall reported so far; thus, we named them " pulvinar slits." Notably, we predominantly detected de-methyl esterified homogalacturonan inside pulvinar slits, with a low deposition of highly methyl-esterified homogalacturonan inside the slits, as with cellulose. In addition, Fourier-transform infrared spectroscopy analysis indicated that the cell wall composition of pulvini is different from that of other axial organs, i.e. petioles or stems. Moreover, monosaccharide analysis showed that pulvini are pectin-rich organs like developing stems. Computer modeling suggested that pulvinar slits facilitate anisotropic extension in the direction perpendicular to the slits in the presence of turgor pressure. Mechanical tests revealed that the proximo-distal extensibility of pulvini is superior to that of the other axial organs. In this study, we thus established the distinctive cell wall structure of CMCs that contribute to plant movement, adding new knowledge to the structural diversity and function of the plant cell wall.


title: Improving an rRNA depletion protocol with statistical design of experiments

abstract:
rRNA depletion is the most expensive step in prokaryotic RNA-seq library preparation. rRNA is so abundant that small increases in depletion efficiency lead to large changes in mRNA sequencing coverage. A variety of commercial and home-made methods exist to lower the cost or increase the efficiency of rRNA removal. Many of these techniques are suboptimal when applied to new species of bacteria or when the protocol or reagents need to be changed. Re-optimizing a protocol by trial-and-error is an expensive and laborious process. Systematic frameworks like the statistical design of experiments (DOE) can improve processes by exploring the quantitative relationship between multiple factors. DOE allows experimenters to find factor interactions that may not be apparent when factors are studied in isolation.

We used DOE to optimize an rRNA depletion protocol by updating reagents and identifying factors that maximize rRNA removal and minimize cost. The optimized protocol more efficiently removes rRNA, uses fewer reagents, and is less expensive than the original protocol. Our optimization required only 17 experiments and identified two significant interactions among three factors. Overall, our approach demonstrates the utility of a rational, DOE framework for improving complex protocols.

category: systems biology


title: Use of Avian Adeno-Associated Virus for the Delivery of Transgenes to Pekin Duck Cells in vitro and in vivo

abstract:
The Pekin duck was domesticated between 4000 and 10,000 years ago from the Mallard duck and is the predominate meat type duck in the world (Cherry and Morris, 2008). The global production of waterfowl is a rapidly growing industry. Total meat duck production increased from 2.9 million tons in 2000 to nearly 4.4 million tons in 2013, a growth rate of 3.2% per year, and further increased to 7.2 million tons in 2018, and valued at $19B in 2019 (IndexBox, 2019). Pekin ducks (Anas platyrhynchus domesticus) are the fastest growing poultry species growing from hatch to 4.5 kg market weight in as little as 28 days (Blois et al., 2019; Campbell et al., 2015; Cherry and Morris, 2008). Thus, there is a need to study the growth and reproduction of this economically important species. While Pekin duck biology is being explored by many researchers, there are fewer molecular tools available for duck compared to other poultry species and many fewer compared to mammalian systems. For example, one molecular tool commonly used to interrogate other systems are adeno-associated virus (AAV) vectors. AAV vectors are being utilized by many researchers to deliver transgenes to target tissues or cells and for genetic manipulation. Recently, avian adeno-associated virus (A3V) has been used to deliver genes to the cells and neurons of the domestic chicken (Gallus gallus domesticus). Here, we show that A3V can be used to deliver genes to Pekin duck neurons and cells in culture. Together, these results suggest that A3V will be a useful molecular tool in Pekin duck research.

Summary StatementHere we demonstrate the use of viral vectors to deliver transgenes to Pekin duck cells. These vectors can be used to advance understanding of reproduction in this economically important species.

category: zoology


title: New distribution data and phylogenetic approach reveal bioregionalization of the western Palearctic ants

abstract:
AimBiogeographic regionalization has fascinated biogeographers and ecologists for centuries and is endued with new vitality by evolutionary perspectives. However, progress is scant for most insect groups due to shortfalls in distribution and phylogenetic information, namely Wallacean and Darwinian shortfalls respectively. Here, we used the western Palearctic ants as the case to tackle these shortfalls and test their biogeographic structure through novel distribution data and phylogenetic approaches.

LocationWestern Palearctic realm.

TaxonAnts (Formicidae).

MethodsFirstly, we developed a refined database integrating the occurrences of 747 ant species across 207 regions of the western Palearctic realm, based on newly expert-validated records derived from the existing global ant biodiversity informatics. Using range estimates for these species derived from polygons and species distribution modelling, we produced species assemblages in 50 x 50 km grid cells. We calculated taxonomic and phylogenetic turnover of ant assemblages, performing hierarchical clustering analysis using the Simpson dissimilarity index to delineate biogeographic structure.

ResultsAt both the regional list- and grid assemblage-levels, the Mediterranean has higher turnover and more biogeographic regions than northern Europe, both taxonomically and phylogenetically. Delineations based on grid assemblages detected more detailed biogeographic transitions, while those based on regional lists showed stronger insularity in biogeographic structure. The phylogenetic regionalization suggested closer but varied affinities between assemblages in comparison to the taxonomic approach.

