An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be up-regulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results indicate that pKFDV02 did not keep its promises as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.

Key messagepPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.
Chemomechanically voxelated niches for programmable histogenesis Stem-cell derived tissue models are commonly cultured under globally-delivered stimuli that trigger histogenesis via self-organizing activity. However, the culture of such tissue models is prone to stochastic behavior, limiting the reproducibility of cellular composition and resulting in non-physiological architectures. To overcome these shortcomings, we developed a method for printing cell niche microenvironments with microstructured cues that mediate local histogenic processes, including mechanosensing and differentiation of selected cell types. Microstructured cues include independently tunable mechano-chemical properties, with conjugated peptides, proteins, and morphogens across a range of Youngs moduli. By rationally designing niches, we mediate the structure of tissues derived from stem-cell-progenitor sources, including a bone-fat assembly from stromal mesenchyme, and embryonic tissues derived from hiPSC. We show that microstructured cues can recapitulate mechano-chemical signals resembling early embryonic histogenesis. This outcome includes a role for niche mechanics in human embryonic organization, where soft niche mechanics bias markers of mesendodermal differentiation and epithelial-to-mesenchymal-transition (EMT), as well as a demonstration of a material-mediated morphogen signaling centers able to induce foci of mesenchymal and EMT differentiation. Thus, microstructured materials can mediate local histogenic processes to enhance the structure and composition of tissue models.
The Rise of West Nile Virus in Southern andSoutheastern Europe: a spatial-temporal analysisinvestigating the combined effects of climate, land use and economic changes West Nile Virus (WNV) has recently emerged as a major public health concern in Europe, its recent expansion also coincided with some remarkable socio-economic and environmental changes, including an economic crisis and some of the warmest temperatures on record. Here we empirically investigate the drivers of this phenomenon at a European wide scale by constructing and analyzing a unique spatial-temporal data-set, that includes data on climate, land-use, the economy, and government spending on environmental related sectors. Drivers and risk factors of WNV were identified by building a conceptual framework, and relationships were tested using a Generalized Additive Model (GAM), which could capture complex non-linear relationships and also account for the spatial and temporal auto-correlation. Some of the key risk factors identified in our conceptual framework, such as a higher percentage of wetlands and arable land, climate factors (higher summer rainfall and higher summer temperatures) were positive predictors of WNV infections. Interestingly, winter temperatures of between 2 {degrees}C and 6 {degrees}C were among some of the strongest predictors of annual WNV infections, one possible explanation for this result is that successful overwintering of infected adult mosquitoes (likely Culex pipiens) is key to the intensity of outbreaks for a given year. Furthermore, lower surface water extent over the summer is also associated with more more intense outbreaks, suggesting that drought, which is known to induce positive changes in WNV prevalence in mosquitoes is also contributing to the upward trend in WNV cases in affected regions. Our indicators representing the economic crisis were also strong predictors of WNV infections, suggesting there is an association between austerity and cuts to key sectors, which could have benefited vector species and the virus during this crucial period. These results, taken in the context of recent winter warming due to climate change, and more frequent droughts, may offer an explanation of why the virus has become so prevalent in Europe. Evolution of class-structured populations in fluctuating environments What is the influence of periodic environmental fluctuations on life-history evolution? We present a general theoretical framework to understand and predict the long-term evolution of lifehistory traits under a broad range of ecological scenarios. Specifically, we investigate how periodic fluctuations affect selection when the population is also structured in distinct classes. This analysis yields time-varying selection gradients that clarify the influence of the fluctuations of the environment on the competitive ability of a specific life-history mutation. We use this framework to analyse the evolution of key life-history traits of pathogens. We examine three different epidemiological scenarios and we show how periodic fluctuations of the environment can affect the evolution of virulence and transmission as well as the preference for different hosts. These examples yield new and testable predictions on pathogen evolution, and illustrate how our approach can provide a better understanding of the evolutionary consequences of time-varying environmental fluctuations in a broad range of scenarios. 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. Accounting for motion in resting-state fMRI: What part of the spectrum are we characterizing in autism spectrum disorder? The exclusion of high-motion participants can reduce the impact of motion in functional Magnetic Resonance Imaging (fMRI) data. However, the exclusion of high-motion participants may change the distribution of clinically relevant variables in the study sample, and the resulting sample may not be representative of the population. Our goals are two-fold: 1) to document the biases introduced by common motion exclusion practices in functional connectivity research and 2) to introduce a framework to address these biases by treating excluded scans as a missing data problem. We use a study of autism spectrum disorder in children without an intellectual disability to illustrate the problem and the potential solution. We aggregated data from 545 children (8-13 years old) who