Design of Specific Primer Sets for the Detection of SARS-CoV-2 Variants of Concern B.1.1.7, B.1.351, P.1, B.1.617.2 using Artificial Intelligence As the COVID-19 pandemic continues, new SARS-CoV-2 variants with potentially dangerous features have been identified by the scientific community. Variant B.1.1.7 lineage clade GR from Global Initiative on Sharing All Influenza Data (GISAID) was first detected in the UK, and it appears to possess an increased transmissibility. At the same time, South African authorities reported variant B.1.351, that shares several mutations with B.1.1.7, and might also present high transmissibility. Earlier this year, a variant labelled P.1 with 17 non-synonymous mutations was detected in Brazil. Recently the World Health Organization has raised concern for the variants B.1.617.2 mainly detected in India but now exported worldwide. It is paramount to rapidly develop specific molecular tests to uniquely identify new variants. Using a completely automated pipeline built around deep learning and evolutionary algorithms techniques, we designed primer sets specific to variants B.1.1.7, B.1.351, P.1 and respectively. Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for each variant. In-silico tests show that the sequences in the primer sets present high accuracy and are based on 2 mutations or more. In addition, we present an analysis of key mutations for SARS-CoV-2 variants. Finally, we tested the designed primers for B.1.1.7 using RT-PCR. The presented methodology can be exploited to swiftly obtain primer sets for each new variant, that can later be a part of a multiplexed approach for the initial diagnosis of COVID-19 patients.
How many markers are needed to robustly determine a cell's type? Our understanding of cell types has advanced considerably with the publication of single cell atlases. Marker genes play an essential role for experimental validation and computational analyses such as physiological characterization through pathway enrichment, annotation, and deconvolution. However, a framework for quantifying marker replicability and picking replicable markers is currently lacking. Here, using high quality data from the Brain Initiative Cell Census Network (BICCN), we systematically investigate marker replicability for 85 neuronal cell types. We show that, due to dataset-specific noise, we need to combine 5 datasets to obtain robust differentially expressed (DE) genes, particularly for rare populations and lowly expressed genes. We estimate that 10 to 200 meta-analytic markers provide optimal performance in downstream computational tasks. Replicable marker lists condense single cell atlases into interpretable and generalizable information about cell types, opening avenues for downstream applications, including cell type annotation, selection of gene panels and bulk data deconvolution.
Darwin's small and medium ground finches might have taste preferences, but not for human foods Urbanization is rapidly changing ecological niches. On the inhabited Galapagos Islands, Darwins finches consume human-introduced foods preferentially; however, it remains unclear why. Here we presented pastry with flavour profiles typical of human foods (oily, salty, sweet) to small ground finches (Geospiza fuliginosa) and medium ground finches (Geospiza fortis) to test if latent taste preferences might drive selection of human foods. If human-food flavours were consumed more than a neutral or bitter control only at sites with human foods, then we predicted tastes were acquired after urbanization; however, if no site-differences were found then this would indicate latent taste preferences. Contrary to both predictions, we found little evidence that human-food flavours were preferred compared to control flavours at any site. Instead, finches showed a weak aversion to oily foods, but only at remote (no human foods present) sites. This was further supported by behavioural responses, with beak wiping occurring more often at remote sites after finches tasted flavours associated with human foods. Our results suggest, therefore, that while Darwins finches may have acquired a tolerance to the flavours of human food, latent taste preferences are unlikely to have played a major role in their dietary response to increased urbanization. Common Dynamic Determinants Govern Quorum Quenching Activity in N-terminal Serine Hydrolases Growing concerns about microbial antibiotic resistance have motivated extensive research into ways of overcoming antibiotic resistance. Quorum quenching (QQ) processes disrupt bacterial communication via quorum sensing, which enables bacteria to sense the surrounding bacterial cell density and markedly affects their virulence. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and may thus avoid inducing resistance. Although many enzymes display QQ activity against various bacterial signaling molecules, their mechanisms of action are poorly understood, limiting their potential optimization as QQ agents. Here we evaluate the capacity of three N-terminal serine hydrolases to degrade N-acyl homoserine lactones that serve as signaling compounds for Gram-negative bacteria. Using molecular dynamics simulations of the free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics molecular dynamics simulations of their initial catalytic steps, we clarify the molecular processes underpinning their QQ activity. We conclude that all three enzymes degrade bacterial signaling molecules via similar reaction mechanisms. Moreover, we experimentally confirmed the activity of two penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), adding these biotechnologically well-optimized enzymes to the QQ toolbox. We also observed enzyme- and substrate-dependent differences in the catalytic actions of these enzymes, arising primarily from the distinct structures of their acyl-binding cavities and the dynamics of their molecular gates. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate had an elevated energy barrier because its shallow acyl binding site could not accommodate a productive substrate-binding configuration. Conversely, aPGA in complex with both substrates exhibited unfavorable energetics in both reaction steps due to the dynamics of the residues gating the acyl binding cavity entrance. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to the unique positioning of Arg297{beta} in this enzyme. The discovery of these dynamic determinants will guide future efforts to design robust QQ agents capable of selectively controlling virulence in resistant bacterial species. A General Biphasic-Bodyweight Model for Scaling Basal Metabolic Rate, Glomerular Filtration Rate and Drug Clearance from Birth to Adulthood Understanding the maturation process of human physiology and metabolism has broad medical and pharmaceutical implications. Age and bodyweight are frequently considered as separate variables in modeling the dynamical changes of human organ functions and of drug clearance from birth to adulthood. The objective of this study is to propose a unified, continuous and bodyweight-only equation to quantify the changes of human basal metabolic rate (BMR), glomerular filtration rate (GFR) and drug clearance (CL) from infancy to adulthood. The BMR datasets were retrieved from a comprehensive historical database of male and female subjects (0.02 to 64 years). The CL datasets for 17 drugs and the GFR dataset were generated from age-incorporated maturation-and-growth models with reported parameter values. The model used in the simulation is independent of the proposed model. A statistical approach was used to simulate the model generated CL and GFR data for a hypothetical population with 26 age groups (ranging from 0 to 20 years). Besides, individual CL data for one drug, and sparse PBPK-modeled CL values for two drugs were also included for analysis. A 4-parameter, mixed-allometry equation with two power-law functions of bodyweight was proposed and evaluated as a general model using nonlinear regression and dimensionless analysis. All datasets universally reveal biphasic curves with two distinct linear segments on log-log plots. Compared with simple allometry, the biphasic model fits satisfactorily to all datasets (based on Akaikes Information Criterion and residual plots). The biphasic equation consists of two reciprocal allometric terms that asymptotically determine the overall curvature. The fitting results show a superlinear scaling phase (slope >1; ca. 1.5 - 3.5) below the characteristic bodyweight at the phase transition; and above which, a sublinear scaling phase (slope <1; ca. 0.5 - 0.7) is evident. The phase-transition bodyweight is ranging from 5 to 20 kg (corresponding to 0.5 - 9 years) and the mean value is around 10 kg ([~]2 years) for all data sets. The dimensionless analysis generalizes, and offers quantitative realization of, the maturation and growth process. In conclusion, the proposed mixed-allometry equation is a generic model that quantitatively describes the phase transition occurring in the human maturation process of BMR, GFR and drug CL. Population bottleneck has only marginal effect on fitness evolution and its repeatability in dioecious C. elegans The predictability of evolution is expected to depend on the relative contribution of deterministic and stochastic processes. This ratio is modulated by effective population size. Smaller effective populations harbor less genetic diversity and stochastic processes are generally expected to play a larger role, leading to less repeatable evolutionary trajectories. Empirical insight into the relationship between effective population size and repeatability is limited and focused mostly on asexual organisms. Here, we tested whether fitness evolution was less repeatable after a population bottleneck in obligately outcrossing populations of Caenorhabditis elegans. Replicated populations founded by 500, 50, or 5 individuals (no/moderate/strong bottleneck) were exposed to a novel environment with a different bacterial prey. As a proxy for fitness, population size was measured after one week of growth before and after 15 weeks of evolution. Surprisingly, we found no significant differences among treatments in their fitness evolution. Even though the strong bottleneck reduced the relative contribution of selection to fitness variation, this did not translate to a significant reduction in the repeatability of fitness evolution. Thus, although a bottleneck reduced the contribution of deterministic processes, we conclude that the predictability of evolution may not universally depend on effective population size, especially in sexual organisms. Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold. Alphaviruses such as Ross River virus (RRV), chikungunya virus, Sindbis virus (SINV), and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 and 1.9 [A]. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogendeuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Key PointsO_LICrystal structures of alphavirus nsP4 RNA polymerase domain from RRV and SINV.