Main conclusionsHere, we integrated expert-validated regional lists, species distribution modelling, and a recent phylogeny to tackle Wallacean and Darwinian shortfalls for an important insect group by developing a next-generation map of biogeographic regionalization for the western Palearctic ants. The results of this study suggest strong constraints from geographic barriers and potential effects of climatic history on ant distributions and evolutionary history, and also provide baseline spatial information for future investigations of regional insect distributions.


title: Familial ALS-associated SFPQ variants promote the formation of SFPQ cytoplasmic aggregates that reduce surface AMPA receptor expression in primary neurons

abstract: SFPQ is a nuclear RNA-binding protein that is involved in a wide range of physiological processes including neuronal development and homeostasis. However, the mislocalization and cytoplasmic aggregation of SFPQ are associated with the pathophysiology of amyotrophic lateral sclerosis (ALS). We have previously reported that zinc mediates SFPQ polymerization and promotes the formation of cytoplasmic aggregates in neurons. Here we characterize two familial ALS (fALS)-associated SFPQ variants, which cause amino acid substitutions in the proximity of the SFPQ zinc-coordinating center (N533H and L534I). Both mutants display increased zinc-binding affinities, which can be explained by the presence of a secondary zinc-binding site revealed by the 1.83[A] crystal structure of the human SFPQ L534I mutant. Overexpression of these fALS-associated mutants significantly increases the number of SFPQ cytoplasmic aggregates in primary neurons. Although they do not affect the density of dendritic spines, the presence of SFPQ cytoplasmic aggregates causes a marked reduction in the levels of the GluA1, but not the GluA2 subunit of AMPA-type glutamate receptors on the neuronal surface. Taken together, our data demonstrate that fALS-associated mutations enhance the propensity of SFPQ to bind zinc and form aggregates, leading to the dysregulation of AMPA receptor subunit composition, which may contribute to neuronal dysfunction in ALS.

category: neuroscience


title: Species-specific chromatin landscape determines how transposable elements shape genome evolution

abstract:
Transposable elements (TEs) are selfish genomic parasites that increase their copy number at the expense of host fitness. The "success," or genome-wide abundance, of TEs differs widely between species. Deciphering the causes for this large variety in TE abundance has remained a central question in evolutionary genomics. We previously proposed that species-specific TE abundance could be driven by the inadvertent consequences of host-direct epigenetic silencing of TEs--the spreading of repressive epigenetic marks from silenced TEs into adjacent sequences. Here, we compared this TE-mediated "epigenetic effect" in six species in the Drosophila melanogaster subgroup to dissect step-by-step the role of such effect in determining genomic TE abundance.

We found that TE-mediated spreading of repressive marks is prevalent and substantially varies across and even within species. While this TE-mediated effect alters the epigenetic states of adjacent genes, we surprisingly discovered that the transcription of neighboring genes could reciprocally impact this spreading. Importantly, our multi- species analysis provides the power and appropriate phylogenetic resolution to connect species-specific host chromatin regulation, TE-mediated epigenetic effects, the strength of natural selection against TEs, and genomic TE abundance unique to individual species. Our findings point towards the importance of host chromatin landscapes in shaping genome evolution through the epigenetic effects of a selfish genetic parasite.


title: THE STABILIZATAION OF C-MYC BY THE NOVEL CELL CYCLE REGULATOR, SPY1, DECREASES EFFICACY OF BREAST CANCER TREATMENTS

abstract:
Purposec-Myc is frequently upregulated in breast cancers, however, targeting c-Myc has proven to be a challenge. Targeting of downstream mediators of c-Myc, such as the  cyclin-like cell cycle regulator Spy1, may be a viable therapeutic option in a subset of breast cancer subtypes.

MethodsMouse mammary tumour cells isolated from MMTV-Myc mice and human breast cancer cell lines were used to manipulate Spy1 levels followed by tamoxifen or chemotherapeutic treatment with a variety of endpoints. Patient samples from TNBC patients were obtained and constructed into a TMA and stained for c-Myc and Spy1 protein levels.

ResultsOver time, MMTV-Myc cells show a decreased response to tamoxifen treatment with increasing levels of Spy1 in the tamoxifen-resistant cells. shRNA against Spy1 re-establishes tamoxifen sensitivity. Spy1 was found to be highly elevated in human TNBC cell and patient samples, correlating to c-Myc protein levels. c-Myc was found to be stabilized by Spy1 and knocking down Spy1 in TNBC cells shows a significant increase in response to chemotherapy treatments.

ConclusionsUnderstanding the interplay between protein expression level and response to treatment is a critical factor in developing novel treatment options for breast cancer patients. These data have shown a connection between Spy1 and c-Myc protein levels in more aggressive breast cancer cells and patient samples. Furthermore, targeting c-Myc has proven difficult, these data suggest targeting Spy1 even when c-Myc is elevated can confer an advantage to current chemotherapies.


title: ATR-I774Yfs*5 promotes genomic instability through micronuclei formation

abstract: Although mismatch repair (MMR) defects are associated with high risk of malignancy, the specific oncogenic drivers pertinent to MMR-affected cancers are poorly characterized. The heterozygous ATR-I774Yfs*5 mutation, the result of strand slippage in a poly-A tract of the Ataxia Telangiectasia and Rad3 related (ATR) gene, is overexpressed in MMR-defective malignancies including colorectal carcinoma (CRC) and is the most common ATR mutation in cancer. Here, we explore the contribution of ATR-I774Yfs*5 to genomic integrity. Using heterozygous ATR-I774Yfs*5 HCT-116 cells to mimic the native mutation, we found this mutation reduced ATR activity as measured by damage-induced Chk1 phosphorylation at S317 and ATR autophosphorylation ATR at T1989. ATR-I774Yfs*5 expression impaired genomic stability as visualized by the appearance of micronuclei in two stable expression models as well as in cell lines transfected with ATR-I774Yfs*5. Micronucleus development was dependent on replication and independent of ATR copy number. ATR-I774Yfs*5 expression did not alter cellular viability, cell cycle progression, or replicative rate, suggesting this mutation is well-tolerated despite its destabilizing effect on the genome. Taken together, these data suggest that the ATR-I774Yfs*5, whose development is favored in the context of MMR deficiency, may represent an important driver of a mutator phenotype by promoting genomic instability.