participated in resting-state fMRI studies at Kennedy Krieger Institute (173 autistic and 372 typically developing) between 2007 and 2020. We found that autistic children were more likely to be excluded than typically developing children, with 28.5% and 16.1% of autistic and typically developing children excluded, respectively, using a lenient criterion and 81.0% and 60.1% with a stricter criterion. The resulting sample of autistic children with usable data tended to be older, have milder social deficits, better motor control, and higher intellectual ability than the original sample. These measures were also related to functional connectivity strength among children with usable data. This suggests that the generalizability of previous studies reporting naive analyses (i.e., based only on participants with usable data) may be limited by the selection of older children with less severe clinical profiles because these children are better able to remain still during an rs-fMRI scan. We adapt doubly robust targeted minimum loss based estimation with an ensemble of machine learning algorithms to address these data losses and the resulting biases. The proposed approach selects more edges that differ in functional connectivity between autistic and typically developing children than the naive approach, supporting this as a promising solution to improve the study of heterogeneous populations in which motion is common. Tatajuba -- Exploring the distribution of homopolymer tracts Length variation of homopolymeric tracts, which induces phase variation, is known to regulate gene expression leading to phenotypic variation in a wide range of bacterial species. There is no specialised bioinformatics software which can, at scale, exhaustively explore and describe these features from sequencing data. Identifying these is non-trivial as sequencing and bioinformatics methods are prone to introducing artefacts when presented with homopolymeric tracts due to the decreased base diversity. We present tatajuba, which can automatically identify potential homopolymeric tracts and their putative phenotypic impact, allowing for rapid investigation. We use it to detect all tracts in two separate datasets, one of Campylobacter jejuni and one of three Bordetella species, and to highlight those tracts that are polymorphic across samples. With this we confirm homopolymer tract variation with phenotypic impact found in previous studies and additionally find many more with potential variability. The software is written in C and is available under the open source license GNU GPL version 3 from https://github.com/quadram-institute-bioscience/tatajuba. Catheterized bladder environment induces Candida albicans hyphal formation and promotes colonization and persistence through Als1. When Candida is found in the urine, physicians face the dilemma to treat or not, since its presence could represent commensalism or infection, resulting in inappropriate management. Candida spp., specifically Candida albicans, are the second most common cause of catheter-associated urinary tract infections (CAUTIs). Yet, the cellular and molecular details of C. albicans CAUTI are poorly understood. With the steady increase in catheter usage, combined with fungal infection mismanagement and treatment challenges, this question is of utmost public health importance. Here, for the first time, we characterized fungal mechanisms during CAUTI, finding that the catheterized bladder environment triggers Candida virulent programing, inducing Efg1-dependent hyphal morphogenesis, required for CAUTI establishment. Moreover, Als1, an Efg1-downstream effector, is necessary for in vitro and in vivo fibrinogen-biofilm formation and Als1 expression levels were crucial for fungal persistence during CAUTI. These findings unveil the mechanisms for fungal CAUTIs and identify potential treatment targets. Geometric machine learning informed by ground truth: Recovery of conformational continuum from single-particle cryo-EM data of biomolecules This work is based on the manifold-embedding approach to study biological molecules exhibiting continuous conformational changes. Previous work established a method capable of reconstructing 3D movies and accompanying energetics of atomic-level structures from single-particle cryo-EM images of macromolecules displaying multiple conformational degrees of freedom. Here, we introduce an unsupervised geometric machine learning approach that is informed by detailed heuristic analysis of manifolds formed by simulated heterogeneous cryo-EM datasets generated from an atomic structure. These simulated data were generated with increasing complexity to account for multiple conformational motions, state occupancies and typical microscope parameters in a wide range of signal-to-noise ratios. Using these datasets as ground-truth, we provide detailed exposition of our findings using several conformational motions while exploring the available parameter space. Guided by these insights, we build a framework to leverage the high-dimensional geometric information obtained towards reconstituting a quasi-continuum of conformational states in the form of a free-energy landscape and respective 3D density maps for all states therein. As shown by a direct comparison of results, this framework offers substantial improvements relative to the previous work. SARS-CoV-2 Can Infect Human Embryos The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. We note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. Using fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, we observe robust infection of trophectoderm cells, and this permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath. A positive correlation between GC content and growth temperature in prokaryotes BackgroundGC pairs are generally more stable than AT pairs; GC-rich genomes were proposed to be more adapted to high temperatures than AT-rich genomes. Previous studies consistently showed positive correlations between growth temperature and the GC contents of structural RNA genes. However, for the whole genome sequences and the silent sites of the codons in protein-coding genes, the relationship between GC content and growth temperature is in a long-lasting debate.