C_LIO_LInsP4 protein is highly dynamic with an intrinsically disordered N-terminal domain.
C_LIO_LIOptimized RNA elongation activity assay to facilitate antiviral discovery.
C_LI Microbiota-instructed regulatory T cell differentiation is mediated by a distinct RORγt+ antigen presenting cell subset The mutualistic relationship of gut-resident microbiota and cells of the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a poised, but largely non-aggressive, immune cell compartment1, 2. Consequences of disturbing this balance, by environmental or genetic factors, include proximal inflammatory conditions, like Crohns disease, and systemic illnesses, both metabolic and autoimmune. One of the means by which this equilibrium is achieved is through induction of both effector and suppressor or regulatory arms of the adaptive immune system. In mice, Helicobacter species induce regulatory (iTreg) and follicular helper (Tfh) T cells in the colon-draining mesenteric lymph nodes under homeostatic conditions, but can instead induce inflammatory Th17 cells when iTreg cells are compromised3, 4. How Helicobacter hepaticus and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here, we investigated which cells and molecular components are required to convey the microbial instruction for the iTreg differentiation program. We found that antigen presentation by cells expressing ROR{gamma}t, rather than by classical dendritic cells, was both required and sufficient for iTreg induction. These ROR{gamma}t+ cells, likely to be type 3 innate lymphoid cells (ILC3) and/or a recently-described population of Aire+ cells termed Janus cells5, require the MHC class II antigen presentation machinery, the chemokine receptor CCR7, and v integrin, which activates TGF-{beta}, for iTreg cell differentiation. In the absence of any of these, instead of iTreg cells there was expansion of microbiota-specific pathogenic Th17 cells, which were induced by other antigen presenting cells (APCs) that did not require CCR7. Thus, intestinal commensal microbes and their products target multiple APCs with pre-determined features suited to directing appropriate T cell differentiation programs, rather than a common APC that they endow with appropriate functions. Our results illustrate the ability of microbiota to exploit specialized functions of distinct innate immune system cells, targeting them to achieve the desired composition of equipoised T cells, thus maintaining tolerance. Robust and annotation-free analysis of alternative splicing using short-read scRNA-seq data Although alternative splicing is a fundamental and pervasive aspect of gene expression in higher eukaryotes, it is often omitted from single-cell studies due to quantification challenges inherent to commonly used short-read sequencing technologies. Here, we undertake the analysis of alternative splicing across numerous diverse murine cell types from two large-scale single-cell datasets--the Tabula Muris and BRAIN Initiative Cell Census Network--while accounting for understudied technical artifacts and unannotated isoforms. We find strong and general cell-type-specific alternative splicing, complementary to total gene expression but of similar discriminatory value, and identify a large volume of novel isoforms. We specifically highlight splicing variation across different cell types in primary motor cortex neurons, bone marrow B cells, and various epithelial cells; and show that the implicated transcripts include many genes which do not display total expression differences. To elucidate the regulation of alternative splicing, we build a custom predictive model based on splicing factor activity, recovering several known interactions while generating new hypotheses, including potential regulatory roles for novel alternative splicing events in critical genes including Khdrbs3 and Rbfox1. We make our results available using public interactive browsers to spur further exploration by the community. Existence of log-phase Escherichia coli persisters and lasting memory of a starvation pulse The vast majority of a bacterial population is quickly killed when treated with a lethal concentration of antibiotics. The time scale of this killing is often comparable with the bacterial generation time before addition of antibiotics. Yet, a small subpopulation typically survives for an extended period. However, the long-term killing dynamics