Variation in female songbird state determines signal strength needed to evoke copulation It is the female response to male signals that determines courtship success. In most songbirds, females control reproduction via the copulation solicitation display (CSD), an innate, stereotyped posture produced in direct response to male displays. Because CSD can be elicited in the absence of males by the presentation of recorded song, CSD production enables investigations into the effects of underlying signal features and behavioral state on female mating preferences. Using computer vision to quantify CSD trajectory in female brown-headed cowbirds (Molothrus ater), we show that both song quality and a females internal state predict CSD production, as well as the onset latency and duration of the display. We also show that CSD can be produced in a graded fashion based on both signal strength and internal state. These results emphasize the importance of underlying receiver state in determining behavioral responses and suggest that female responsiveness acts in conjunction with male signal strength to determine the efficacy of male courtship.
Electrocommunication signals indicate motivation to compete during dyadic interactions of an electric fish Animals across species compete for limited resources. While in some species competition behavior is solely based on own abilities, others assess their opponents to facilitate these interactions. Using cues and communication signals, contestants gather information about their opponent, adjust their behavior accordingly, and can thereby avoid high costs of escalating fights. We tracked electrocommunication signals, in particular "rises", and agonistic behaviors of the gymnotiform electric fish Apteronotus leptorhynchus in staged competition experiments. A larger body size relative to the opponent was the sole significant predictor for winners. Sex and the frequency of the continuously emitted electric field were only mildly influencing competition outcome. In males, correlations of body-size and winning were stronger than in females and, especially when losing against females, communication and agonistic interactions were enhanced, hinting towards males being more motivated to compete. Fish that lost competitions emitted the majority of rises in dependence on the competitors relative size and sex. The emission of rises was costly since it provoked ritualized biting or chasing behaviors by the other fish. Despite winners being accurately predictable based on rise numbers already after the initial 25 minutes, losers continued to emit rises. The number of rises emitted by losers and the duration of chasing behaviors depended in similar ways on physical attributes of contestants. The detailed evaluation of these correlations hint towards A. leptorhynchus adjusting their competition behavior according to mutual assessment, where rises could signal a losers motivation to continue assessment through ritualized fighting.
Population differences in the length and early-life dynamics of telomeres among European pied flycatchers Telomere length and shortening rate are increasingly used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between- population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the non-breeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north- south gradient (Finland, Estonia, England, and Spain) predicting negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from mid-latitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level. The compound of baicalein, wogonin, and oroxylin-A inhibits EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Combined Lineage Tracing and scRNA-seq Reveals Unexpected First Heart Field Predominance of Human iPSC Differentiation During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF-progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation. The Point Mutations in SARS-CoV-2 Variants Induce Long-Range Dynamical Perturbations in Neutralizing Antibodies Monoclonal antibodies are emerging as a viable treatment for the coronavirus disease 19 (COVID-19). However, newly evolved variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can reduce the efficacy of currently available antibodies and can diminish vaccine-induced immunity. Here, we demonstrate that the microscopic dynamics of neutralizing monoclonal antibodies can be profoundly modified by the mutations present in the spike proteins of the SARS-COV-2 variants currently circulating in the world population. The dynamical perturbations within the antibody structure, which alter the thermodynamics of antigen recognition, are diverse and can depend both on the nature of the antibody and on the spatial location of the spike mutation. The correlation between the motion of the antibody and that of the spike receptor binding domain (RBD) can also be changed, modulating binding affinity. Using protein-graph-connectivity networks, we delineated the mutant-induced modifications in the information-flow along allosteric pathway throughout the antibody. Changes in the collective dynamics were spatially distributed both locally and across long-range distances within the antibody. On the receptor side, we identified an anchor-like structural element that prevents the detachment of the antibodies; individual mutations there can significantly affect the antibody binding