ResultsWith a dataset much larger than previous studies (681 bacteria and 155 archaea with completely assembled genomes), our phylogenetic comparative analyses showed positive correlations between optimal growth temperature (Topt) and GC content both in bacterial and archaeal structural RNA genes and in bacterial whole genome sequences, chromosomal sequences, plasmid sequences, core genes, and accessory genes. However, in the 155 archaea, we did not observe a significant positive correlation of Topt with whole-genome GC content (GCw) or GC content at four-fold degenerate sites. We randomly drew 155 samples from the 681 bacteria for 1000 rounds. In most cases (> 95%), the positive correlations between Topt and genomic GC contents became statistically nonsignificant (P > 0.05). This result suggested that the small sample sizes might account for the lack of positive correlations between growth temperature and genomic GC content in the 155 archaea and the bacterial samples of previous studies. Comparing the GC content among four categories (psychrophiles/psychrotrophiles, mesophiles, thermophiles, and hyperthermophiles) also revealed a positive correlation between GCw and growth temperature in bacteria. By including the GCw of incompletely assembled genomes, we expanded the sample size of archaea to 303. Positive correlations between GCw and Topt appear especially after excluding the halophilic archaea whose GC contents might be are strongly shaped by intense UV radiation.

ConclusionsThis study explains the previous contradictory observations and ends a long debate. Prokaryotes growing in high temperatures have higher GC contents. Thermal adaptation is one possible explanation for the positive association. Meanwhile, we propose that the elevated efficiency of DNA repair in response to heat mutagenesis might have the by-product of increasing GC content like that happens in intracellular symbionts and marine bacterioplankton. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. SARS-CoV-2 infection induces soluble platelet activation markers and PAI-1 in the early moderate stage of COVID-19 IntroductionCoagulation dysfunction and thromboembolism emerge as strong comorbidity factors in severe COVID-19. However, it is unclear when particularly platelet activation markers and coagulation factors dysregulated during the pathogenesis of COVID-19. Here, we sought to assess the levels of coagulation and platelet activation markers at moderate and severe stages of COVID-19 to understand the pathogenesis.