of bacterial cells has not been fully quantified even in well-controlled laboratory conditions. We constructed a week-long killing assay and followed the survival fraction of Escherichia coli K12 exposed to a high concentration of ciprofloxacin. We found that long-term survivors were formed during exponential growth, with some cells surviving at least 7 days. The long-term dynamics contained at least three timescales, which greatly enhances predictions of the population survival time compared to the biphasic extrapolation from the short term behavior. Furthermore, we observed a surprisingly long memory effect of a brief carbon starvation pulse, which was dependent on the (p)ppGpp synthase relA. Specifically, one hour of carbon starvation prior to antibiotics exposure increased the surviving fraction by nearly 100-fold even after 4 days of ciprofloxacin treatment. P5A-ATPase controls the ER translocation of Wnt in neuronal migration Wnt family are conserved secretory proteins required for developmental patterning and tissue homeostasis. The mechanisms underlying intracellular maturation and intercellular signal transduction of Wnt proteins have been extensively studied. However, how Wnt is targeted to the endoplasmic reticulum (ER) for processing and secretion remains elusive. Here we report that CATP-8/P5A-ATPase directs neuronal migration non-cell autonomously in C. elegans by regulating EGL-20/Wnt biogenesis. CATP-8 functions as a translocase to translocate EGL-20/Wnt nascent polypeptide into the ER by interacting with the hydrophobic core region of EGL-20 signal sequence. Such regulation of Wnt biogenesis by P5A-ATPase is conserved in human cells. These findings reveal physiological roles of P5A-ATPase in neural development and identify Wnt proteins as direct substrates of P5A-ATPase for ER translocation. Classification of Pacemaker Dynamics in the Mouse Intestine by Field Potential Microimaging The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals. Mutations in coral soma and sperm imply lifelong stem cell renewal and cell lineage selection In many animals, the germline differentiates early in embryogenesis, so only mutations that accumulate in germ cells are inherited by offspring1. Exceptions to this developmental process may indicate that other mechanisms have evolved to limit the effects of deleterious mutation accumulation2. Stony corals are animals that can live for hundreds of years3 and have long been thought to produce gametes from somatic tissue4. To clarify conflicting evidence about germline-soma distinction in corals, we sequenced high coverage, full genomes with technical replicates for parent coral branches and their sperm pools. We identified post-embryonic single nucleotide variants (SNVs) unique to each parent branch, then checked if each SNV was shared by the respective sperm pool: 26% of post-embryonic SNVs were shared by the sperm but 74% were not. We also identified germline SNVs, those that were present in the sperm but not in the parent. These data suggest that self-renewing stem cells in corals differentiate into germ and soma throughout the adult life of the colony, with SNV rates and patterns differing markedly in stem, soma, and germ lineages. In addition to informing the important place in the evolutionary spectrum from non-Weismannian to Weismmanian animals that corals occupy, these insights inform how corals may generate adaptive diversity necessary in the face of global climate change. AFFORDABLE, REPLICABLE ROBOTIC MINIRHIZOTRON SAMPLING FOR ROOT DYNAMICS STUDIES O_LIMinirhizotrons (paired camera systems and buried observatories) are the best current method to make repeatable measurements of fine root dynamics in the field. Automating data collection is the only way to assess fine-scale temporal changes in fine roots in natural systems. When paired with above-ground proximal remote sensing, automatic minirhizotron data could reveal coordination between above and below ground phenology, supporting both plant allocation and carbon exchange understanding. However, there are many challenges to automating minirhizotrons for field experiments. First, instruments must be cheap enough to replicate given shallow field of view and variable natural environments. Second, automated analysis must be robust to changeable soil and root conditions because image properties extracted must have biological meaning.