propensity. Our study provides insight into how virus neutralization by monoclonal antibodies can be impacted by local mutations in the epitope via a change in dynamics. This realization adds a new layer of sophistication to the efforts for rational design of monoclonal antibodies against new variants of SARS-CoV2, taking the allostery in the antibody into consideration. Identification of differentially distributed gene expression and distinct sets of cancer-related genes identified by changes in mean and variability There is increasing evidence that changes in the variability or overall distribution of gene expression are important both in normal biology and in diseases, particularly cancer. Genes whose expression differs in variability or distribution without a difference in mean are ignored by traditional differential expression-based analyses. Using a Bayesian hierarchical model that provides tests for both differential variability and differential distribution for bulk RNA-seq data, we report here an investigation into differential variability and distribution in cancer. Analysis of eight paired tumour-normal datasets from The Cancer Genome Atlas confirms that differential variability and distribution are able to identify cancer-related genes. We further demonstrate that differential variability identifies cancer-related genes that are missed by differential expression analysis, and that differential expression and differential variability identify functionally distinct sets of genes. These results suggest that differential variability analysis may provide insights into genetic aspects of cancer that would not be revealed by differential expression, and that differential distribution analysis may allow for more comprehensive identification of cancer-related genes than analyses based on changes in mean or variability alone. A tool for assessing alignment of biomedical data repositories with open, FAIR, citation and trustworthy principles Increasing attention is being paid to the operation of biomedical data repositories in light of efforts to improve how scientific data is handled and made available for the long term. Multiple groups have produced recommendations for functions that biomedical repositories should support, with many using requirements of the FAIR data principles as guidelines. However, FAIR is but one set of principles that has arisen out of the open science community. They are joined by principles governing open science, data citation and trustworthiness, all of which are important aspects for biomedical data repositories to support. Together, these define a framework for data repositories that we call OFCT: Open, FAIR, Citable and Trustworthy. Here we developed an instrument using the open source PolicyModels toolkit that attempts to operationalize key aspects of OFCT principles and piloted the instrument by evaluating eight biomedical community repositories listed by the NIDDK Information Network (dkNET.org). Repositories included both specialist repositories that focused on a particular data type or domain, in this case diabetes and metabolomics, and generalist repositories that accept all data types and domains. The goal of this work was both to obtain a sense of how much the design of current biomedical data repositories align with these principles and to augment the dkNET listing with additional information that may be important to investigators trying to choose a repository, e.g., does the repository fully support data citation? The evaluation was performed from March to November 2020 through inspection of documentation and interaction with the sites by the authors. Overall, although there was little explicit acknowledgement of any of the OFCT principles in our sample, the majority of repositories provided at least some support for their tenets. Analyzing Assay Specificity in Metabolomics using Unique Ion Signature Simulations Targeted, untargeted and data-independent acquisition (DIA) metabolomics workflows are often hampered by ambiguous identification based on either MS1 information alone or relatively few MS2 fragment ions. While DIA methods have been popularized in proteomics, it is less clear whether they are suitable for metabolomics workflows due to their large precursor isolation windows and complex co-isolation patterns. Here, we quantitatively investigate the conditions necessary for unique metabolite detection in complex backgrounds using precursor and fragment ion mass-to-charge separation, comparing three benchmarked MS methods (MS1, MRM, DIA). Our simulations show that DIA outperformed MS1-only and MRM-based methods with regards to specificity by a factor of ~2.8-fold and ~1.8-fold, respectively. Additionally, we show that our results are not dependent on the number of transitions used or the complexity of the background matrix. Finally, we show that collision energy is an important factor in unambiguous detection and that a single collision energy setting per compound cannot achieve optimal pairwise differentiation of compounds. Our analysis demonstrates the power of using both high resolution precursor and high resolution fragment ion m/z for unambiguous compound detection. This work also establishes DIA as an emerging MS acquisition method with high selectivity for metabolomics, outperforming both DDA and MRM with regards to unique compound identification potential.