title: Taxonomic investigation of the zooplanktivorous Lake Malawi cichlids Copadichromis mloto (Iles) and C. virginalis (Iles).

abstract: The taxonomic status of the zooplanktivorous cichlids Copadichromis mloto and C. virginalis has been confused since their original descriptions by lles in 1960. While two forms of C. virginalis, Kaduna and Kajose, were distinguished in the type material, C. mloto has not been positively identified since its original description. Here we re-examined the types as well as 54 recently collected specimens from multiple sampling locations. Genome sequencing of 51 recent specimens revealed two closely related but reciprocally monophyletic clades. Geometric morphological analysis indicated that one clade morphologically encompasses the type specimens of C. virginalis identified by Iles as the  Kaduna form, including the holotype, while the other clade encompasses not only the paratypes identified as the  Kajose form, but also the type series of C. mloto. Given that all three forms have the same type locality, that there are no meristic or character states to differentiate them and that there are no records of adult male C. mloto in breeding colours, we conclude that the  Kajose form previously identified as C. virginalis represents relatively deeper bodied sexually active or maturing individuals of C. mloto.


title: Comparing the potential of microglia preparations generated using different human iPSC-based differentiation methods to model microglia-mediated mechanisms of amyotrophic lateral sclerosis pathophysiology

abstract: Microglia are the resident immune cells of nervous system. In healthy conditions, microglia actively patrol neural tissues in a homeostatic state, which can rapidly change to an activated state in response to local injury/disease. Dysregulated microglia activation is a hallmark of disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The elucidation of the roles of microglia in human biology and disease has recently benefitted from the development of human induced pluripotent stem cell (iPSC)-based approaches to generate microglia-like cells. Microglia represent a heterogeneous group of cells with spatial diversity in both health and disease. This situation poses a considerable challenge along the path towards establishing the most pathologically-relevant human iPSC-derived microglia preparations to investigate the complex roles of microglia in ALS and other neurological diseases. The success of these approaches must account for microglia diversity in different regions of the brain and spinal cord. In this study, we compared the transcriptomes of human iPSC-derived microglia generated using different methods to determine whether or not separate strategies can be used to generate microglia with distinct transcriptional signatures in vitro. We show that different derivation methods give rise to preparations comprising human microglia with distinct transcriptomic signatures resembling the gene profiles of specific microglia subpopulations in vivo. These findings suggest that a careful, and coordinated, implementation of multiple microglia differentiation methods from human iPSCs can be an effective approach towards the goal of generating multiple microglia subtypes that will offer enhanced model systems to account for microglia heterogeneity in vivo. Spatially-defined human iPSC-derived microglia would represent an enhanced tool to study the multiple levels of involvement of microglia in mechanisms of motor neuron degeneration in ALS, as well as other neurological diseases and disorders.


title: Purinergic P2X7 Receptor-mediated inflammation precedes PTSD-related Behaviors in Rats

abstract: Clinical evidence has linked increased peripheral pro-inflammatory cytokines with post-traumatic stress disorder (PTSD) symptoms. However, whether inflammation contributes to or is a consequence of PTSD is still unclear. Previous research shows that stress can activate P2X7 receptors (P2X7Rs) on microglia to induce inflammation and behavioral changes. In this investigation, we examined whether P2X7Rs contribute to the development of PTSD-like behaviors induced by single prolonged stress (SPS) exposure in rats. Consistent with the literature, exposing adult male and female rats to SPS produced a PTSD-like phenotype of impaired fear extinction and increased anxiety-like behavior one week after exposure. In addition, SPS-exposed animals had more Iba1-positive microglia expressing the P2X7R in the ventral hippocampus, a structure that regulates fear extinction and anxiety-like behavior. Next, we examined if inflammation precedes the behavioral manifestations. Three days after SPS exposure, increased inflammatory cytokines were found in the blood and hippocampal microglia showed increased expression of the P2X7R, IL-1{beta}, and TNF-, suggesting increased peripheral and central inflammation before behavioral testing. To determine whether P2X7Rs contribute to the PTSD-related behaviors induced by SPS exposure, we gave ICV infusions of the P2X7R antagonist, A-438079, for one week starting the day of SPS exposure. Blocking P2X7Rs prevented the SPS-induced impaired fear extinction and increased anxiety-like behaviors in male and female rats, suggesting that SPS activates P2X7Rs which increase inflammation to produce a PTSD-like phenotype.


title: The pore-forming protein gasdermin D is a cellular redox sensor

abstract: Reactive oxygen species (ROS) affect inflammation and immunity in a multitude of ways, in particular in the context of the signaling pathways that determine cell fate. Inflammasomes are multiprotein cytoplasmic complexes whose pyroptosis-inducing activities are controlled by ROS. Our knowledge of how ROS mediates inflammasome activities is largely based on studies of the initiating events in these pathways. Herein, we show that ROS controls the terminal events in the pyroptosis pathways. We found that ROS oxidizes the protein gasdermin D (GSDMD) and promotes its assembly into a death-inducing pore forming complex. Mechanistically, ROS enhances GSDMD-mediated pyroptosis in an intrinsic manner, likely through oxidative modification of a specific cysteine residue (C192). Diverse ROS sources promote GSDMD oxidation, ranging from homeostatic control via the Ragulator-Rag complex, to inducible control via diverse microbial products and environmental toxins. These findings expand the steps in the inflammasome pathway that are controlled by ROS and suggest that GSDMD operates as a pyroptosis-inducing redox sensor.