C_LIO_LIHere, we demonstrate an automatic minirhizotron scheme, built with off-the-shelf parts and capable to sample at sub-daily resolution paired with a neural network-based image analysis method. As a proof of concept, we tested the instruments and the methodology in a mesocosm study and in two short real-world field trials alongside datasets from other instruments (e.g. proximal remote sensed leaf dynamics).
C_LIO_LIWe show that the method produces robust daily time-series of root dynamics in the mesocosm and the field. We applied the same model across multiple instruments, demonstrating good reproducibility of measurements. Temporal variations of root cover were a strong driver of soil CO2 efflux in mesocosm, and vegetation dynamics from above ground proximal remote sensing and minirhizotron data differed both in mesocosm and field. We found sensitivity of root cover extracted to soil moisture and sometimes time of day (potentially relating to soil moisture), which may only affect high frequency imagery and not commonly reported as encountered when training neural networks on traditional, time-distinct minirhizotron studies.
C_LIO_LIWe discuss avenues for dealing with such issues in future field applications of minirhizotrons. If such issues are dealt with to a satisfactory manner in the field, timeseries of root biomass and traits from replicated instruments can add a new dimension to phenology studies at the ecosystem level by understanding the dynamics of root properties and traits.
C_LI Trade-offs in biosensor optimization for dynamic pathway engineering Recent progress in synthetic biology allows the construction of dynamic control circuits for metabolic engineering. This technology promises to overcome many challenges encountered in traditional pathway engineering, thanks to their ability to self-regulate gene expression in response to bioreactor perturbations. The central components in these control circuits are metabolite biosensors that read out pathway signals and actuate enzyme expression. However, the construction of metabolite biosensors is a major bottleneck for strain design, and a key challenge is to understand the relation between biosensor dose-response curves and pathway performance. Here we employ multiobjective optimization to quantify performance trade-offs that arise in the design and calibration of metabolite biosensors. Our approach reveals strategies for tuning dose-response curves along an optimal trade-off between production flux and the cost of an increased expression burden on the host. We explore properties of control architectures built in the literature, and identify their advantages and caveats in terms of performance and robustness to growth conditions and leaky promoters. We demonstrate the optimality of a control circuit for glucaric acid production in Escherichia coli, which has been shown to increase titer by 2.5-fold as compared to static designs. Our results lay the groundwork for the automated design of control circuits for pathway engineering, with applications in the food, energy and pharmaceutical sectors. Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible. In support of this, RNA polymerase transcribed a looped segment more slowly than an unlooped segment and paused more briefly at LacI obstacles on positively supercoiled templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase appears to facilitate elongation along topologically-complex, protein-coated templates. Motor attention to the tactile modality induces sensory attenuation for sounds Self-produced stimuli appear attenuated compared to stimuli generated by others. This effect is known as sensory attenuation (Blakemore et al., 1999). Here, we asked whether an attentional prioritization for the tactile modality during a goal-directed hand movement leads to sensory attenuation for sounds. We presented stimuli in a virtual reality setup that allowed to manipulate the time of tactile feedback when pressing a virtual button. We found that a tactile impulse was perceived as more intense in the moment the hand pushed the button (N = 29). In a second experiment (N = 25), participants pushed a button and estimated the intensity of sounds. We found sensory attenuation for sounds only when tactile feedback was provided at the time the movement goal was reached. These data indicate that sensory attenuation for sounds occurs because tactile attention is boosted towards the movement goal, leaving reduced attentional resources for the auditory modality. Genetic instability from a single S-phase after whole genome duplication Diploid and stable karyotypes are associated with health and fitness in animals. In contrast, whole genome duplications (WGDs) - doubling full chromosome content - are linked to genetic instability (GIN) and frequently found in human