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org.highwire.dtl.DTLVardef@142b100org.highwire.dtl.DTLVardef@1728a1corg.highwire.dtl.DTLVardef@c953c8org.highwire.dtl.DTLVardef@19b856a_HPS_FORMAT_FIGEXP  M_FIG C_FIG Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Targeted Gene Editing in Plants using CRISPR-Cas9, Single-Stranded DNA Oligonucleotides, and Protoplast Regeneration Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6-bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants. Modelling porcine reproductive and respiratory syndrome virus dissemination dynamics to quantifythe contribution of multiple modes of transmission: between-farm pig and vehicle movements, farmto-farm proximity, feed ingredients, and re-break Accounting for multiple modes of livestock disease dissemination in epidemiological models remains a challenge. We developed and calibrated a mathematical model for transmission of porcine reproductive and respiratory syndrome virus (PRRSV), tailored to fit nine modes of between-farm transmission pathways including: farm-to-farm proximity (local transmission), contact network of batches of pigs transferred between farms (pig movements), re-break probabilities for farms with previous PRRSV outbreaks, with the addition of four different contact networks of transportation vehicles (vehicles to transport pigs to farms, pigs to markets, feed and crew) and the amount of animal by-products within feed ingredients (e.g. animal fat or meat and bone meal). The model was calibrated on weekly PRRSV outbreaks data. We assessed the role of each transmission pathway considering the dynamics of specific types of production (i.e., sow, nursery). Although our results estimated that the networks formed by transportation vehicles were more densely connected than the network of pigs transported between-farms, pig movements and farm proximity were the main PRRSV transmission routes regardless of farm types. Among the four vehicle networks, vehicles transporting pigs to farms explained a large proportion of infections, sow = 20.9%; nursery = 15%; and finisher = 20.6%. The animal by-products showed a limited association with PRRSV outbreaks through descriptive analysis, and our model results showed that the contribution of animal fat contributed only 2.5% and meat and bone meal only 0.03% of the infected sow farms. Our work demonstrated the contribution of multiple routes of PRRSV dissemination, which has not been deeply explored before. It also provides strong evidence to support the need for cautious, measured PRRSV control strategies for transportation vehicles and further research for feed by-products modeling. Finally, this study provides valuable information and opportunities for the swine industry to focus effort on the most relevant modes of PRRSV between-farm transmission. Estimating the rate of plasmid transfer with an adapted Luria-Delbrück fluctuation analysis To increase our basic understanding of the ecology and evolution of conjugative plasmids, we need a reliable estimate of their rate of transfer between bacterial cells. However, accurate estimates of plasmid transfer have remained elusive due to biological and experimental complexity. Current methods to measure transfer rate can be confounded by many factors. A notable example involves plasmid transfer between different strains or species where the rate that one type of cell donates the plasmid is not equal to the rate at which the other cell type donates. Asymmetry in these rates has the potential to bias or constrain current transfer estimates, thereby limiting our capabilities for estimating transfer in microbial communities. Inspired by the classic fluctuation analysis of Luria and Delbruck, we develop a novel approach, the Luria-Delbruck method ( LDM), for estimating plasmid transfer rate. Our new approach embraces the stochasticity of conjugation departing from the current deterministic population dynamic methods. In addition, the LDM overcomes obstacles of traditional methods by not being affected by different growth and transfer rates for each population within the assay. Using stochastic simulations and experiments, we show that the LDM has high accuracy and precision for estimation of transfer rates compared to the most widely used methods, which can produce estimates that differ from the LDM estimate by orders of magnitude.

Significance StatementConjugative plasmids play significant roles in the ecology and evolution of microbial communities. Notably, antibiotic resistance genes are often encoded on conjugative plasmids. Thus, conjugation--the transfer of a plasmid copy from one cell to another--is a common way for antibiotic resistance to spread between important clinical pathogens. For both public health modeling and a basic understanding of microbial population biology, accurate estimates of this fundamental rate are of great consequence. We show that widely used methods can lead to biased estimates, deviating from true values by several orders of magnitude. Therefore, we developed a new approach, inspired by the classic fluctuation analysis of Luria and Delbruck, for accurately assessing the rate of plasmid conjugation under a variety of conditions. Deficiency of the paternally inherited gene Magel2 alters the development of separation-induced vocalization and maternal behavior in mice The behavior of offspring results from the combined expression of maternal and paternal genes. Genomic imprinting silences some genes in a parent-of-origin specific manner, a process that, among all animals, occurs only in mammals. How genomic imprinting affects the behavior of mammalian offspring, however, remains poorly understood. Here, we studied how the loss of the paternally inherited gene Magel2 in mouse pups affects the emission of separation-induced ultrasonic vocalizations (USV). Using quantitative analysis of more than one hundred thousand USVs, we characterized the rate of vocalizations as well as their spectral features from postnatal days 6 to 12 (P6-P12), a critical phase of mouse development during which pups fully depend on the mother for survival. Our analyses show that Magel2 deficient offspring emit separation-induced vocalizations at lower rates and with altered spectral features. We also show that dams display altered behavior towards their own Magel2 deficient offspring. In a test to compare the retrieval of two pups, dams retrieve wildtype control pups first and faster than Magel2 deficient offspring. These results suggest that the loss of Magel2 impairs the expression of separation-induced vocalization in pups as well as maternal behavior, both of which support the pups growth and development. Rec8 cohesin-mediated axis-loop chromatin architecture is required for meiotic recombination During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion. Neutralization of SARS-CoV-2 Omicron BA.2 by Therapeutic Monoclonal Antibodies Monoclonal antibody therapy for the treatment of SARS-CoV-2 infection has been highly successful in decreasing disease severity; however, the recent emergence of the heavily mutated Omicron variant has posed a challenge to this treatment strategy. The Omicron variant BA.1 has been found to evade neutralization by several of the therapeutic monoclonal antibodies authorized for emergency use, while Vir-7831 and a cocktail consisting of monoclonal antibodies AZD8895+AZD1061 retain significant neutralizing activity. A newly emerged variant, Omicron BA.2, containing some of the BA.1 mutations plus an additional 6 mutations and 3 deletions, 3 of which lie in the receptor binding domain, has been found to be spreading with increased transmissibility. We report here, using spike protein-pseudotyped lentiviruses, decreased neutralization of BA.2 by several therapeutic monoclonal antibodies but that the mixture of AZD8895+AZD1061 retained substantial neutralizing activity against BA.2. Combining Plasma Extracellular Vesicle Let-7b-5p, miR-184 and Circulating miR-22-3p Levels for NSCLC Diagnosis and Drug Resistance Prediction Low-dose computed tomography (LDCT) Non-Small Cell Lung (NSCLC) screening is associated with high false-positive rates, leading to unnecessary expensive and invasive follow ups. There is a need for minimally invasive approaches to improve the accuracy of NSCLC diagnosis. Plasma extracellular vesicle (EV) and circulating microRNA (miRNA) have been proposed as cancer screening biomarkers. However, the identification of highly sensitive and broadly predictive core miRNA signatures remains a challenge. Also, how these systemic and diverse miRNAs impact cancer drug response is not well understood. Using an integrative approach, we examined plasma EV and circulating miRNA isolated from NSCLC patients versus screening controls with a similar risk profile. We found that combining EV (Hsa-miR-184, Let-7b-5p) and circulating (Hsa-miR-22-3p) miRNAs abundance robustly discriminates between NSCLC patients and high-risk cancer-free controls. Diagnosed NSCLC patients harboring sensitizing mutations in epidermal growth factor receptor EGFR (T790M, L578R) are treated with Osimertinib, a potent tyrosine kinase inhibitor (TKI). Nearly all patients develop TKI resistance via complex mechanisms and progress. We found that Hsa-miR-22-3p, Hsa-miR-184, and Let-7b-5p functionally converge on WNT/{beta}catenin and mTOR/AKT signaling axes, known cancer therapy resistance signals. Targeting Hsa-miR-22-3p and Hsa-miR-184 desensitized EGFR-mutated (T790M, L578R) NSCLC cells to Osimertinib. These findings suggest that the expression levels of circulating hsa-miR-22-3p combined with EV hsa-miR-184 and Let-7b-5p levels potentially define a core biomarker signature for improving the accuracy of NSCLC diagnosis. Importantly, these biomarkers have the potential to enable prospective identification of patients who are at risk of responding poorly to Osimertinib alone but likely to benefit from Osimertinib/AKT blockade combination treatments. Localization of signaling receptors maximizes cellular informationacquisition in spatially-structured natural environments Cells in natural environments like tissue or soil sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions, sculpted by processes such as fluid flow and substrate adhesion. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. We develop an information-theoretic framework for computing the optimal spatial organization of a sensing system for a given signaling environment. We find that receptor localization maximizes information acquisition in simulated natural contexts, including tissue and soil. Receptor localization extends naturally to produce a dynamic protocol for continuously redistributing signaling receptors, which when implemented using simple feedback, boosts cell navigation efficiency by 30-fold. Broadly, our work shows how cells can maximize the fidelity of information transfer by adapting the spatial organization of signaling molecules to the spatial structure of the environment. Sensing Local Field Potentials with a Directional and Scalable Depth Array: the DISC array A variety of electrophysiology tools are available to the neurosurgeon for diagnosis, functional therapy, and neural prosthetics. However, no tool can currently address these three critical needs: (i) access to all cortical regions in a minimally invasive manner; (ii) recordings with microscale, mesoscale, and macroscale resolutions simultaneously; and (iii) access to spatially distant multiple brain regions that constitute distributed cognitive networks. We present a novel device for recording local field potentials (LFPs) with the form factor of a stereo-electroencephalographic electrode but combined with radially positioned microelectrodes and using the lead body to shield LFP sources, enabling directional sensitivity and scalability, referred to as the DISC array. As predicted by our electro-quasistatic models, DISC demonstrated significantly improved signal-to-noise ratio, directional sensitivity, and decoding accuracy from rat barrel cortex recordings during whisker stimulation. Critically, DISC demonstrated equivalent fidelity to