category: immunology


title: Evolutionary Principles of Bacterial Signaling Capacity and Complexity

abstract: Microbes rely on signal transduction systems to sense and respond to environmental changes for survival and reproduction. It is generally known that niche adaptation plays an important role in shaping the signaling repertoire. However, the evolution of bacterial signaling capacity lacks systematic studies with a temporal direction. Particularly, it is unclear how complexity evolved from simplicity or vice versa for signaling networks. Here we examine the evolutionary processes of major signal transduction systems in Campylobacterota (formerly Epsilonproteobacteria), a phylum with sufficient evolutionary depth and ecological diversity. Evolution of signaling systems within Campylobacterota shows two opposite trends. During niche expansion, signaling complexity increases with gene expansions through horizontal gene transfer (HGT), gene duplication, fusion and fission, which create opportunities for genetic innovation and pathway integration. In contrast, as the lineages adapt to a specialized niche, complexity decreases with massive gene losses that lead to the decline or disappearance of pathways mediated by multiple transmitters. Overall, signaling capacity and complexity arise and drop together in Campylobacterota, determined by sensory demand, genetic resources and co-evolution within the genomic context. These findings reflects plausible evolutionary principles for other cellular networks and genome evolution of the Bacteria domain.


title: Identification of unusually disulphide-bonded insulin forms using mass spectrometry and thermolysin cleavage

abstract: Insulin is an essential hormone made by the pancreatic beta-cells in the islets of Langerhans. Beta-cells produce more insulin protein than virtually all other cellular proteins combined. Dysfunction in the process of insulin synthesis can lead to disease, including rare forms of monogenic diabetes. Specifically, aberrant intra-insulin and inter-insulin disulphide bonds have been implicated in the pathology of type 1 diabetes and type 2 diabetes, respectively. In type 1 diabetes, misprocessed insulin isoforms may be neoepitopes that kick-start and/or exacerbate the auto-immune response. In type 2 diabetes, aberrant disulphides form insulin dimers that can clog the endoplasmic reticulum and contribute to beta cell dysfunction. To facilitate the study of novel and known insulin neoepitopes and dimers, we present an unbiased and rapid technique for identifying insulin disulphide patterns from pancreatic islet extracts. The basis of this method is the cleavage between insulins cysteine residues with the metalloprotease, thermolysin, and subsequent identification of cysteine containing fragments and their partner peptides by LC-MS/MS. Using this technique, we identify 6 aberrant disulphide bonded insulin species, including a previously described type 1 diabetes neoepitope, as well as inter-chain disulphide bonded insulin dimers. Furthermore, using the endoplasmic stress inducer, thapsigargin, we observe increased disulphide errors in a patient donor sample. This approach lays foundations to identify the scope and cause of aberrant insulin disulphide formation in health and disease.


title: Interstrand Crosslinks in Donor DNA Boost Gene Editing in Human Cells

abstract: Co-introduction of targeted nucleases and DNA/RNA templates encoding new genomic sequence is the basis for rapid, effective, and iterable gene editing workflows for therapeutic, agricultural, and basic science applications. Extensive optimization of reagent delivery and nuclease activity have improved genome editing workflows, but comparatively few efforts have been made to alter the gene editing activity of template molecules. Here, we report template DNA modified with interstrand crosslinks (ICLs) - xHDRTs - increases editing frequencies in Cas9-directed gene editing workflows by up to five-fold. xHDRTs increase gene editing frequencies independent of DNA template topology, amount of sequence added, or cell type. Gene editing using xHDRTs requires the DNA repair kinase, ATR, and partially requires Fanconi Anemia proteins, including FANCA, but is independent of other ICL-repair pathways. Covalent modification of donor DNA thus presents a compelling opportunity to improve nonviral gene editing workflows.


title: Ecdysone signaling controls early embryogenesis in the short-germ hemimetabolous insect Blattella germanica.

abstract: Steroid hormone signaling is a central regulator of insect development especially during the post-embryonic period. However, its role during embryonic development is less understood, particularly in short-germ band hemimetabolous insects. Here, we used the short-germ band hemimetabolous insect Blattella germanica to analyse the functions of 20-hydroxyecdysone (20E), the most active insect steroid, signaling during the embryonic stage. We show that the heterodimeric 20E receptor, BgEcR-A and BgRXR, is present from the beginning of embryogenesis and that a stereotypical 20E-dependent cascade of nuclear receptors is first detected at the early blastoderm stage. Using parental RNAi, we show that both receptors are required for the proper formation of the germ band by controlling cell proliferation during the blastoderm stage. In addition, they are also required for the coordinated induction of the 20E-induced cascade of nuclear receptors. Finally, we show that two of these nuclear receptors, BgHR3 and BgFTZ-F1, have relevant roles in germ band formation. Whereas BgHR3 controls the formation and development of the cephalic region of the germ band, BgFTZ-F1 stripped expression suggested a role in the segmentation of the germ band. In summary, our results show that 20E-signaling is required much earlier in short-germ band hemimetabolous than in long-germ band holometabolous embryogenesis, which raises the possibility that the loss of 20E-signaling during the initial stages of embryogenesis could be a key feature in the evolution of the different types of insect development.

category: developmental biology


title: Robustness of field studies evaluating biodiversity responses to invasive species management in New Zealand