MethodsTo understand this, hospitalized COVID-19 patients with (severe cases that required intensive care) or without pneumonia (moderate cases) were recruited. Phenotypic and molecular characterizations were performed employing basic coagulation tests including PT, APTT, D-Dimer and TFPI. The flow cytometry-based multiplex assays were performed to assess FXI, anti-thrombin, prothrombin, fibrinogen, FXIII, P-selectin, sCD40L, plasminogen, tissue-plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and D-Dimer.

ResultsThe investigations revealed induction of plasma P-selectin and CD40 ligand (sCD40L) in moderate COVID-19 cases which were significantly abolished with the progression of COVID-19 severity. Moreover, a profound reduction in plasma tissue factor pathway inhibitor (TFPI) and FXIII were identified particularly in the severe COVID-19. Further analysis revealed fibrinogen induction in both moderate and severe patients. Interestingly, an elevated PAI-1 more prominently in moderate, and tPA particularly in severe COVID-19 cases were observed. Particularly, the levels of fibrinogen and tPA directly correlated with the severity of the disease.

ConclusionsIn summary, induction of soluble P-selectin, sCD40L, fibrinogen and PAI-1 in moderate COVID-19 cases suggests the activation of platelets and coagulation system before patients require intensive care. These findings would help in designing better thromboprophylaxis to limit the COVID-19 severity. Can double PPO mutations exist in the same allele and are such mutants functional? BackgroundResistance to PPO-inhibiting herbicides is primarily endowed by target site mutations at the PPO2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus ssp. PPO2, the most prevalent target mutations are deletion of the G210 codon and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same ppo2 allele.

ResultsPlants carrying R128G+/+ {Delta}G210+/-, where + indicates presence of the mutation, were crossed with each other. The ppo2 of the offspring was subjected to pyrosequencing and E. coli- based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G {Delta}G210 can occur only in one allele; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying {Delta}G210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions.

ConclusionsHomozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele.

NomenclaturePalmer amaranth, Amaranthus palmeri S. Wats.; protoporphyrinogen oxidase, PPO; tall waterhemp, Amaranthus tuberculatus (Moq.) J.D.Sauer Force profile of the two-handed hardstyle kettlebell swing in novice older adults: an exploratory profile BackgroundUnderstanding the force profile of an exercise increases clinical confidence when assessing the benefits and potential risks of a prescribed exercise. This exploratory study presents the force profile of the hardstyle kettlebell swing in novice older adults and compares peak force with kettlebell deadlifts. These data will help inform healthcare providers and coaches who are considering prescribing kettlebell exercises for older adults.

MethodsThirty-five community-dwelling males and females (59-79 years) were recruited, from applicants to participate in the BELL trial. Two-handed hardstyle swings were performed with 8-16 kg. Deadlifts were performed with 8-24 kg and 8-32 kg for females and males, respectively. Ground reaction force was obtained from a floor-mounted force platform. Pairwise comparisons of peak force, forward force, rate of force development, swing cadence, sex, and kettlebell mass, were investigated for the kettlebell swing, with representative force-time curves described. Pairwise comparisons of peak force, sex and kettlebell mass were investigated for the deadlift, with comparisons of peak force, kettlebell mass, and sex, between swings and deadlifts.

ResultsFor kettlebells up to 16 kg, paired samples T-tests show a large exercise effect ({delta} > 1.4) with peak force higher for swings than deadlifts. Data shows: (i) higher peak force during swings than deadlifts ({delta} = 1.77), reaching 4.5 (1.0) N.kg-1, (ii) peak force during an 8 kg swing was greater than a 32kg deadlift, (iii) negligible difference in normalised peak force between males and females performing kettlebell swings, but a moderately large effect size during deadlifts (males > females, {delta} = 0.69), (iv) mean rate of force development of 19.9 (4.7) N.s-1.kg-1 with a very weak, positive correlation with kettlebell mass (y = 14.4 + 0.32x), and trivial effect of sex, (v) mean forward force equal to 5.5% of vertical force during swings, increasing from 3.8 (1.6) % with 8 kg to 7.1 (2.6) % with 16 kg.

