Competitive dynamics underlie cognitive improvements during sleep. We provide evidence that human sleep is a competitive arena where cognitive domains vie for limited resources. Using pharmacology and effective connectivity analysis, we demonstrate that long-term memory and working memory are served by distinct offline neural mechanisms that are mutually antagonistic. Specifically, we administered zolpidem to increase central sigma activity and demonstrated targeted suppression of autonomic vagal activity. With effective connectivity, we determined the central activity has greater causal influence over autonomic activity, and the magnitude of this influence during sleep produced a behavioral trade-off between offline long-term and working memory processing. These findings show the first evidence of a sleep switch mechanism that toggles between central sigma-dependent long-term memory and autonomic vagal-dependent working memory processing.

Significant StatementSleep facilitates both long-term episodic memory consolidation and short-term working memory functioning. However, the mechanism by which the sleeping brain performs both complex feats, and which sleep features are associated with these processes remain unclear. Using a pharmacological approach, we demonstrate that long-term and working memory are served by distinct offline neural mechanisms, and that these mechanisms are mutually antagonistic. We propose a Sleep Switch model in which the brain toggles between the two memory processes via a complex interaction at the synaptic, systems, and mechanistic level, with implications for research on cognitive disturbances observed in neurodegenerative disorders such as Alzheimers and Parkinsons disease, both of which involve the decline of sleep.
A causal test of affect processing bias in response to affect regulation In this study we merged methods from machine learning and human neuroimaging to causally test the role of self-induced affect processing states in biasing the affect processing of subsequent image stimuli. To test this causal relationship we developed a novel paradigm in which (n=40) healthy adult participants observed affective neural decodings of their real-time functional magnetic resonance image (rtfMRI) responses as feedback to guide explicit regulation of their brain (and corollary affect processing) state towards a positive valence goal state. By this method individual differences in affect regulation ability were controlled. Attaining this brain-affect goal state triggered the presentation of pseudo-randomly selected affectively congruent (positive valence) or incongruent (negative valence) image stimuli drawn from the International Affective Picture Set. Separately, subjects passively viewed randomly triggered positively and negatively valent image stimuli during fMRI acquisition. Multivariate neural decodings of the affect processing induced by these stimuli were modeled using the task trial type (state- versus randomly-triggered) as the fixed-effect of a general linear mixed-effects model. Random effects were modeled subject-wise. We found that self-induction of a positive valence brain state significantly positively biased valence processing of subsequent stimuli. As a manipulation check, we validated affect processing state induction achieved by the image stimuli using independent psychophysiological response measures of hedonic valence and autonomic arousal. We also validated the predictive fidelity of the trained neural decoding models using brain states induced by an out-of-sample set of image stimuli. Beyond its contribution to our understanding of the neural mechanisms that bias affect processing this work demonstrated the viability of novel experimental paradigms triggered by pre-defined cognitive states. This line of individual differences research potentially provides neuroimaging scientists with a valuable tool for causal exploration of the roles and identities of intrinsic cognitive processing mechanisms that shape our perceptual processing of sensory stimuli.
Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Linked machine learning classifiers improve species classification of fungi when using error-prone long-reads on extended metabarcodes BackgroundThe increased usage of error-prone long-read sequencing for metabarcoding of fungi has not been matched with adequate public databases and concomitant analysis approaches. We address this gap and present a proof-of-concept study for classifying fungal taxa using linked machine learning classifiers. We demonstrate the capability of linked machine learning classifiers to accurately classify species and strains using real-world and simulated fungal ribosomal DNA datasets, including plant and human pathogens. We benchmark our new approach in comparison to current alignment and k-mer based methods based on synthetic mock communities. We also assess real world applications of species identification in complex unlabelled datasets.

ResultsOur machine learning approach assigned individual nanopore long-read amplicon sequences to fungal species with high recall rates and low false positive rates. Importantly, our approach successfully distinguished between closely-related species and strains when individual read errors were higher than the genetic distance between individual taxa, which the alignment and k-mer methods could not do. The machine learning approach showed an ability to identify key species with high recall rates, even in complex samples of unknown species composition.