abstract: Benefits of invasive species management for terrestrial biodiversity are widely expected and promoted in New Zealand. Evidence for this is presented in policy and scientific reviews of the literature, but the robustness and repeatability of the underpinning evidence-base remains poorly understood. We evaluated the design of field-based studies assessing biodiversity responses to invasive species management in 155 peer-reviewed articles published across 46 journals from 2010 - 2019. Each study was assessed against nine principles of experimental design, covering robustness of sampling and avoidance of bias. These principles are important in New Zealand to detect treatment effects from environmental variability driven by underlying gradients such as soil fertility, climate and disturbance. Fifty two percent of studies defined a sampling universe and 68% of studies specified the treatment. Whereas, 54%, 74%, and 50% of studies did not utilise replication, representatively sample the universe, or quantify invasive species, respectively. Ninety five percent of studies quantified biodiversity responses, although a high proportion of these did not representatively sample replicates. Initial conditions and accounting for effects of experimental implementation were not utilised in 57% and 84% of studies respectively. No studies avoided observer/analyst bias using blinding methods, despite this being widely adopted in other fields. Ordinal logistic regression showed these principles varied in how robustly they were applied among categories of biodiversity responses and invasive species. Our findings suggest that greater attention to experimental design principles is desirable: supported by researchers, funding agencies, reviewers, and journal editors. Greater resources is not necessarily a solution to these design issues. Undertaking fewer studies, that are individually more expensive because they better adhere to experimental design principles, is one alternative. Our intent in this article is to improve the robustness of future field studies for at least some principles. Robust designs have enduring value, reduce uncertainty and increase our understanding of when, where and how often the impacts of invasive species on biodiversity are indeed reversible.

category: ecology


title: Anatomical, ontogenetic, and genomic homologies guide reconstructions of the teeth-to-baleen transition in mysticete whales

abstract: The transition in Mysticeti (Cetacea) from capture of individual prey using teeth to bulk filtering batches of small prey using baleen ranks among the most dramatic evolutionary transformations in mammalian history. We review phylogenetic work on the homology of mysticete feeding structures from anatomical, ontogenetic, and genomic perspectives. Six characters with key functional significance for filter-feeding behavior are mapped to cladograms based on 11 morphological datasets to reconstruct evolutionary change across the teeth-to-baleen transition. This comparative summary within a common parsimony framework reveals extensive conflicts among independent systematic efforts but also broad support for the newly named clade Kinetomenta (Aetiocetidae + Chaeomysticeti). Complementary anatomical studies using CTscans and ontogenetic series hint at commonalities between the developmental programs for teeth and baleen, lending further support for a  transitional chimaeric feeder scenario that best explains current knowledge on the transition to filter feeding. For some extant mysticetes, the ontogenetic sequence in fetal specimens recapitulates the inferred evolutionary transformation: from teeth, to teeth and baleen, to just baleen. Phylogenetic mapping of inactivating mutations reveals mutational decay of  dental genes related to enamel formation before the emergence of crown Mysticeti, while  baleen genes that were repurposed or newly derived during the evolutionary elaboration of baleen currently are poorly characterized. Review and meta-analysis of available data suggest that the teeth-to-baleen transition in Mysticeti ranks among the best characterized macroevolutionary shifts due to the diversity of data from the genome, the fossil record, comparative anatomy, and ontogeny that directly bears on this remarkable evolutionary transformation.

category: evolutionary biology


title: Dynamic evolution of recently duplicated genes in Caenorhabditis elegans

abstract:
As a major origin of evolutionary novelties, gene duplication is a widespread phenomenon across species. However, the evolutionary force that determines the fate of duplicate genes is still under debate. Here, we studied the functional evolution of duplicate genes at both macroevolution and microevolution scales using the genomic sequences of eleven Caenorhabditis species and 773 C. elegans wild isolates. We found that compared to older duplicate genes and single-copy genes, recently duplicated gene copies showed rapid turnover, large genetic diversity, and signs of balancing and positive selection within the species. Young duplicate genes have low basal expression restricted to a few tissues but show highly responsive expression towards pathogenic infections. Recently duplicated genes are enriched in chemosensory perception, protein degradation, and innate immunity, implicating their functions in enhancing adaptability to external perturbations. Importantly, we found that young duplicate genes are rarely essential, while old duplicate genes have the same level of essentiality as singletons, suggesting that essentiality develops over a long time. Together, our work in C. elegans demonstrates that natural selection shapes the dynamic evolutionary trajectory of duplicate genes.

SignificanceThe "evolution by gene duplication" theory suggests that gene duplications provide the genetic materials for mutation and selection to act upon, expand the repertoire of molecular functions, and enable evolutionary novelty. Although various models were proposed to describe the fate of duplicate genes, empirical evidence for these models is limited. We analyzed gene duplications in eleven nematode Caenorhabditis species and studied the intraspecific variation of these duplicate genes among C. elegans wild strains. We found that compared to older duplicate gens and single-copy genes, recently duplicated genes show rapid turnover, large genetic diversity, and strong signs of balancing and positive selection but rarely develop essential functions. Our results describe the evolutionary trajectory of duplicate genes shaped by natural selection.


title: Variation in cytonuclear expression accommodation among allopolyploid plants

abstract: Cytonuclear coevolution is a common feature among plants, which coordinates gene expression and protein products between the nucleus and organelles. Consequently, lineage-specific differences may result in incompatibilities between the nucleus and cytoplasm in hybrid taxa. Allopolyploidy is also a common phenomenon in plant evolution. The hybrid nature of allopolyploids may result in cytonuclear incompatibilities, but the massive nuclear redundancy created during polyploidy affords additional avenues for resolving cytonuclear conflict (i.e., cytonuclear accommodation). Here we evaluate expression changes in organelle-targeted nuclear genes for six allopolyploid lineages that represent four genera (i.e., Arabidopsis, Arachis, Chenopodium, and Gossypium) and encompass a range in polyploid ages. Because incompatibilities between the nucleus and cytoplasm could potentially result in biases toward the maternal homoeolog and/or maternal expression level, we evaluate patterns of homoeolog usage, expression bias, and expression level dominance in cytonuclear genes relative to the background of non-cytonuclear expression changes and to the diploid parents. Although we find subsets of cytonuclear genes in most lineages that match our expectations of maternal preference, these observations are not consistent among either allopolyploids or categories of organelle-targeted genes. Our results indicate that cytonuclear expression accommodation may be a subtle and/or variable phenomenon that does not capture the full range of mechanisms by which allopolyploid plants resolve nuclear-cytoplasmic incompatibilities.


