Light activation of Orange Carotenoid Protein reveals initial C8'-C7' double bond trans/cis isomerization Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation1,2. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows carotenoid trans/cis isomerization at the C7-C8double bond and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven cis/trans isomerization that recovers to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. The isomerization and its transient nature are confirmed in OCP crystals and solution by FTIR and UV/Vis spectroscopy. This study reveals the isomerization of the carotenoid and subsequent thermal structural reactions as the basis of OCP photoreception. Understanding and potentially controlling the OCP dynamics offers the prospect of novel applications in biomass engineering3 as well as in optogenetics and bioimaging.
Phenotypic Heterogeneity Facilitates Survival While Hindering the Evolution of Drug Resistance Due to Intraspecific Competition Rising rates of resistance to antimicrobial drugs threatens the effective treatment of infections across the globe. Drug resistance has been established to emerge from non-genetic mechanisms, such as "persistence" in quiescent microbes and fluctuations in gene expression in actively replicating cells, as well as from genetic mutations. However, it is still unclear how non-genetic drug resistance affects the evolution of genetic drug resistance. We develop deterministic and stochastic population models that incorporate resource competition to quantitatively investigate the transition from non-genetic to genetic resistance during the exposure to static and cidal drugs. We find that non-genetic resistance facilitates the survival of cell populations during drug treatment, but that it hinders the development of genetic resistance due to the competition between the non-genetically and genetically resistant subpopulations. Non-genetic drug resistance in the presence of subpopulation competition is found to increase the first-appearance and fixation times of drug resistance mutations, while increasing the probability of mutation before population extinction during cidal drug treatment. Intense intraspecific competition during drug treatment leads to extinction of the susceptible and non-genetically resistant subpopulations. These findings advance our fundamental understanding of the evolution of drug resistance and may guide novel treatment strategies for patients with drug-resistant infections.

SIGNIFICANCEDrug resistance is predicted to kill as many as 10 million people per year and cost over 100 trillion USD in cumulative lost production globally by 2050. To mitigate these socio-economic costs, we need to fundamentally understand the drug resistance process. We investigate the effect that different forms of resistance have on the evolution of drug resistance using mathematical modeling and computer simulations. We find that the presence of non-genetically drug-resistant cells (whose resistance is temporary and not encoded in a genetic mutation) allows the population to survive drug treatment, while competition between these subpopoulations simultaneously slows down the evolution of permanent genetic drug resistance and in some cases drives them extinct. These findings have important implications for advancing evolutionary theory and for developing effective "resistance-proof" treatments.
A network approach to genetic circuit designs As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information. 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. Entorhinal cortex epigenome-wide association study highlights four novel loci showing differential methylation in Alzheimer's disease BackgroundStudies on DNA methylation (DNAm) in Alzheimers disease (AD) have recently highlighted several genomic loci showing association with disease onset and progression.

MethodsHere, we conducted an epigenome-wide association study (EWAS) using DNAm profiles in entorhinal cortex (EC) from 149 AD patients and control brains and combined these with two previously published EC datasets by meta-analysis (total n=337).

ResultsWe identified 12 cytosine-phosphate-guanine (CpG) sites showing epigenome-wide significant association with either case-control status or Braaks tau-staging. Four of these CpGs, located in proximity to CNFN/LIPE, TENT5A, PALD1/PRF1, and DIRAS1, represent novel findings. Integrating DNAm levels with RNA sequencing-based mRNA expression data generated in the same individuals showed significant DNAm-mRNA correlations for 6 of the 12 significant CpGs. Lastly, by calculating rates of epigenetic age acceleration using two recently proposed "epigenetic clock" estimators we found a significant association with accelerated epigenetic aging in AD patients vs. controls.