ConclusionDuring kettlebell swings, there is negligible difference in normalised net peak force between novice males and females using the same absolute loads. Where ground reaction force is a therapeutic target, kettlebell swings with an 8 kg kettlebell could have similar effects to much heavier deadlifts (>24 kg). Kettlebell swings performed with lighter loads, could provider similar therapeutic value to much heavier deadlifts, and may be a more appealing, affordable, and convenient option for older adults. Changes in Behavior and Salivary Serum Amyloid A in cows with Subclinical Mastitis The aim of this study was to identify detailed changes in behavior, and in salivary serum amyloid A (SAA), associated with subclinical mastitis. This included standard sickness behaviors (which form part of an adaptive response to conserve energy, minimize heat loss and facilitate recovery following infection and inflammation) and lesser-studied behaviors, that are biologically predicted to change at low-level infection, and therefore particularly relevant for detecting subclinical mastitis (here labelled  luxury behaviors). SAA is a physiological marker of systemic inflammation, with levels in milk and serum already known to increase during subclinical mastitis. We investigated whether the same was true for SAA in cow saliva. Data were collected for n = 34 commercial barn-housed dairy cows, tested in 17 matched-pairs. Each pair comprised a cow with subclinical mastitis (SCM) and a healthy control (CTRL), identified using somatic cell count (SCC) (SCM: SCC >200 x1000 cells/ml; CTRL: SCC <100 x1000 cells/ml). SCM cows were selected for study ad-hoc, at which point they were paired with a CTRL cow, based upon parity and calving date; consequently, the full data set was accrued over several months. Data were collected for each pair over 3 days: SCC (l4:00-l5:00h) Day 1; behavior (24h from 00:00h) Day 2; salivary serum amyloid-A (SAA) Day 3. We report, for the first time, that an increase in salivary SAA occurs during subclinical mastitis; SAA was higher in SCM cows and demonstrated a positive (weak) correlation with SCC. The behavioral comparisons revealed that SCM cows had reductions in activity (behavioral transitions and distance moved), social exploration, social reactivity (here: likelihood to move away/be displaced following receipt of agonism), performance of social grooming and head butts, and the receipt of agonistic non-contact challenges. In addition, SCM cows received more head swipes, and spent a greater proportion of time lying with their head on their flank than CTRL cows. SCM cows also displayed an altered feeding pattern; they spent a greater proportion of feeding time in direct contact with two conspecifics, and a lower proportion of feeding time at self-locking feed barriers, than CTRL cows. Behavioral measures were found to correlate, albeit loosely, with serum SAA in a direction consistent with predictions for sickness behavior. These included positive correlations with both lying duration and the receipt of all agonistic behavior, and negative correlations with feeding, drinking, the performance of all social and all agonistic behavior, and social reactivity. We conclude that changes in salivary SAA, social behavior, and activity offer potential in the detection of subclinical mastitis and recommend further investigation to substantiate and refine our findings. Climate change and chill accumulation: implications for tree fruit production in cold-winter regions Winter chill accumulation is critical for the productivity and profitability of perennial tree fruit systems. Several studies have quantified the impacts of global warming on chill accumulation in the warmer production regions of the world, where insufficient chill events occur and their frequency is increasing. In contrast, we focus on a region with relatively cold winters-the Pacific Northwest United States (PNW)-where insufficient chill events are currently absent, and quantify the potential for introduction of these risks under climate change. Results identified spatial variation within the PNW, with chill accumulation projected to increase in some areas but decrease in others. There was also spatiotemporal variation in the driving factors of changes to chill accumulation. Even with decreases in chill accumulations, there are likely minimal issues with insufficient chill accumulation. However, delayed chill accumulation in combination with advances in the onset of heat accumulation can potentially shift the region from one where spring phenology is primarily forcing-driven to one where the dynamic interplay between chilling and forcing processes become important. These interactions might create production risks for varieties with high chill requirements, post mid-21st-century under high emissions scenarios. Future work should focus on understanding, modeling, and projecting responses across these overlapping chilling and forcing processes. Additionally, given significant spatial differences across a relatively small geographic range, it is also critical to understand and model these dynamics at a local landscape resolution for regions such as the PNW. High school science fair: Experiences of two groups of undergraduate bioscience students Science fairs offer potential opportunities for students to learn first-hand about the practices of science. Over the past six years we have been carrying out voluntary and anonymous surveys with regional and national groups of high school and post high school students to learn about their high school science fair experiences regarding help received, obstacles encountered, and opinions about the value and impact of science fair. Understanding what students think about science fairs will help educators make science fairs more effective learning opportunities. In this paper, we focus on the findings with two national groups of post high school students - undergraduate research fellows (SURF students) who did research at UT Southwestern Medical Center during 2014-2019 and undergraduates biology students attending the 2019 Howard Hughes Medical Institute Science Education Alliance (SEA) summer symposium. About 25% of the students who completed surveys indicated that they had participated in high school science fair, but more than half attended high schools where science fairs were unavailable. Effectively, 6 out of every 10 students participated in science fair if available. Students who could have participated in high school science fair but chose not to do so identified not enough time and coming up with their project idea as major reasons why not. About half the SURF students favored requiring non-competitive science fair regardless whether they themselves had participated in science fair. On the other hand, less than 1 in 5 thought that competitive science fair should be required. Introduction to the scientific process and general learning were mentioned most frequently as the reasons to require non-competitive science fair; these reasons were mentioned rarely in connection with competitive science fair. Unlike the national cohort of high school students we surveyed previously, who mostly did science fair in 9th and 10th grades, SURF students participated in science fair throughout high school and were twice as likely as high school students to have carried out science fair more than once. In conclusion, our findings suggest that participation of the undergraduate bioscience majors in high school science fairs occurs far more frequently than recognized previously. The findings emphasize further the importance of incentivizing rather than requiring science fair participation, especially in 9th and 10th grades, and the potential value of developing non-competitive science fairs. Centriole distal-end proteins CP110 and Cep97 influence centriole cartwheel growth at the proximal-end Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. We previously showed that an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. Here, we show that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, entrained by the core Cdk/Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110/Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, we find that changing the levels of CP110/Cep97 alters the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which may help to coordinate centriole growth. Titers of antibodies the receptor-binding domain (RBD) of ancestral SARS-CoV-2 are predictive for levels of neutralizing antibodies to multiple variants Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring. The Consequences of Differential Origin Licensing Dynamics in Distinct Chromatin Environments MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with under-licensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability. Atrazine induced In vivo immunotoxicity in Bivalves Atrazine is ubiquitously used broad-spectrum herbicide to control the weeds in agriculture. The present study aimed to evaluate the acute toxicity and immunotoxicity of Atrazine in two ecologically and economically important bivalves. Acute toxicity of atrazine evaluated in triplicates by taking control and six experimental groups each comprising of 30 animals and treated with a range of atrazine from 2 PPM to 12 PPM for 96 hours. Mortalities were recorded for every 24 hours until 96 hours and data analyzed by one-way ANOVA and Dunnett T-test. The results indicated a significant increase in mortalities with increase in dose and time of exposure in both species. The values of LC50 were determined as 6.10 PPM and 4.90 PPM respectively for Perna viridis and Paphia malabarica. Furthermore, the immunotoxic potential of atrazine assessed by treating mussels and clams with the five sub-lethal doses of atrazine for 14 days and quantifying the viability of hemocytes by using simple yet reliable Tryphan blue exclusion assay. The results of the present study suggest atrazine-induced immunotoxicity by decreasing the number of viable hemocytes in bivalves. Hemocytes with phagocytic function are indispensable to confer innate immunity in bivalves, decreased viability of these cells leads to compromised immunity. This study is first of its kind to implicate atrazine with the immunotoxicity in bivalves. An exploratory study on the role of the stiffness of breast cancer cells in their detachment from spheroids and migration in 3D collagen matrices BackgroundTumour-cell detachment is a critical early event in the metastatic cascade. However, the role of the cells mechanical properties in detachment and migration is not well understood. This exploratory study aimed to assess how intracellular stiffness changes these processes.

MethodsMDA-MB-231 cells were embedded as 10,000-cell spheroids in 2 and 4 mg/ml collagen matrices. Intracellular stiffness was assessed with mitochondria tracking microrheology of cells that migrated distances equivalent to four and six times the cell diameter (dC) from the spheroid and compared to cells at the spheroid surface (0dC), representing medium, high and no migration, respectively.

Results and discussionThe mitochondrial mean square displacement and intracellular stiffness decreased during detachment and migration for both collagen concentrations (i.e. rigidities). The mean square displacement of 4dC and 6dC cells was similar, whereas cell stiffness was lower for 4dC than for 6dC cells. With increasing matrix rigidity, the intracellular stiffness decreased for 0dC cells and did not change for 4dC and 6dC cells. It is proposed that decreased cell stiffness drives detachment and migration and increased matrix rigidity physically hinders migration, and cells need to become softer or remodel the environment to migrate. The independence of the stiffness of migrated cells from matrix rigidity suggests that cells remodel their environment through matrix proteins cleavage to migrate.

ConclusionsThe study revealed the collective effects of enhanced migratory conditions and increased matrix rigidity on the mechanical properties of the cells. The expression of matrix metalloproteinases and transforming growth factor {beta} and the role of cell volume on detachment and migration in matrices with varying pore sizes are proposed targets for further studies on metastatic cancer cells. Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats implicated unique Lewis X epitopes in mammals The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the glycan structures, the Lexis X antigen was detected on O-fucose glycans. Our results refined the putative consensus sequence for the EOGT-dependent extracellular O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans. Sequence-dependent model of genes with dual σ factor preference Escherichia coli uses {sigma} factors to quickly control large gene cohorts during stress conditions. While most of its genes respond to a single {sigma} factor, approximately 5% of them have dual {sigma} factor preference. The most common are those responsive to both {sigma}70, which controls housekeeping genes, and {sigma}38, which activates genes during stationary growth and stresses. Using RNA-seq and flow-cytometry measurements, we show that  {sigma}70+38 genes are nearly as upregulated in stationary growth as  {sigma}38 genes. Moreover, we find a clear quantitative relationship between their promoter sequence and their response strength to changes in {sigma}38 levels. We then propose and validate a sequence dependent model of {sigma}70+38 genes, with dual sensitivity to {sigma}38 and {sigma}70, that is applicable in the exponential and stationary growth phases, as well in the transient period in between. We further propose a general model, applicable to other stresses and {sigma} factor combinations. Given this, promoters controlling {sigma}70+38 genes (and variants) could become important building blocks of synthetic circuits with predictable, sequence-dependent sensitivity to transitions between the exponential and stationary growth phases. A pilot study on the home range and movement patterns of the Andean Fox Lycalopex culpaeus (Molina 1782) in Cotopaxi National Park, Ecuador This study reports movement patterns and home range estimates of an Andean fox (Lycalopex culpaeus) in Cotopaxi National Park in Ecuador, representing the first GPS-tagging of the species. The GPS functioned well during the 197-day tracking period. Home range sizes ranged between 4.9 - 8.1 km2, depending on the estimation method. Movement speeds averaged 0.17 km/hr at day vs. 0.87 km/hr. at night, and distance traveled averaged 0.23 km at day vs. 0.89 km at night. These preliminary results highlight the importance of collecting unbiased, high-quality data which enables an enhanced understanding on mammal behavior and human/animal interaction.