title: Opiate responses are controlled by interactions of Oprm1 and Fgf12 loci in the murine BXD family: Correspondence to human GWAS finding

abstract: We analyzed time-dependent behavioral responses to morphine and naloxone obtained from a large family of young adult BXD mice (n = 70 strains, including C57BL/6J and DBA/2J parents, 4-9 cases per strain) using the latest whole genome sequencing (WGS)-based genetic markers. These data include quantitative locomotor and behavior responses measured three hours after an acute morphine injection (50 mg/kg i.p.), followed by naloxone-induced withdrawal, obtained by Philip et al (2010). Locomotor data were analyzed in 15 min bins and mapped jointly for both sexes or independently for males and females. We confirmed a highly significant association between locomotor response and a genomic region that overlaps with Oprm1 on Chr 10 at 6.8 Mb (LOD maximum of 11.4) between 15-105 min, with a peak at 75 min. Effects were modestly dependent of sex. Strains that were B homozygotes run 76 meters farther than those that are D homozygotes after a morphine injection. We discovered a novel association between a locus on Chr 16 and a late phase locomotor response (after 150 min) in both sexes. This locus had a significant but transient epistatic interaction with the Oprm1 locus between 45-90 min, well before the main effect was detectable. The Chr 16 locus includes one compelling candidate--fibroblast growth factor 12 (Fgf12). Null mutation of Fgf12 has been shown to cause locomotor deficits (e.g., ataxia) in mice. Analysis of genes correlated with both OPRM1 and FGF12 in six human brain regions (GTEx, v8) demonstrated an enrichment of genetic signals associated with SUD phenotypes, and a modest corroboration of variants in the FGF12 loci on Chr 3q28. To the best of our knowledge this is the first demonstration of a transient time-dependent epistatic interaction modulating drug response in mammals--a finding with interesting mechanistic implications. Finally, this work demonstrates how high-quality FAIR+ data can be used with newly acquired data sets to yield striking results, and how joint mouse and human neurogenomic and mapping data can be merged at gene and network levels for bidirectional validation of potential SUD variants and molecular networks.

category: genetics


title: Retained introns in long RNA-seq reads are not reliably detected in sample-matched short reads

abstract: There is growing interest in retained introns in a variety of disease contexts including cancer and aging. Many software tools have been developed to detect retained introns from short RNA-seq reads, but reliable detection is complicated by overlapping genes and transcripts as well as the presence of unprocessed or partially processed RNAs. We compared introns detected by 5 tools using short RNA-seq reads with introns observed in long RNA-seq reads from the same biological specimens and found: (1) significant disagreement among tools (Fleiss{kappa} = 0.231) such that 52.4% of all detected intron retentions were not called by more than one tool; (2) that no tool achieved greater than 20% precision or 35% recall under generous conditions; and (3) that retained intron detectability was adversely affected by greater intron length and overlap with annotated exons.


title: Protein arginine and lysine methylation in Trypanosoma cruzi

abstract:
Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein interaction and protein-nucleic acid. Mass spectrometry-based proteomics has allowed a large-scale identification of protein methylation - mainly in arginine residues-, in different organisms, including some trypanosomes. However, the presence and role of protein methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. In this work, we applied mass spectrometry and described the arginine and lysine methylproteome of T. cruzi. In epimastigotes, 1336 methylsites (657 methyl-arginines and 679 methyl-lysines) in 878 methylated proteins were identified by LC-MS/MS. Our functional and interaction analyzes show that protein methylation impacts different biological processes, with emphasis on translation. Separately, protein arginine methylation is related to oxireduction and carbohydrate metabolism, while lysine methylation impacts the protein synthesis. In addition, 50 methylated proteins have been previously described with phosphorylation sites in T. cruzi, represented by RNA binding proteins, sterol methyltransferase activity and calpain peptidases, indicating the possibility of crosstalk in the regulation of these proteinsThis work represents the first proteomic analysis of T. cruzi methylproteome and is the first to characterize lysine methylation in trypanosomatids. Collectively, these data inform about new fundamental biological aspects of this organism impacted by protein methylation, that can contribute to the identification of pathways and key pieces in the biology of this human pathogen. Keywords: Trypanosoma cruzi. Protein methylation. Proteomics. LC-MS/MS.

Biological significanceTrypanosoma cruzi is a protozoan parasite that causes Chagas disease in humans and throughout its life cycle faces different environment changes. Protein methylation is an important post-translational modification by which cells respond and adapt to the environment. To understand the importance of protein methylation in T.cruzi biology, we applied a mass spectrometry-based proteomics (GeLC-MS/MS) and report the first proteomic analysis of both arginine and lysine methylproteome in T. cruzi, being the first large-scale characterization of lysine methylation in trypanosomes. Our data demonstrate that the methylation of proteins in T. cruzi is broad and impacts different cellular processes. This study represents a significant advance on the importance of protein methylation in trypanosomes.

