Recurrent Connections in the Primate Ventral Visual Stream Mediate a Tradeoff Between Task Performance and Network Size During Core Object Recognition The computational role of the abundant feedback connections in the ventral visual stream (VVS) is unclear, enabling humans and non-human primates to effortlessly recognize objects across a multitude of viewing conditions. Prior studies have augmented feedforward convolutional neural networks (CNNs) with recurrent connections to study their role in visual processing; however, often these recurrent networks are optimized directly on neural data or the comparative metrics used are undefined for standard feedforward networks that lack these connections. In this work, we develop task-optimized convolutional recurrent (ConvRNN) network models that more correctly mimic the timing and gross neuroanatomy of the ventral pathway. Properly chosen intermediate-depth ConvRNN circuit architectures, which incorporate mechanisms of feedforward bypassing and recurrent gating, can achieve high performance on a core recognition task, comparable to that of much deeper feedforward networks. We then develop methods that allow us to compare both CNNs and ConvRNNs to fine-grained measurements of primate categorization behavior and neural response trajectories across thousands of stimuli. We find that high performing ConvRNNs provide a better match to this data than feedforward networks of any depth, predicting the precise timings at which each stimulus is behaviorally decoded from neural activation patterns. Moreover, these ConvRNN circuits consistently produce quantitatively accurate predictions of neural dynamics from V4 and IT across the entire stimulus presentation. In fact, we find that the highest performing ConvRNNs, which best match neural and behavioral data, also achieve a strong Pareto-tradeoff between task performance and overall network size. Taken together, our results suggest the functional purpose of recurrence in the ventral pathway is to fit a high performing network in cortex, attaining computational power through temporal rather than spatial complexity.
Sensitivity to the slope of the amplitude spectrum is dependent on the spectral slopes of recently viewed environments: A visual adaptation study in modified reality Scenes contain many statistical regularities that could benefit visual processing if accounted for by the visual system. One such statistic is the orientation-averaged slope () of the amplitude spectrum of natural scenes. Human observers show different discrimination sensitivity to : sensitivity is highest for  values between 1.0 and 1.2 and decreases as  is steepened or shallowed. The range of  for peak discrimination sensitivity is concordant with the average  of natural scenes, which may indicate that visual mechanisms are optimized to process information at  values commonly encountered in the environment. Here we explore the association between peak discrimination sensitivity and the most viewed s in natural environments. Specifically, we verified whether discrimination sensitivity depends on the recently viewed environments. Observers were immersed, using a Head-Mounted Display, in an environment that was either unaltered or had its average  steepened or shallowed by 0.4. Discrimination thresholds were affected by the average shift in , but this effect was most prominent following adaptation to a shallowed environment. We modeled these data with a Bayesian observer and explored whether a change in the prior or a change in the likelihood best explained the psychophysical effects. Change in discrimination thresholds following adaptation could be explained by a shift in the central tendency of the prior concordant with the shift of the environment, in addition to a change in the likelihood. Our findings suggest that expectations on the occurrence of  that result from a lifetime of exposure remain plastic and able to accommodate for the statistical structure of recently viewed environments.
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. Microvesicles Transfer Mitochondria and Increase Mitochondrial Function in Brain Endothelial Cells We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage- derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.