ConclusionIn summary, our study represents the hitherto most comprehensive EWAS in AD using EC and highlights several novel differentially methylated loci with potential effects on gene expression. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. Comprehensive analysis of pyroptosis-associated in molecular classification, immunity and prognostic of glioma Integrative analysis was performed in the Chinese Glioma Genome Atlas and The Cancer Genome Atlas to describe the pyroptosis-associated molecular classification and prognostic signature in glioma. Pyroptosis-related genes were used for consensus clustering and to develop a prognostic signature. The immune statuses, molecular alterations and clinical features of differentially expressed genes were analyzed among different subclasses and risk groups. A lncRNA-miRNA-mRNA network was built, and drug sensitivity analysis was used to identify small molecular drugs for the identified genes. Glioma can be divided into two subclasses using 30 pyroptosis-related genes. Cluster 1 displayed high immune signatures and poor prognosis as well as high immune-related function scores. A prognostic signature based on 15 pyroptosis-related genes of the CGGA cohort can predict the overall survival of glioma and was well validated in the TCGA cohort. Cluster 1 had higher risk scores. The high-risk group had high immune cell and function scores and low DNA methylation of pyroptosis-related genes. The differences in pyroptosis-related gene mutations and somatic copy numbers were significant between the high-risk and low-risk groups. The ceRNA regulatory network uncovered the regulatory patterns of different risk groups in glioma. Nine pairs of target genes and drugs were identified. In vitro, CASP8 promotes the progression of glioma cells. Pyroptosis-related genes can reflect the molecular biological and clinical features of glioma subclasses. The established prognostic signature can predict prognosis and distinguish molecular alterations in glioma patients. Our comprehensive analyses provide valuable guidelines for improving glioma patient management and individualized therapy. 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. Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbBs are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the new class of inner membrane energy machinery formed by ExbB,ExbD and HasB. The Hippocampus uses cryptographic principles rather than plasticity to enable animals to secretly cache and retrieve their food For animals, the ability to hide and retrieve valuable information, such as the location of food, can mean the difference between life and death. Here, we propose that to achieve this, their brain uses spatial cells similarly to how we utilize encryption for data security. Some animals are able to cache hundreds of thousands of food items annually by each individual and later retrieve most of what they themselves stashed. Rather than memorizing their cache locations as previously suggested, we propose that they use a single cryptographic-like mechanism during both caching and retrieval. The model we developed is based on hippocampal spatial cells, which respond to an animals positional attention, such as when the animal enters a specific region (place-cells) or gazes at a particular location (spatial-view-cells). We know that the region that activates each spatial cell remains consistent across subsequent visits to the same area but not between areas. This remapping, combined with the uniqueness of cognitive maps, produces a persistent crypto-hash function for both food caching and retrieval. We also show that the model stores temporal information that helps animals in food caching order preference as previously observed. This behavior, which we refer to as crypto-taxis, might also explain consistent differences in decision-making when animals are faced with a large number of alternatives such as in foraging. Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics. Neuronal Activation of the Gastrointestinal Tract Shapes the Gut Environment in Mice The enteric nervous system (ENS) coordinates responses that shape the intestinal milieu, and participates in bi-directional communication with various organs including the brain. Tools to study, and knowledge of, ENS development and function lag behind brain research. Herein, we deploy recombinant adeno-associated viral (rAAV) vectors with enhanced tropism for the gut to map and activate gut associated neurons in mice with spatial and temporal resolution. rAAV-mediated fluorescent labelling coupled with whole-tissue clearing methods in the small intestine and colon reveal novel neuronal architectures in the gut of mice. Viral delivery of genetically-encoded calcium indicators enable in vivo measurement of ENS activity. Further, we employed chemogentics to specifically activate gut neurons that express choline acetyltransferase (ChAT+) or tyrosine hydroxylase (TH+). Targeted activation of ChAT+ or TH+ neuronal populations associated with the gastrointestinal (GI) tract alters the intestinal transcriptome, mouse and microbial proteomes, metagenome, and fecal metabolome. We reveal previously unknown and broad roles for enteric neurons in modulating GI physiology, mucosal immunity, and microbiome structure, and provide evidence for novel functions by the ENS such as regulating fungal colonization and shaping of bile acid profiles in the gut. These advanced experimental tools and rich datasets with multi-parameter characterization of the gut ecosystem may facilitate expanded understanding and study of the ENS. Stress-dependent cell stiffening by tardigrade tolerance proteins through reversible formation of cytoskeleton-like filamentous network and gel-transition Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed "Desolvataion-induced ReversiblY condensing Proteins (DRYPs)". Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRYPs, 3 of which were major components of DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. CAHS filamentation increases cell stiffness to resist deformation and improves resistance to dehydration-like stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and also contribute to the exceptional physical stability in a dehydrated state. Low photorespiratory capacity is sufficient to block the induction of photosynthetic capacity in high light The induction of high photosynthetic capacity in high light (HL) is a common response among many herbaceous dicot plants, however, the signals that control this response remain largely unknown. Here, multiple independent lines of evidence are presented in support of the conclusion that low photorespiratory capacity acts a negative signal to limit photosynthetic capacity acclimation in HL in Arabidopsis thaliana. Using a panel of natural accessions, primary nitrogen (N) assimilation and photorespiration rates early after a shift to growth in HL, as well as activities for key enzymes in these pathways, were shown to positively correlate with the magnitude of the subsequent induction of photosynthetic capacity, which occurred several days later. Time-resolved metabolomic data during acclimation to HL were collected using a strongly acclimating ecotype and a weakly acclimating ecotype, revealing in greater detail the differences in N assimilation, photorespiration, and triose-phosphate utilization pathways underlying efficient photosynthetic capacity acclimation. When shifted into HL growth conditions under non-photorespiratory conditions, weakly acclimating ecotypes and even photorespiratory mutants gained the ability to strongly induce high photosynthetic capacity in HL. Thus, a negative, photorespiration-dependent signal early in the HL shift appears to block photosynthetic capacity acclimation in accessions with low photorespiratory capacity, whereas accessions with high photorespiratory capacity are licensed to increase photosynthetic capacity. Cortical network structure mediates response to stimulation: An optogenetic study in non-human primate cortex As aberrant network-level functional connectivity underlies a variety of neural disorders, the ability to induce targeted functional reorganization would be a profound development towards therapies for neural disorders. Brain stimulation has been shown to alter large-scale network-wide functional connectivity, but the mapping from stimulation to the modification is unclear. Here, we leverage advances in neural interfaces, interpretable machine learning, and graph theory to arrive at a model which accurately predicts stimulation-induced network-wide functional reorganization. The model jointly considers the stimulation protocol and the cortical network structure, departing from the standard approach which only considers the stimulation protocol. We validate our approach in the primary sensorimotor cortex of non-human primates using paired optogenetic stimulation through a large-scale optogenetic interface. We observe that the stimulation protocol only predicts a small portion of the induced functional connectivity changes while the network structure predicts much more, indicating that the underlying network is the primary mediator of the response to stimulation. We extract the relationships linking the stimulation and network characteristics to the functional connectivity changes and observe that the mappings diverge over frequency bands and successive stimulations. Finally, we uncover shared processes governing real-time and longer-term effects of stimulation. Our framework represents a paradigm shift for targeted neural stimulation and can be used to interrogate, improve, and develop stimulation-based interventions for neural disorders.

TeaserBrain stimulation rewires the brain, but the pre-existing network structure of the brain controls the rewiring. First detection of herpesvirus and frequency of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. 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. A map of binary SARS-CoV-2 protein interactions implicates host immune regulation and ubiquitination Key steps in viral propagation, immune suppression, and pathology are mediated by direct, binary, physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary interactions between viral and host proteins, complementing previous co-complex association maps by conveying more direct mechanistic understanding and potentially enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, and confirming high quality of these interactions via a calibrated orthogonal assay. Host proteins interacting with SARS-CoV-2 proteins are enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The binary virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly. 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. The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 Beta (B.1.351) and other variants of concern in preclinical studies. There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), high titre binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) are induced. In addition, a strong and polyfunctional T cell response was measured in these booster regimens. These data support the ongoing clinical development and testing of this new variant vaccine. The ribose methylation enzyme FTSJ1 has a conserved role in neuron morphology and learning performance FTSJ1 is a conserved human 2-O-methyltransferase (Nm-MTase) that modifies several transfer RNAs (tRNAs) at position 32 and the wobble position 34 in the AntiCodon Loop (ACL). Its loss of function has been linked to Non-Syndromic X-Linked Intellectual Disability (NSXLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here we report a novel FTSJ1 pathogenic variant from a NSXLID patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation (Nm) on all tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neuronal progenitor cells (NPC) into neurons displays long and thin spine neurites compared to control cells. These defects are also observed in Drosophila and are associated with long term memory deficit in this organism. Altogether, our study adds insight into FTSJ1 pathologies in human by the identification of novel FTSJ1 targets and the defect in neuron morphology. Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. Despite extensive work on the formation and functions of EVs in cultured cells, studies of EVs in vivo have remained scarce. We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. We defined two distinct EV subpopulations, one of which contains the Munc13-4 homologue Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion events. The formation of Stac-positive luminal EVs depends on the tip-cell-specific GTPase Arl3, which is also required for the formation of Stac-positive multivesicular bodies, suggesting that Stac-EVs derive from fusion of Stac-MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. We propose that Stac-MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4. Modelling porcine reproductive and respiratory syndrome virus dissemination dynamics to quantifythe contribution of multiple modes of transmission: between-farm pig and vehicle movements, farmto-farm proximity, feed ingredients, and re-break Accounting for multiple modes of livestock disease dissemination in epidemiological models remains a challenge. We developed and calibrated a mathematical model for transmission of porcine reproductive and respiratory syndrome virus (PRRSV), tailored to fit nine modes of between-farm transmission pathways including: farm-to-farm proximity (local transmission), contact network of batches of pigs transferred between farms (pig movements), re-break probabilities for farms with previous PRRSV outbreaks, with the addition of four different contact networks of transportation vehicles (vehicles to transport pigs to farms, pigs to markets, feed and crew) and the amount of animal by-products within feed ingredients (e.g. animal fat or meat and bone meal). The model was calibrated on weekly PRRSV outbreaks data. We assessed the role of each transmission pathway considering the dynamics of specific types of production (i.e., sow, nursery). Although our results estimated that the networks formed by transportation vehicles were more densely connected than the network of pigs transported between-farms, pig movements and farm proximity were the main PRRSV transmission routes regardless of farm types. Among the four vehicle networks, vehicles transporting pigs to farms explained a large proportion of infections, sow = 20.9%; nursery = 15%; and finisher = 20.6%. The animal by-products showed a limited association with PRRSV outbreaks through descriptive analysis, and our model results showed that the contribution of animal fat contributed only 2.5% and meat and bone meal only 0.03% of the infected sow farms. Our work demonstrated the contribution of multiple routes of PRRSV dissemination, which has not been deeply explored before. It also provides strong evidence to support the need for cautious, measured PRRSV control strategies for transportation vehicles and further research for feed by-products modeling. Finally, this study provides valuable information and opportunities for the swine industry to focus effort on the most relevant modes of PRRSV between-farm transmission. 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. Mitofusin 1 and 2 regulation of mitochondrial DNA content is a critical determinant of glucose homeostasis The dynamin-like GTPases Mitofusin 1 and 2 (Mfn1 and Mfn2) are essential for mitochondrial function, which has been principally attributed to their regulation of fission/fusion dynamics. Here, we report that Mfn1 and 2 are critical for glucose-stimulated insulin secretion (GSIS) primarily through control of mitochondrial DNA (mtDNA) content. Whereas Mfn1 and Mfn2 individually were dispensable for glucose homeostasis, combined Mfn1/2 deletion in {beta}-cells reduced mtDNA content, impaired mitochondrial morphology and networking, and decreased respiratory function, ultimately resulting in severe glucose intolerance. Importantly, gene dosage studies unexpectedly revealed that Mfn1/2 control of glucose homeostasis was dependent on maintenance of mtDNA content, rather than mitochondrial structure. Mfn1/2 maintain mtDNA content by regulating the expression of the crucial mitochondrial transcription factor Tfam, as Tfam overexpression ameliorated the reduction in mtDNA content and GSIS in Mfn1/2-deficient {beta}-cells. Thus, the primary physiologic role of Mfn1 and 2 in {beta}-cells is coupled to the preservation of mtDNA content rather than mitochondrial architecture, and Mfn1 and 2 may be promising targets to overcome mitochondrial dysfunction and restore glucose control in diabetes. The ecological consequences and evolution of retron-mediated suicide as a way to protect bacteria from being killed by phage In 1984, retrons were described as DNA sequences that code for a reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). It would not be until 2020 that a function was shown, when compelling evidence was presented that retrons can protect bacteria from infections by lytic phage via an abortive infection mechanism. When infected with the virulent mutant of the phage lambda, {lambda}VIR, and to a lesser extent other phages, a retron designated Ec48 is activated, the Escherichia coli bearing this element dies and the infecting phage is lost. With the aid of a mathematical model, we explored the a priori conditions under which retrons will protect bacterial populations from invasion by phage and when retron-bearing bacteria will evolve in populations without this element. Using E. coli with and without Ec48, and {lambda}VIR we estimated the parameters of this model and tested the hypotheses generated from our analysis of its properties. Our models and experiments suggest cells expressing retron-mediated abortive infection have no advantage over cells lacking this system as a defense against phage, and we question whether the protection against phage infection is the unique selective force responsible for their evolution in bacteria.