HighlightsO_LIFirst methylproteome description of human parasite T. cruzi and first of lysine methylation in trypanosomes;
C_LIO_LIProtein arginine and lysine methylation is widely found in T. cruzi epimastigotes;
C_LIO_LIDifferent processes are impacted by protein methylaton in T. cruzi, mainly the protein synthesis;
C_LIO_LI50 methylated proteins have been previously described with phosphorylation sites in T. cruzi.
C_LI


title: Snaq: A Dynamic Snakemake Pipeline for Microbiome data analysis with QIIME2

abstract: Optimizing a protocol for 16S microbiome data analysis with QIIME2 is a challenging task for biologists with no programming experience. It involves a multi-step process, and multiple parameters and options that need to be tested and determined. In this article, we describe Snaq, a snakemake pipeline that helps automate and optimize 16S data analysis using QIIME2. Snaq offers an informative file naming system and automatically performs the analysis of a data set by downloading and installing the required databases and classifiers, all through a single command-line instruction. It works natively on Linux and Mac and on Windows through the use of containers, and is potentially extendable by adding new rules. This pipeline will substantially reduce the efforts in sending commands and prevent the confusion caused by the accumulation of analysis results due to testing multiple parameters.


title: Identifying transcription factor-DNA interactions using machine learning

abstract: Machine learning approaches have been applied to identify transcription factor (TF)-DNA interaction important for gene regulation and expression. However, due to the enormous search space of the genome, it is challenging to build models capable of surveying entire reference genomes, especially in species where models were not trained. In this study, we surveyed a variety of methods for classification of epigenomics data in an attempt to improve the detection for 12 members of the Auxin Response Factor (ARF) binding DNAs from maize and soybean as assessed by DNA Affinity Purification and sequencing (DAP-seq). We used the classification for prediction by minimizing the genome search space by only surveying unmethylated regions (UMRs). For identification of DAP-seq binding events within the UMRs, we achieved 93.54% accuracy, 6.2% false positive, and a 43.29% false negative rate across 12 members of ARFs of maize on average by encoding DNA with count vectorization for k-mer with a logistic regression classifier with up-sampling and feature selection. Importantly, feature selection helps to uncover known and potentially novel ARF binding motifs. This demonstrates an independent method for identification of transcription factor binding sites. Finally, we tested the model built with maize DAP-seq data and applied it directly to the soybean genome and found unacceptably high false positive rates, which accounted for more than 40% across the ARF TFs tested. The findings in this study suggest the potential use of various methods to predict TF-DNA interactions within and between species with varying degrees of success.


title: EZH2 endorses cell plasticity to carcinoma cells facilitating mesenchymal to epithelial transition and tumour colonization

abstract: Reversible transition between the epithelial and mesenchymal states are key aspects of carcinoma cell dissemination and the metastatic disease, and thus, characterizing the molecular basis of the epithelial to mesenchymal transition (EMT) is crucial to find druggable targets and more effective therapeutic approaches in cancer. Emerging studies suggest that epigenetic regulators might endorse cancer cells with the cell plasticity required to conduct dynamic changes in cell state during EMT. However, epigenetic mechanisms involved remain mostly unknown. Polycomb Repressive Complexes (PRCs) proteins are well-established epigenetic regulators of development and stem cell differentiation, but their role in different cancer systems is inconsistent and sometimes paradoxical. In this study, we have analysed the role of the PRC2 protein EZH2 in lung carcinoma cells. We found that besides its described role in CDKN2A-dependent cell proliferation, EZH2 upholds the epithelial state of cancer cells by repressing the transcription of hundreds of mesenchymal genes. Chemical inhibition or genetic removal of EZH2 promotes the residence of cancer cells in the mesenchymal state during reversible epithelial-mesenchymal transition. In fitting, analysis of human patient samples and tumour xenograft models indicate that EZH2 is required to efficiently repress mesenchymal genes and facilitate tumour colonization in vivo. Overall, this study discloses a novel role of PRC2 as a master regulator of EMT in carcinoma cells. This finding has important implications for the design of therapies based on EZH2 inhibitors in human cancer patients.

category: cancer biology


title: The GHB analogue HOCPCA improves sensorimotor function after MCAO via CaMKIIα

abstract: Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented GHB ligands as the first small molecules selectively targeting the CaMKII hub, a domain primarily responsible for holoenzyme oligomerisation, with an emerging functional role. Here, we report that the GHB ligand, HOCPCA, improves sensorimotor function after experimental stroke in mice when administered at clinically relevant time and in combination with alteplase. We observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated the ischemia-specific expression of a constitutively active CaMKII kinase fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. HOCPCAs selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKII hub domain as an attractive neuroprotective strategy.


title: Transcutaneous electrical nerve stimulation modulates corticospinal excitability while preserving motor unit discharge properties during isometric contractions

abstract:
AimTranscutaneous electrical nerve stimulation (TENS) aims to supplement sensory feedback to improve force steadiness or motor function. In this study, we directly assessed potential changes in corticospinal excitability and motor unit discharge characteristics from the first dorsal interosseous (FDI) muscle due to TENS by using transcranial magnetic stimulation (TMS) and high-density surface electromyography (HDsEMG).

MethodsEleven healthy young adults performed a series of submaximal isometric index abductions. We estimated i) motor evoked potential (MEP) amplitudes, ii) persistent inward current amplitudes (PIC, i.e., delta F), iii) motor unit recruitment thresholds and discharge rates, and iv) common synaptic input to motor units before and after TENS.

ResultsTENS did not affect force steadiness (2.5 {+/-} 0.9% and 3.3 {+/-} 1.9% (p = 0.010)). MEP amplitudes decreased at 110% of the resting motor threshold (rMT; 0.72 {+/-} 0.66 mV vs. 0.59 {+/-} 0.63 mV; p < 0.001), increased at 130% rMT (1.18 {+/-} 1.10 mV vs. 1.41 {+/-} 1.29 mV; p < 0.001). Delta F increased after TENS (3.7 {+/-} 2.2 pps vs. 4.5 {+/-} 2.6 pps; p = 0.010). We did not find a change in the level of common synaptic input or in the temporal variability of motor unit discharge rates after the session of TENS.