HighlightsO_LIMicrovesicles transfer mitochondria to endothelial cells and brain slice neurons
C_LIO_LIMitochondrial transfer increased ATP in ischemic brain endothelial cells (BECs)
C_LIO_LITransfer of microvesicles increased mitochondrial function in hypoxic BECs
C_LIO_LITransfer of exosomes did not affect mitochondrial function in hypoxic BECs
C_LIO_LIHomotypic BEC-derived EVs result in greater ATP levels in the recipient BECs
C_LI Unravelling Rubber Tree Growth by Integrating GWAS and Biological Network-Based Approaches Hevea brasiliensis (rubber tree) is a large tree species of the Euphorbiaceae family with inestimable economic importance. Rubber tree breeding programs currently aim to improve growth and production, and the use of early genotype selection technologies can accelerate such processes, mainly with the incorporation of genomic tools, such as marker-assisted selection (MAS). However, few quantitative trait loci (QTLs) have been used successfully in MAS for complex characteristics. Recent research shows the efficiency of genome-wide association studies (GWAS) for locating QTL regions in different populations. In this way, the integration of GWAS, RNA-sequencing (RNA-Seq) methodologies, coexpression networks and enzyme networks can provide a better understanding of the molecular relationships involved in the definition of the phenotypes of interest, supplying research support for the development of appropriate genomic based strategies for breeding. In this context, this work presents the potential of using combined multiomics to decipher the mechanisms of genotype and phenotype associations involved in the growth of rubber trees. Using GWAS from a genotyping-by-sequencing (GBS) Hevea population, we were able to identify molecular markers in QTL regions with a main effect on rubber tree plant growth under constant water stress. The underlying genes were evaluated and incorporated into a gene coexpression network modelled with an assembled RNA-Seq-based transcriptome of the species, where novel gene relationships were estimated and evaluated through in silico methodologies, including an estimated enzymatic network. From all these analyses, we were able to estimate not only the main genes involved in defining the phenotype but also the interactions between a core of genes related to rubber tree growth at the transcriptional and translational levels. This work was the first to integrate multiomics analysis into the in-depth investigation of rubber tree plant growth, producing useful data for future genetic studies in the species and enhancing the efficiency of the species improvement programs. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Effects of hydroelectric dam construction on spatio-temporal changes of land use land cover in Bui National Park, Ghana The construction of hydroelectric dams in forest reserves has become a matter of concern for biodiversity conservationists. Visibly among which is the potential to cause changes in climate and land cover and subsequently affect fauna and flora composition. Spatio-temporal changes of climate and land cover in the Bui National Park was analyzed using indices calculations of the landscape based on land cover maps obtained from Landsat satellite images for pre-and post-dam construction periods. Significant changes in land cover following the dam construction were observed. Notable changes include the built-up areas and water body, which recorded an increase of 315.64 % and 4593.43 % respectively, while the forest area decreased. Significant reduction in rainfall (U = 24,{rho} < 0.05) and increase in temperature (U = 22.5,{rho} < 0.05) were observed between the pre-and post-dam construction periods. Increased human activities such as illegal mining, indiscriminate tree felling, uncontrolled cattle grazing and charcoal production within the reserve results from inadequate monitoring and law enforcement after the dam construction could likely compound the changes in land cover. A novel Boolean network inference strategy to model early hematopoiesis aging Hematopoietic stem cell (HSC) aging is a multifactorial event that leads to changes in HSC properties and function. These changes are intrinsically coordinated and affect the early hematopoiesis, involving hematopoietic stem and progenitor cells (HSPCs). The objective of this work is to better understand the mechanisms and factors controlling these changes. We have therefore developed an original strategy to construct a Boolean network of genes explaining the priming and homeostasis of HSCs (graphical abstract). Based on our previous scRNA-seq data, we performed an exhaustive analysis of the transcriptional network and identified active transcription modules or regulons along the differentiation trajectory of selected HSPC states. This global view of transcriptional regulation led us to focus on 15 components, 13 selected TFs (Tal1, Fli1, Gata2, Gata1, Zfpm1, Egr1, Junb, Ikzf1, Myc, Cebpa, Bclaf1, Klf1, Spi1) and 2 complexes regulating the ability of HSC to cycle (CDK4/6 - Cyclin D and CIP/KIP). We then defined the connections controlling the differentiation dynamics of HSC states and constructed an influence graph between the TFs involved in the dynamics by mixing observations from our scRNA-seq data and knowledge from the literature. Then, using answer set programming (ASP) and in silico perturbation analysis, we obtained a Boolean model which is the solution of a Boolean satisfiability problem. Finally, perturbation of the model based on age-related changes revealed important regulations, such as the overactivation of Egr1 and Junb or the loss of Cebpa activation by Gata2, which were found to be relevant for the myeloid bias of aged HSC. Our work shows the efficiency of the combination of manual and systematic methods to elaborate a Boolean model. The developed strategy led to the proposal of new regulatory mechanisms underlying the differentiation bias of aged HSCs, explaining the decreased transcriptional priming of HSCs to all mature cell types except megakaryocytes. Characterization of methylation profiles in spontaneous preterm birth placental villous tissue Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing. Impact of glycosylation on a broad-spectrum vaccine against SARS-CoV-2 A major challenge to end the pandemic caused by SARS-CoV-2 is to develop a broadly protective vaccine. As the key immunogen, the spike protein is frequently mutated with conserved epitopes shielded by glycans. Here, we reveal that spike glycosylation has site-differential effects on viral infectivity and lung epithelial cells generate spike with more infective glycoforms. Compared to the fully glycosylated spike, immunization of spike protein with N-glycans trimmed to the monoglycosylated state (Smg) elicits stronger immune responses and better protection for hACE2 transgenic mice against variants of concern. In addition, a broadly neutralizing monoclonal antibody was identified from the Smg immunized mice, demonstrating that removal of glycan shields to better expose the conserved sequences is an effective and simple approach to broad-spectrum vaccine development.