conventional electrodes at the macroscale and uniquely, revealed stereoscopic information about current source density. Directional sensitivity of LFPs may significantly improve brain-computer interfaces and many diagnostic procedures, including epilepsy foci detection and deep brain targeting. Development of a Novel PROTAC using the Nucleic Acid Aptamer as a Targeting Ligand for Tumor Selective Degradation of Nucleolin PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and have poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs. RNA polymerase II is recruited to DNA double-strand breaks for dilncRNA transcription in Drosophila DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells. Trade-offs in biosensor optimization for dynamic pathway engineering Recent progress in synthetic biology allows the construction of dynamic control circuits for metabolic engineering. This technology promises to overcome many challenges encountered in traditional pathway engineering, thanks to their ability to self-regulate gene expression in response to bioreactor perturbations. The central components in these control circuits are metabolite biosensors that read out pathway signals and actuate enzyme expression. However, the construction of metabolite biosensors is a major bottleneck for strain design, and a key challenge is to understand the relation between biosensor dose-response curves and pathway performance. Here we employ multiobjective optimization to quantify performance trade-offs that arise in the design and calibration of metabolite biosensors. Our approach reveals strategies for tuning dose-response curves along an optimal trade-off between production flux and the cost of an increased expression burden on the host. We explore properties of control architectures built in the literature, and identify their advantages and caveats in terms of performance and robustness to growth conditions and leaky promoters. We demonstrate the optimality of a control circuit for glucaric acid production in Escherichia coli, which has been shown to increase titer by 2.5-fold as compared to static designs. Our results lay the groundwork for the automated design of control circuits for pathway engineering, with applications in the food, energy and pharmaceutical sectors. Arabidopsis AtPME2 has a pH-dependent processivity and control cell wall mechanical properties Pectin methylesterases (PMEs) modify homogalacturonans (HG) chemistry and play a key role in regulating primary cell wall mechanical properties. How PME activity can fine-tune pectin structure in the growing plant has remained elusive. Here we report on the Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum suitable for detecting recombinant and native enzyme independent of species source. At neutral pH AtPME2 is preferentially active on pectins with a degree of 55-70% methylesterification and can be inhibited by PME inhibitor protein (PMEI). We show that the mode of action for AtPME2 can switch from full processivity (at pH 8), creating large blocks of unmethylated galacturonic acid, to low processivity (at pH 5) and relate these observations to the differences in electrostatic potential of the protein at acidic and alkaline pH. To assess the role of AtPME2 in development, we characterized two knock-out lines. We show that in the context of acidified apoplast, low-processive demethylesterification by AtPME2 can loosen the cell wall, with consequent increase in cell elongation and etiolated hypocotyl length. Our study brings insights into how the pH-dependent regulation by PME activity could affect pectin structure and associated cell wall mechanical properties in expansion.

One sentence summaryThe processivity of AtPME2, a pectin methylesterase that fine-tunes cell wall pectins is modulated by pH in vitro and impacts the mechanical properties of the wall, affecting development in planta. Fatal COVID-19 ARDS associated with incomplete AEC1 differentiation from the transitional state without senescence or fibrosis ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. The Skeletal Muscle Circadian Clock Regulates Titin Splicing Through RBM20 Circadian rhythms are maintained by a cell autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression through both RNAseq and LC-MS. This alteration in titins spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. Finally, we identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies. Late-life fitness gains explain the absence of a selection shadow in ants A key hypothesis for the occurrence of senescence is a decrease in the selection strength because of low late-life fitness - the so-called selection shadow. However, in social insects, aging is considered a plastic trait and senescence seems to be absent. By life-long tracking of 102 ant colonies, we find that queens increase the production of sexuals in late life regardless of their absolute lifespan or worker investment. This indicates a genetically accommodated adaptive shift towards increasingly queen-biased caste ratios over the course of a queens life. Furthermore, mortality decreased with age, supporting the hypothesis that aging is adaptive. We argue that selection for late life reproduction diminishes the selection shadow of old age and leads to the apparent absence of senescence in ants, in contrast to most iteroparous species.

Significance StatementSocial insects are extraordinary with regard to the absolute age of queens of some species as well as the age differences between queens and short-lived workers. Yet, ultimate causes explaining these aging patterns remain poorly understood. By manipulating the investment ratio into queens and workers we studied the effect on lifespan and temporal reproductive investment of queens. We show that queens shift to the production of sexuals late in life, independent of social context (colony size) or individual quality (reproductive output and lifespan). Such late fitness gains result in maintenance in the selection strength with age, and thus explains the absence of senescence in queens.