cancers 1-3. It has been established that WGDs fuel chromosome instability through abnormal mitosis 4-8, however, the immediate consequences of tetraploidy in the first interphase are not known. This is an essential question because single WGD events such as cytokinesis failure can promote tumorigenesis 9. Here, we found that newly born tetraploid human cells undergo high rates of DNA damage during DNA replication in the first S-phase. Using DNA combing and single cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we found that these defects result from the lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. Cytokinin oxidase/dehydrogenase family genes exhibit functional divergence and overlap in rice growth and development Cytokinins play key roles in plant growth and development; hence, cytokinin biosynthesis and degradation have been extensively studied. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes that regulate oxidative cleavage to maintain cytokinin homeostasis. In rice, 11 OsCKX genes have been identified to date; however, most of their functions remain unknown. Here, we comprehensively analyzed the expression patterns and functions of OsCKX genes. Using CRISPR/Cas9 technology, we constructed mutants of all OsCKX genes to determine the functions of OsCKXs in rice development. The results revealed that the single osckx and higher-order osckx4 osckx9 mutant lines showed functional overlap and subfunctionalization. Notably, the osckx1 osckx2 and osckx4 osckx9 double mutants displayed contrasting phenotypic changes in tiller number and panicle size compared to the wild type. Moreover, we identified several genes with significantly altered expression in osckx4 and osckx9 single and double mutant plants. Many differentially expressed genes were found to be associated with auxin and cytokinin pathways. Additionally, the cytokinins in osckx4 osckx9 mutants were increased compared to the wild type. Overall, our findings provide new insights into the functions of OsCKX genes in rice growth and may be used as a foundation for future studies aimed at improving rice yield. Key words: Cytokinin, expression pattern, OsCKX, panicle, phenotype, rice, tiller

HighlightThe osckx4 osckx9 double mutant had a significantly greater number of tillers, whereas the osckx1 osckx2 double mutant showed the opposite phenotypic change, compared to the wild type Relative contributions of sex hormones, sex chromosomes, and gonads to sex differences in tissue gene regulation Sex differences in physiology and disease in mammals result from the effects of three classes of factors that are inherently unequal in males and females: reversible (activational) effects of gonadal hormones, permanent (organizational) effects of gonadal hormones, and cell-autonomous effects of sex chromosomes, as well as genes driven by these classes of factors. Often, these factors act together to cause sex differences in specific phenotypes, but the relative contribution of each and the interactions among them remain unclear. Here, we used the Four Core Genotypes (FCG) mouse model with or without hormone replacement to distinguish the effects of each class of sex-biasing factors on transcriptome regulation in liver and adipose tissues. We found that the activational hormone levels have the strongest influence on gene expression, followed by the organizational gonadal sex effect and, lastly, sex chromosomal effect, along with interactions among the three factors. Tissue specificity was prominent, with a major impact of estradiol on adipose tissue gene regulation, and of testosterone on the liver transcriptome. The networks affected by the three sex-biasing factors include development, immunity and metabolism, and tissue-specific regulators were identified for these networks. Furthermore, the genes affected by individual sex-biasing factors and interactions among factors are associated with human disease traits such as coronary artery disease, diabetes, and inflammatory bowel disease. Our study offers a tissue-specific account of the individual and interactive contributions of major sex-biasing factors to gene regulation that have broad impact on systemic metabolic, endocrine, and immune functions. Applied tutorial for the design and fabrication of biomicrofluidic devices by resin 3D printing Stereolithographic (SL) 3D printing, especially digital light processing (DLP) printing, is a promising rapid fabrication method for bio-microfluidic applications such as clinical tests, lab-on-a-chip devices, and sensor integrated devices. The benefits of 3D printing lead many to believe this fabrication method will accelerate the use of microfluidics, but there are a number of potential obstacles to overcome for bioanalytical labs to fully utilize this technology. For commercially available printing