One-Sentence SummaryRemoving glycan shields to expose conserved epitopes is an effective approach to develop a broad-spectrum SARS-CoV-2 vaccine. Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals. Nonideal polar residues in the core of the coiled coil of FtsLB are critical for modulating its stability and activation in cell division The FtsLB complex is a key regulator of bacterial cell division, existing in either an off or on state which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a "detuned" structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction. Generalizing predictions to unseen sequencing profiles via deep generative models Predictive models trained on sequencing profiles often fail to achieve expected performance when externally validated on unseen profiles. While many factors such as batch effects, small data sets, and technical errors contribute to the gap between source and unseen data distributions, it is a challenging problem to generalize the predictive models across studies without any prior knowledge of the unseen data distribution. Here, this study proposes DeepBioGen, a sequencing profile augmentation procedure that characterizes visual patterns of sequencing profiles, generates realistic profiles based on a deep generative model capturing the patterns, and generalizes the subsequent classifiers. DeepBioGen outperforms other methods in terms of enhancing the generalizability of the prediction models on unseen data. The generalized classifiers surpass the state-of-the-art method, evaluated on RNA sequencing tumor expression profiles for anti-PD1 therapy response prediction and WGS human gut microbiome profiles for type 2 diabetes diagnosis. Growth and division mode plasticity is dependent on cell density in marine-derived black yeasts The diversity and ecological contribution of the fungus kingdom in the marine environment remain under-studied. A recent survey in the Atlantic (Woods Hole, MA, USA) brought to light the diversity and unique biological features of marine fungi. The study revealed that black yeast species undergo an unconventional cell division cycle, which has not been documented in conventional model yeast species such as Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast). The prevalence of this unusual property is unknown. Here, I collected and identified 65 marine fungi species across 40 genera from the surface ocean water, sediment, and the surface of macroalgae (seaweeds) in the Pacific (Sugashima, Toba, Japan). The Sugashima collection largely did not overlap with the Woods Hole collection and included several unidentifiable species, further illustrating the diversity of marine fungi. Three black yeast species were isolated, two of which were commonly found in Woods Hole (Aureobasidium pullulans, Hortaea werneckii). Surprisingly, their cell division mode was dependent on cell density, and the previously reported unconventional division mode was reproduced only at a certain cell density. For all three black yeast species, cells underwent filamentous growth with septations at low cell density and immediately formed buds at high cell density. At intermediate cell density, two black yeasts (H. werneckii and an unidentifiable species) showed rod cells undergoing septation at the cell equator. In contrast, all eight budding yeast species showed a consistent division pattern regardless of cell density. This study suggests the plastic nature of the growth/division mode of marine-derived black yeast. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. 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. A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. Snrpb, the gene mutated in CCMS, is haploinsufficient and required in neural crest cells for proper splicing of developmentally important genes essential for craniofacial morphogenesis Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for Cerebrocostomandibular Syndrome (CCMS). We show that Snrpb heterozygous embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon-skipping and intron-retention in association with increased 5 splice site strength. We found increased exon-skipping in negative regulators of the P53-pathway, increased levels of nuclear P53, P53-target genes. However, removing TrP53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon-skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53-activity and craniofacial-specific genes both contribute to craniofacial malformations. Tobramycin-induced secretion of P. aeruginosa 5' tRNA-fMet halves suppresses lung inflammation via AGO2 gene silencing Although inhaled tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of P. aeruginosa in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is unclear. Here, we demonstrate that tobramycin increases the abundance of two 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by P. aeruginosa and that the 5' tRNA-fMet halves reduce IL-8 secretion by CF bronchial epithelial cells (CF-HBECs). In mouse lung, the 5' tRNA-fMet halves attenuate KC secretion and neutrophil recruitment. We also report that the 5' tRNA-fMet halves suppress pro-inflammatory network gene expression by an Argonaut 2 (AGO2)-mediated gene silencing mechanism, thereby reducing IL-8 secretion in CF-HBECs. Moreover, tobramycin reduces the IL-8 concentration and neutrophil content in bronchoalveolar lavage fluid of pwCF. Thus, we conclude that tobramycin improves lung function in part by reducing chronic inflammation and neutrophil-mediated lung damage in pwCF. Agonist efficiency from concentration-response curves: structural implications and applications Agonists are evaluated by a concentration-response curve (CRC), with a midpoint (EC50) that indicates potency, a high-concentration asymptote that indicates efficacy and a low-concentration asymptote that indicates constitutive activity. A third agonist attribute, efficiency ({eta}), is the fraction of binding energy that is applied to the conformational change that activates the receptor. We show that {eta} can be calculated from EC50 and the asymptotes of a CRC derived from either single-channel or whole-cell responses. For 20 agonists of skeletal muscle nicotinic receptors, the distribution of {eta} values is bimodal with population means at 51% (including ACh, nornicotine and DMPP) and 40% (including epibatidine, varenicline and cytisine). The value of {eta} is related inversely to the size of the agonists head-group, with high-versus low-efficiency ligands having an average volume of 70 [A]3 versus 102 [A]3. Most binding site mutations have only a small effect on ACh efficiency except for Y190A (35%), W149A (60%) and those at G153 (42%). If {eta} is known, the midpoint and high-concentration asymptote can be calculated from each other. Hence, an entire CRC can be estimated from the response to a single agonist concentration, and efficacy can be estimated from EC50 of a CRC that has been normalized to 1. Given {eta}, the level of constitutive activity can be estimated from a single CRC.

Statement of significanceReceptors are molecular machines that convert chemical energy from agonist binding into mechanical energy of a global conformational change that generates a cell response. Agonists are distinguished by their potency (proportional to affinity) and efficacy, but also by the efficiency at which their binding energy is applied to receptor activation. Here we show that agonist efficiency can be estimated from a single concentrationresponse curve, and estimate efficiencies of 20 nicotinic receptor agonists. These have a bimodal distribution with larger agonists belonging to the lower-efficiency population. We further show that mutations of some binding site residues alter efficiency, and that knowledge of agonist efficiency simplifies and extends dose-response curve analysis. 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. One-carbon pathway metabolites are altered in the plasma of subjects with Down syndrome: relation to chromosomal dosage Down syndrome (DS) is the most common chromosomal disorder and it is caused by trisomy of chromosome 21 (Hsa21). Subjects with DS show a large heterogeneity of phenotypes and the most constant clinical features present are typical facies and intellectual disability (ID). Several studies demonstrated that trisomy 21 causes an alteration in the metabolic profile, involving among all one-carbon cycle. We performed enzyme-linked immunosorbent assays (ELISAs) to identify the concentration of 5 different intermediates of the one-carbon cycle, namely tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-methyl-THF), 5-formyltetrahydrofolate (5-formyl-THF), S-adenosyl-homocysteine (SAH) and S-adenosyl-methionine (SAM) in plasma samples obtained from a total of 164 subjects with DS compared to 54 euploid subjects. Results highlight specific alterations of THF with a median concentration ratio DS/control of 2:3, a decrease of a necessary molecule perfectly consistent with a chromosomal dosage effect. Moreover, SAM and SAH show a ratio of 2:1 and 3.6:1, respectively. The relevance of these results for the biology of intelligence and its impairment in trisomy 21 is discussed, leading to the final proposal of 5-methyl-THF as the best candidate for a clinical trial aimed at restoring the dysregulation of one-carbon cycle in trisomy 21, possibly improving cognitive skills of subjects with DS. 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. Thirteen independent genetic loci associated with preserved processing speed in a study of cognitive resilience in 330,097 individuals in the UK Biobank Cognitive resilience is the ability to withstand the negative effects of stress on cognitive functioning and is important for maintaining quality of life while aging. UK Biobank (UKB) does not have direct measurements of the same cognitive phenotype at distal timepoints. Therefore, we used number of education years (EY) as a proxy phenotype for past cognitive performance. Current cognitive performance was determined based on processing speed. This approach captured an average time span of 40 years between past and current cognitive performance in 330,097 individuals. A confounding factor was that EY is highly polygenic and masked the genetics of resilience. To overcome this, we employed Genomics Structural Equation Modelling (GenomicSEM) to perform a GWAS-by-subtraction using two GWAS, one GWAS of EY and resilience and a second GWAS of EY but not resilience. Subtracting one from the other generated a GWAS of Resilience. Replication of this approach was shown using independent discovery and replication samples within UKB and the full UKB GWAS results show significant genetic correlation with a GWAS of cognitive change in the independent Health and Retirement Study (N=9,526; P=1.5x10-3). We found 13 independent genetic loci for Resilience. Functional analyses showed enrichment in several brain regions and involvement of specific cell types, including GABAergic neurons (P=6.59x10-8) and glutamatergic neurons (P=6.98x10-6) in the cortex. Gene-set analyses implicated the biological process "neuron differentiation" (P=9.7x10-7) and the cellular component "synaptic part" (P=2.14x10-6). Mendelian randomization analysis showed a causative effect of white matter volume on cognitive resilience. These results enhance neurobiological understanding of resilience. Nelfinavir induces cytotoxicity towards high-grade serous ovarian cancer cells, involving induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction of HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction of clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. In addition, dose- and time-dependent phosphorylation of H2AX indicated NFV-mediated DNA damage, which was associated with decreased survival and proliferation signals driven by the AKT and ERK pathways. NFV also mediated a dose-dependent increase in endoplasmic reticulum stress-related molecules associated with long-term inhibition of protein synthesis and concurrent cell death; such events were accompanied by a proapoptotic environment, signaled by increased phospho- eIF2, ATF4, and CHOP, increased Bax/Bcl-2 ratio, and cleaved executer caspase-7. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile. Learning and motor inhibitory control in crows and domestic chickens Cognitive abilities allow animals to navigate through complex, fluctuating environments. In the present study, we tested the performance of a captive group of eight crows, Corvus corone, and ten domestic chickens, Gallus gallus domesticus, in the cylinder task, as a test of motor inhibitory control, and reversal learning as a measure of learning ability and behavioural flexibility. Four crows and nine chickens completed the cylinder task, eight crows and six chickens completed the reversal learning experiment. Crows performed better in the cylinder task compared to chickens. In the reversal learning experiment, species did not significantly differ in the number of trials until learning criterion was reached. Performance in the reversal learning experiment did not correlate with performance in the cylinder task in chickens. Our results suggest crows to possess better motor inhibitory control compared to chickens. In contrast, learning performance in a reversal learning task did not differ between the species, indicating similar levels of behavioural flexibility. Interestingly, we describe notable individual differences in performance. We stress the importance not only to compare cognitive performance between species but also between individuals of the same species when investigating the evolution of cognitive skills.