ConclusionsA proof of concept machine learning approach using a tree-descent approach on a decision tree of classifiers can identify known taxa with high accuracy, and precisely detect known target species from complex samples with high recall rates. We propose this approach is suitable for detecting the known knowns of pathogens or invasive species in any environment of mostly unknown composition, including agriculture and wild ecosystems. Pathfinder: A gamified measure to integrate general cognitive ability into the biological, medical, and behavioural sciences Genome-wide association (GWA) studies have uncovered DNA variants associated with individual differences in general cognitive ability (g), but these are far from capturing heritability estimates obtained from twin studies. A major barrier to finding more of this  missing heritability is assessment - the use of diverse measures across GWA studies as well as time and cost of assessment. In a series of four studies, we created a 15-minute (40-item), online, gamified measure of g that is highly reliable (alpha = .78; two-week test-retest reliability = .88), psychometrically valid and scalable; we called this new measure Pathfinder. In a fifth study, we administered this measure to 4,751 young adults from the Twins Early Development Study. This novel g measure, which also yields reliable verbal and nonverbal scores, correlated substantially with standard measures of g collected at previous ages (r ranging from .42 at age 7 to .57 at age 16). Pathfinder showed substantial twin heritability (.57, 95% CIs = .43, .68) and SNP heritability (.37, 95% CIs = .04, .70). A polygenic score computed from GWA studies of five cognitive and educational traits accounted for 12% of the variation in g, the strongest DNA-based prediction of g to date. Widespread use of this engaging new measure will advance research not only in genomics but throughout the biological, medical, and behavioural sciences. 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. The Shigella type III effector protein OspB is a cysteine protease The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and dysenteric disease. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes.

ImportanceShigella spp. are important human pathogens and one of the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS), and definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB has been shown to contribute to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad. Genetic integration of behavioural and endocrine components of the stress response The vertebrate stress response comprises a suite of behavioural and physiological traits that must be functionally integrated to ensure organisms cope adaptively with acute stressors. Natural selection should favour functional integration, leading to a prediction of genetic integration of these traits. Despite the implications of such genetic integration for our understanding of human and animal health, as well as evolutionary responses to natural and anthropogenic stressors, formal quantitative genetic tests of this prediction are lacking. Here we demonstrate that acute stress response components in Trinidadian guppies are both heritable and integrated on the major axis of genetic covariation. This integration could either facilitate or constrain evolutionary responses to selection, depending upon the alignment of selection with this axis. Such integration also suggests artificial selection on the genetically correlated behavioural responses to stress could offer a viable non-invasive route to the improvement of health and welfare in captive animal populations. Beneficial effects of novel strains of Aureobasidium pullulans produced 1,3-1,6 β-glucans on non-esterified fatty acid levels in diabetic KKAy mice Obesity, metabolic syndrome, associated lipotoxicity and its cascade of events contribute to the majority of the burden related to non-communicable diseases globally. Preventive lifestyle changes aside, several beneficial effects have been reported in type II diabetes mellitus and dyslipidaemia patients with biological response modifier glucans (BRMG) produced as an exopolysaccharide by Aureobasidium pullulans. In this study, we compared two strains (AFO-202 and N-163) that produce beta glucans in alleviating lipotoxicity. This study was performed in obese diabetic mice model of KK-Ay mice, in four groups with six subjects in each group - Group 1: sacrificed on Day 0 for baseline values; Group 2: control (drinking water); Group 3: AFO-202 beta glucan--200 mg/kg/day; Group 4: N-163 beta glucan--300 mg/kg/day. The animals in groups 2-4 had the test solutions administered by gavage once daily for 28 consecutive days. Biochemical analyses were conducted of blood glucose, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol and non-esterified fatty acids (NEFA). Group 4 (N-163) had the lowest NEFA levels, as compared to the other groups, and marginally decreased triglyceride levels. The groups had no significant differences in blood glucose, HbA1c, triglycerides, or LDL and HDL cholesterol. N-163 produced by A. pullulans decreased NEFA in a diabetic mice model in 28 days. These results, although modest, warrant further in-depth research into lipotoxicity and associated inflammatory cascades in both healthy and disease affected subjects to develop novel strategies for prevention and management. 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. ScRNA-seq Expression of IFI27 and APOC2 Identifies Four Alveolar Macrophage Superclusters in Healthy BALF Alveolar macrophages (AMs) reside on the luminal surface of the airways and alveoli, ensuring proper gas exchange by ingesting cellular debris and pathogens, and regulating inflammatory responses. Therefore, understanding the heterogeneity and diverse roles played by AMs and recruited monocytes is critical for treating airway diseases. We performed single-cell RNA sequencing on 113,213 bronchoalveolar lavage cells from four healthy and three uninflamed cystic fibrosis subjects and identified four AM superclusters (families) based on the expression of IFI27 and APOC2 genes. These 4 AM families have at least eight distinct functional members (subclusters) named after their differentially expressed gene(s): IGF1, CCL18, CXCL5, Cholesterol, Chemokine, Metallothionein, Interferon and small-cluster AMs. Interestingly, the Chemokine cluster further divides with each subcluster selectively expressing a unique combination of chemokines. One of the most striking observations, besides the heterogeneity, is the conservation of AM family members in relatively equal ratio across all AM superclusters and individuals. Finally, transcriptional data and TotalSeq technology were used to investigate cell surface markers that distinguish resident AMs from recruited monocytes. Overall, functional specializations of the AM subclusters suggest that there are AM niches with defined programming states, highlighting a clear division of labor. Quantifying the Morphology and Mechanisms of Cancer Progression in 3D in-vitro environments: Integrating Experiments and Multiscale Models Mathematical models of cancer growth have become increasingly more accurate both in the space and time domains. However, the limited amount of data typically available has resulted in a larger number of qualitative rather than quantitative studies. In the present study, we provide an integrated experimental-computational framework for the quantification of the morphological characteristics and the mechanistic modelling of cancer progression in 3D environments. The proposed framework allows for the calibration of multiscale, spatiotemporal models of cancer growth using state-of-the-art 3D cell culture data, and their validation based on the resulting experimental morphological patterns. Its implementation enabled us to pursue two goals; first, the quantitative description of the morphology of cancer progression in 3D cultures, and second, the relation of tumour morphology with underlying biophysical mechanisms that govern cancer growth and migration. We applied this framework to the study of the spatiotemporal progression of Triple Negative Breast Cancer cells cultured in Matrigel scaffolds, and validated the hypothesis of chemotactic migration using a multiscale Keller-Segel model. The results revealed transient, non-random spatial distributions of cancer cells that consist of clustered, and dispersion patterns. The proposed model was able to describe the general characteristics of the experimental observations and suggests that cancer cells exhibited chemotactic migration and accumulation, as well as random motion during the examined time period of development. To our knowledge, this is the first time that a multiscale model is used to quantify the relationship between the spatial patterns and the underlying mechanisms of cancer growth in 3D environments. A chromosome-level genome assembly of the European Beech (Fagus sylvatica) reveals anomalies for organelle DNA integration, repeat content and distribution of SNPs The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 Mb and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb of the genome in which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in Arabidopsis thaliana, ribosomal cistrons are present in Fagus sylvatica only in four major regions, in line with FISH studies. On most assembled chromosomes, telomeric repeats were found at both ends, while centromeric repeats were found to be scattered throughout the genome apart from their main occurrence per chromosome. The genome- wide distribution of SNPs was evaluated using a second individual from Jamy Nature Reserve (Poland). SNPs, repeat elements and duplicated genes were unevenly distributed in the genomes, with one major anomaly on chromosome 4. The genome presented here adds to the available highly resolved plant genomes and we hope it will serve as a valuable basis for future research on genome architecture and for understanding the past and future of European Beech populations in a changing climate. Augmenting Recombinant Antibody Production in HEK293E Cells: Optimising Transfection and Culture Parameters BackgroundOptimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations.

MethodsInvestigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements.

ResultsUsing 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 g respectively in a cost-effective manner. With the addition of peptone, {kappa} light chain increased by ~4-fold to 1032 g while whole antibody increased to a lesser extent by ~2.5-fold to 51 g, with benefits potentially for antibodies limited by their light chains in production.

ConclusionsOur optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells.

Statement of SignificanceRecombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~4-fold light chain alone production increase to 1032 g and a 2.5-fold whole antibody production increase to 51 g from 2 mL transfections. Intestine-enriched apolipoprotein b orthologs are required for stem cell differentiation and regeneration in planarians Lipid metabolism plays an instructive role in regulating stem cell state and differentiation. However, the roles of lipid mobilization and utilization in stem cell-driven regeneration are unclear. Planarian flatworms readily restore missing tissue due to injury-induced activation of pluripotent somatic stem cells called neoblasts. Here, we identify two intestine-enriched orthologs of apolipoprotein b, apob-1 and apob-2, which mediate transport of neutral lipid stores from the intestine to target tissues including neoblasts, and are required for tissue homeostasis and regeneration. Inhibition of apob function by RNAi causes head regression and lysis in uninjured animals, and delays body axis re-establishment and regeneration of multiple organs in amputated fragments. Furthermore, apob RNAi causes expansion of the population of differentiating neoblast progeny and dysregulates expression of genes enriched in differentiating and mature cells in eight major cell type lineages. We conclude that intestine-derived lipids serve as a source of metabolites required for neoblast differentiation. Deficits in seizure threshold and other behaviors in adult mice without gross neuroanatomic injury after late gestation transient prenatal hypoxia Intrauterine hypoxia is a common cause of brain injury in children resulting in a broad spectrum of long-term neurodevelopmental sequela, including life-long disabilities that can occur even in the absence of severe neuroanatomic damage. Postnatal hypoxia-ischemia rodent models are commonly used to understand the effects of ischemia and transient hypoxia on the developing brain. Postnatal models, however, have some limitations. First, they do not test the impact of placental pathologies on outcomes from hypoxia. Second, they primarily recapitulate severe injury because they provoke substantial cell death, which is not seen in children with mild hypoxic injury. Lastly, they do not model preterm hypoxic injury. Prenatal models of hypoxia in mice may allow us to address some of these limitations to expand our understanding of developmental brain injury. The published rodent models of prenatal hypoxia employ multiple days of hypoxic exposure or complicated surgical procedures, making these models challenging to perform consistently in mice. Furthermore, large animal models suggest that transient prenatal hypoxia without ischemia is sufficient to lead to significant functional impairment to the developing brain. However, these large animal studies are resource-intensive and not readily amenable to mechanistic molecular studies. Therefore, here we characterized the effect of late gestation (embryonic day 17.5) transient prenatal hypoxia (5% inspired oxygen) on long-term anatomical and neurodevelopmental outcomes in mice. Late gestation transient prenatal hypoxia increased hypoxia-inducible factor 1 alpha protein levels (a marker of hypoxic exposure) in the fetal brain. Hypoxia exposure predisposed animals to decreased weight at postnatal day 2, which normalized by day 8. However, hypoxia did not affect gestational age at birth, litter size at birth, or pup survival. No differences in fetal brain cell death or long-term gray or white matter changes resulted from hypoxia. Animals exposed to prenatal hypoxia did have several long-term functional consequences, including sex-dichotomous changes. Hypoxia exposure was associated with a decreased seizure threshold and abnormalities in hindlimb strength and repetitive behaviors in males and females. Males exposed to hypoxia had increased anxiety-related deficits, whereas females had deficits in social interaction. Neither sex developed any motor or visual learning deficits. This study demonstrates that late gestation transient prenatal hypoxia in mice is a simple, clinically relevant paradigm for studying putative environmental and genetic modulators of the long-term effects of hypoxia on the developing brain. α-catenin links integrin adhesions to F-actin to regulate ECM mechanosensing and rigidity-dependence Both cell-cell and cell-matrix adhesions are regulated by mechanical signals, but the mechanobiological processes that mediate the crosstalk between these structures are poorly understood. Here we show that - catenin, a mechanosensitive protein that is classically associated with cadherin-based adhesions, directly interacts with and regulates integrin adhesions. -catenin is recruited to the edges of mesenchymal cells, where it interacts with F-actin. This is followed by mutual retrograde flow of -catenin and F-actin from the cell edge, during which -catenin interacts with vinculin within integrin adhesions. This interaction affects adhesion maturation, stress-fiber assembly, and force transmission to the matrix. In epithelial cells, -catenin is present in cell-cell adhesions and absent from cell-matrix adhesions. However, when these cells undergo epithelial-to-mesenchymal transition, -catenin transitions to cell-matrix adhesions, where it facilitates proper mechanosensing. This is highlighted by the ability of -catenin-depleted cells to grow on soft matrices. These results suggest a dual role of -catenin in mechanosensing, through both cell-cell and cell-matrix adhesions. Phytoene synthase 2 in tomato fruits remains functional and contributes to abscisic acid formation In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-{beta}-cyclase (CYCB). Though both PSY1 and PSY2 genes express in tomato fruits, the functional role of PSY2 is not known. To decipher whether PSY2-mediated carotenogenesis operates in ripening fruits, we blocked the in vivo activity of lycopene-{beta}-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-{beta}-cyclases. The CPTA-treatment induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even, in psy1 mutants V7 and r that are deficient in fruit-specific carotenoid biosynthesis, CPTA triggered lycopene accumulation but lowered the abscisic acid level. Differing from fruit-specific carotenogenesis, CPTA-treated V7 and r mutant fruits accumulated lycopene but not phytoene and phytofluene. The lack of phytoene and phytofluene accumulation was reminiscent of PSY2-mediated leaf-like carotenogenesis, where phytoene and phytofluene accumulation is never seen. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like crystalline structures. Our study uncovers the operation of a parallel carotenogenesis pathway mediated by PSY2 that provides precursors for abscisic acid biosynthesis in ripening tomato fruits.

Significance statementIt is believed that in ripening tomato fruits phytoene synthase 2 that drives carotenoid biosynthesis in leaves is redundant. Contrary to this, we show that in phytoene synthase 1 mutant fruit that is bereft of lycopene, the chemical inhibition of lycopene {beta}-cyclases triggers lycopene accumulation. Our results uncover that phytoene synthase 2 remains functional in ripening fruits and provides precursors for abscisic acid formation in fruits. Mouse lemur, a new animal model to investigate cardiac pacemaker activity in primates. BackgroundGray mouse lemurs (Microcebus murinus) are one of the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since Microcebus murinus replicates similar senescence processes of humans, it constitutes a useful model also for studying cardiovascular dysfunctions. However, its cardiac physiology is unknown. Thus, we investigated sinoatrial node (SAN) pacemaker activity of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates.

Methods and ResultsWe recorded the heart automaticity in freely moving mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of electrical impulses in the SAN tissue. Conversely, their ventricular depolarization is more similar to large mammals. Mouse lemur SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (ICa,L) and T-type (ICa,T) calcium currents. Moreover, mouse lemur SAN myocytes display spontaneous ryanodine receptor (RyR)-dependent Ca2+ transients. Using muscle-derived stem cells (MDSCs) from the Microcebus murinus skeletal muscle, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45).

ConclusionsIn conclusion, our characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans, and for pharmacologic testing of molecules that could assist in modulating age-related dysfunctions of automaticity. Land use change through the lens of macroecology: insights from Azorean arthropods and the Maximum Entropy Theory of Ecology Human activity and land management practices, in particular land use change, have resulted in the global loss of biodiversity. These types of disturbance affect the shape of macroecological patterns, and therefore analyzing these patterns can provide insights into how ecosystems are affected by land use change. We here use arthropod census data from 96 sites at Terceira Island in the Azores archipelago across four different land uses of increasing management intensity: native forest, exotic forest, semi-natural pasture, and intensive pasture, to examine the effects of land use type on three macroecological patterns: the species abundance distribution, the metabolic rate distribution of individuals, and the species-area relationship. The Maximum Entropy Theory of Ecology (METE) has successfully predicted these patterns across habitats and taxa in undisturbed ecosystems, and thus provides a null expectation for their shapes. Across these patterns, we find that the forest habitats are the best fit by METE predictions, while the semi-natural pasture is consistently the worst fit, and the intensive pasture is intermediately well fit. We show that the direction of failure of the METE predictions at the pasture sites is likely due to the hyper-dominance of introduced spider species present there. We hypothesize that the particularly poor fit for the semi-natural pasture is due to the mix of arthropod communities out of equilibrium, leading to greater heterogeneity in composition and complex dynamics that violate METEs assumption of static state variables. The comparative better fit for the intensive pasture plausibly results from more homogeneous arthropod communities that are well adapted to intensive management, and thus whose state variables are less in flux. Analyzing deviations from theoretical predictions across land use type provides useful information about how land use and disturbance affect ecosystems, and such comparisons could be useful across other habitats and taxa. If Research is Not the Evidence, What is it? The Lack of Research Citations in Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context. Liver Endothelium Promotes HER3-mediated Cell Survival in KRAS Wild-type and Mutant Colorectal Cancer Cells We previously showed that liver endothelial cells (ECs) secreted soluble factors in a paracrine fashion and activated human epidermal growth factor receptor 3 (HER3, also known as ERBB3) for promoting colorectal cancer (CRC) growth and chemoresistance. However, RAS proteins play a critical role in receptor tyrosine kinase signaling pathways, and KRAS mutations mediate CRC resistance to therapies targeting EGFR, another HER protein. Therefore, the role of KRAS mutation status in EC-induced HER3 activation and CRC survival was investigated as it has therapeutic implications. We used CRC cell lines and patient-derived xenografts harboring KRAS wild-type or mutant genes and demonstrated that liver EC-secreted factors promoted HER3-mediated CRC cell growth independent of KRAS mutation status. Also, blocking HER3 in CRC cells by siRNAs or a HER3 antibody seribantumab blocked EC-induced CRC survival. Our findings highlight the potential of utilizing HER3 targeted therapies for treating patients with mCRC independent of RAS mutational status. Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy As of October 2021, coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Due to high efficiency, remarkable stability, resilience to nebulization and low production cost, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status. 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. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. Single-molecule Forster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.

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