ConclusionThese results suggest that TENS can modulate corticospinal excitability through supraspinal and spinal processes and, thus act as a priming technique. At the same time, TENS does not generate short-term changes in the neural control of force in young, healthy adults.

category: physiology


title: Candidatus Liberibacter asiaticus reduces callose and reactive oxygen species production in the phloem

abstract: Huanglongbing (HLB) causes significant economic loss in citrus production worldwide. HLB is caused by Candidatus Liberibacter asiaticus (CLas), a gram-negative bacterium which inhabits the phloem exclusively. CLas infection results in accumulation of callose and reactive oxygen species in the phloem of infected plants, but little is known about the specific processes that take place during infection because of the sparse distribution of bacteria and the inaccessibility of the phloem inside the tree. In this study, we used the seed vasculatures, which accumulate a high number of CLas, as a model tissue to study CLas-host cellular interactions. In vasculature where CLas is abundant, sieve pore callose and H2O2 concentration were reduced compared to healthy seed vasculature. The expression of callose synthases (CalS) and respiratory burst oxidase homolog (RBOH) genes were downregulated in infected seeds compared to healthy ones. In leaves of HLB-infected plants, H2O2 concentration and CalS expression increased compared to uninfected leaves, but cells with CLas had lower levels of sieve plate callose compared to cells without CLas. Our results provide evidence that the bacteria manipulate cell metabolism to disable plant defenses and suggests that HLB disease is the result of a constant arms-race between the pathogen and a defense response, which is ultimately harmful to the host plant.


title: Assessing cats' (Felis catus) sensitivity to human pointing gestures

abstract: A wide range of non-human animal species have been shown to be able to respond to human referential signals, such as pointing gestures. The aim of the present study was to replicate previous findings showing cats to be sensitive to human pointing cues (Miklosi et al. 2005). In our study, we presented two types of human pointing gestures - momentary direct pointing and momentary cross-body pointing. We tested nine rescue cats in a two-way object choice task. On a group level, the success rate of cats was 74.4 percentage. Cats performed significantly above chance level in both the direct pointing and cross-body pointing condition. Trial number, rewarded side and type of gesture did not significantly affect the cats performance in the experiment. On an individual level, 5 out of 7 cats who completed 20 trials, performed significantly above chance level. Two cats only completed 10 trials. One of them succeeded in 8, the other in 6 of these. The results of our study replicate previous findings of cats being responsive to human direct pointing cues and add additional knowledge about their ability to follow cross-body pointing cues. Our results highlight a domestic species, socialised in a group setting, to possess heterospecific communication skills, however we have to consider parsimonious explanations, such as local and stimulus enhancement.


title: The rostral medial frontal cortex is crucial for engagement in consummatory behavior

abstract:
The medial frontal cortex (MFC) in rodents emits rhythmic activity that is entrained to the animals licking cycle during consumption and encodes the value of consumed fluids (Horst and Laubach, 2013; Amarante et al., 2017; Amarante and Laubach, 2021). These signals are especially prominent in the rostral half of the MFC. This region is located above an orbitofrontal region where mu opioid receptors regulate intake (Mena et al., 2011; Castro and Berridge, 2017) and reversible inactivation reduces behavioral measures associated with the incentive value and palatability of liquid sucrose (Parent et al., 2015a). Here, we examined the effects of reversible inactivation and stimulation of mu opioid receptors in rostral MFC on behavior in an incentive contrast licking task. Adult male rats licked to receive access to liquid sucrose, which alternated between high (16%) and low (4%) values over 30 sec periods. Bilateral infusion of muscimol reduced the total number of licks emitted over the 30 min test sessions, the time spent actively consuming sucrose, and the ratio of licks for the higher and lower value fluids. Inactivation did not alter licking frequency or variability or microstructural measures such as the duration of licking bouts that are classically associated with the palatability of a liquid reward. Infusions of DAMGO (1g/L) at the same sites had inconsistent behavioral effects across different subjects. Our findings suggest that the rostral MFC has a distinct role in the control of consummatory behavior and contributes to peristent consumption and not to the expression of palatability.

SIGNIFICANCE STATEMENTThe medial frontal cortex (MFC) of rodents has received attention in recent years and is considered as a singular cortical region with a potential unitary function. Increasing evidence suggests that MFC is composed of distinct subregions, with unique roles in the control of behavior. The present study adds to this literature by showing unique effects of reversibly inactivating the most rostral part of the medial frontal cortex and a lack of consistent effects of stimulating mu opioid receptors in the subregion. Findings are in contrast to previous reports on the more ventral orbitofrontal cortex and caudal medial frontal cortex and are important for understanding the general role of the rodent frontal cortex and how opioids may act to control behavior.


title: The Speech Envelope Following REsponse in Normal and Hearing Impaired Listeners

abstract: The aim of this work was to investigate the perceptual relevance of the frequency following response to the syllable /da/ for speech intelligibility in noise based on age and hearing deficits. Recordings of the auditory evoked potential from young normal hearing (NH) and older individuals with both normal hearing and high-frequency (HF) hearing loss were analyzed. EFR metrics obtained in quiet and noise condition were calculated and correlated with speech reception. The envelope following responses were analyzed in terms of amplitude, latency and noise robustness. The response was first simulated to form predictions on the effect of cochlear synaptopathy and outer hair cell loss on the EFR. The experimental findings were in line with the computational predictions in the found observation that the EFR was reduced as a consequence of ageing and HF hearing loss. Both the audiogram and the speech EFR magnitude fell short in the individual prediction of SRT in stationary noise, but they accounted well for group performance. We also obtained within-group EFR latency with a cross covariance matrix. Validation of the method confirmed that speech EFR latency was predictive of click ABR Wave V peak latency. Moreover, statistical analysis not only showed that the robustness of the EFR obtained in the noise condition was dependent on the degree of high-frequency hearing loss in the older NH adults, but also dependent on the EFR magnitude in the NH younger adults. These findings provide evidence towards the important role of the EFR in speech-in-noise perception.


title: The perceptual relevance of the speech envelope-following response in normal hearing and age-related hearing impaired listeners