materials, this includes challenges in producing prints with the print resolution and mechanical stability required for a particular design, along with cytotoxic components within many SL resins and low optical compatibility for imaging experiments. Potential solutions to these problems are scattered throughout the literature and rarely available in head-to-head comparisons. Therefore, we present here a concise guide to the principles of resin 3D printing most relevant for fabrication of bioanalytical microfluidic devices. Intended to quickly orient labs that are new to 3D printing, the tutorial includes the results of selected systematic tests to inform resin selection, strategies for design optimization, and improvement of biocompatibility of resin 3D printed bio-microfluidic devices. Polycystins are upregulated in fibrotic kidneys and promote renal fibrosis Autosomal polycystic kidney disease (ADPKD) is caused by mutations in PKD1or PKD2 gene, which encodes polycystin-1 or polycystin-2 respectively. Paradoxically, polycystins are up-regulated in human ADPKD kidneys and overexpression of polycystin-1 even induced renal cyst formation in mice, suggesting that up-regulation of polycystin is pathogenic. In this study, we determined the role of polycystin in renal fibrosis and explored its underlying mechanisms. We found that the expression of polycystin-1 or polycystin-2 was positively correlated with the expression of collagen-I in the kidney of three different fibrotic mouse models. Pharmaceutical inhibition or genetic knockout of polycystin-2 reduced the expression of pro-fibrotic markers in fibrotic kidneys of two animal models. Moreover, conditional knockout of Pkd1 gene attenuated renal fibrosis in two mouse models. Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase which plays important role in renal fibrosis. We found that EZH2 was positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of the Ezh2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in fibrotic kidneys.

In conclusion, polycystins are up-regulated in injured kidneys and polycystins promote renal fibrosis through up-regulation of EZH2 in fibrotic kidneys, suggesting that renal fibrosis in polycystic kidneys is not the direct effect of PKD gene mutations.

SignificanceMutations of polycystins cause autosomal polycystic kidney disease (ADPKD) which is associated with increased renal fibrosis in cystic kidneys. However, the role of polycystin in renal fibrosis remains unclear. Here, we showed that the expression of polycystins is positively correlated with the development of renal fibrosis in injured kidneys. Pharmaceutical inhibition or genetic deletion of polycystin-2 attenuated renal fibrosis in fibrotic kidneys. Moreover, genetic deletion of polycystin-1 inhibited renal fibrosis in two different animal models. Finally, we showed that ablation of the methyltransferase EZH2 mitigated the anti-fibrotic effect of polycystin-1 deletion in fibrotic kidneys. We conclude that polycystin promotes through EZH2 and renal fibrosis in cystic kidney is not the direct effect of PKD gene mutations. 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. Preprints in motion: tracking changes between preprint posting and journal publication during a pandemic Amidst the COVID-19 pandemic, preprints in the biomedical sciences are being posted and accessed at unprecedented rates, drawing widespread attention from the general public, press and policymakers for the first time. This phenomenon has sharpened longstanding questions about the reliability of information shared prior to journal peer review. Does the information shared in preprints typically withstand the scrutiny of peer review, or are conclusions likely to change in the version of record? We assessed preprints from bioRxiv and medRxiv that had been posted and subsequently published in a journal through 30th April 2020, representing the initial phase of the pandemic response. We utilised a combination of automatic and manual annotations to quantify how an article changed between the preprinted and published version. We found that the total number of figure panels and tables changed little between preprint and published articles. Moreover, the conclusions of 7.2% of non-COVID-19-related and 17.2% of COVID-19-related abstracts undergo a discrete change by the time of publication, but the majority of these changes do not qualitatively change the conclusions of the paper. A platform of patient-derived microtumors identifies individual treatment responses and therapeutic vulnerabilities in ovarian cancer. BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses.

MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format.

ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach.

Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment.