Biology subjects

Miller, S.

Publications and source records attributed to Miller, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

TGFβ determines morphology and key cellular processes of activated CD4+ T cells

During an immune response, cells are simultaneously exposed to multiple cytokine signals that collectively determine their phenotype. Transforming growth factor {beta} (TGF{beta}) is a pleiotropic cytokine acting as a key regulator of T-cell differentiation with activating and suppressive effects on their immune function. Here, we systematically analyze the cellular responses of CD4+ T cells to TGF{beta} across diverse cytokine environments in the presence or absence of TGF{beta}. We found that TGF{beta} had a profound dominant effect independent of the presence of other cytokines, modulating the expression of more than 4,000 genes. In the presence of TGF{beta}, cells exhibit lower expression of translation-related and apoptosis-related genes, accompanied by increased survival of activated T cells. Notably, cells cultured in the presence of TGF{beta} were smaller in size while preserving their proliferative ability. Accordingly, we identified a dense network of transcription factors that were modulated by TGF{beta}, suggesting a core gene set connecting TGF{beta} signaling to the regulation of T-cell size. We found N-Myc to be at the center of this network, and we directly show that TGF{beta} regulates its gene expression level, protein level, and nuclear localization. Our work provides a system to study cell size control and demonstrate the profound effect of TGF{beta} in the modulation and regulation of T-cell properties, expanding its role beyond guiding their phenotype. Significance StatementTGF{beta} is a key determinant of CD4+ T-cell differentiation; however, understanding its effect on additional aspects of T-cell state is lacking. Here, we systematically studied the role of TGF{beta} in regulating T-cell physiology. Exposing cells to diverse combinations of cytokines enabled us to distill the core effect of TGF{beta}. We found TGF{beta} to have a profound effect on multiple cellular processes critical to T-cell function. Significantly, TGF{beta} induced smaller T-cells both in vitro and in vivo, suggesting that TGF{beta} could skew the population towards tissue infiltration and residency. Furthermore, TGF{beta} can be used to fine-tune T-cell size, providing a system for studying cell size control. Overall, our findings demonstrate the profound effect of TGF{beta} in the regulation of T-cell physiology.

immunology↗

bioRxiv2024

Extracellular Matrix-Induced Genes May Reduce Response to Rapamycin in LAM

RationaleLymphangioleiomyomatosis (LAM) is a rare cystic lung disease driven by nodules containing TSC2-/- LAM cells and recruited LAM associated fibroblasts (LAFs). Although rapamycin reduces lung function loss, some patients continue to decline meaning additional therapies are needed. ObjectivesTo investigate how the LAM nodule environment affects LAM cell proliferation and the response to rapamycin. MethodsChanges in advanced LAM were identified using shotgun proteomics and immunohistochemistry in tissue from carefully phenotyped patients. Genes potentially associated with rapamycin insensitivity of cells grown on LAF-derived extracellular matrix were identified by RNA sequencing and validated using repurposed pharmacologic inhibitors. Main ResultsMore advanced disease was associated with increasing nodules adjacent to lung cysts and greater decline in forced expiratory volume in 1 sec (FEV1) when treated with rapamycin (p=0.005). In late-stage LAM, proteomics identified upregulation of pathways associated with accumulation of activated fibroblasts, including extracellular matrix deposition, glucose metabolism and the actin cytoskeleton. Picrosirius red staining and immunohistochemistry confirmed deposition of extracellular matrix within LAM nodules. The growth of TSC2-/- model LAM cells was increased on LAF-derived extracellular matrix (LAF ECM), and incompletely supressed by rapamycin (p<0.0001). RNA sequencing of cells grown on LAF ECM identified upregulation of pathways driving cell cycle control, transcription and metabolism in cells. Tractable, pro-proliferative, rapamycin insensitive genes included CDK7, GAS6 and PLAU. Repurposed inhibitors of these pathways inhibited LAM cell proliferation and enhanced the anti-proliferative effect of rapamycin. ConclusionsExtracellular matrix deposited by LAM associated fibroblasts upregulates expression of genes which potentially blunt the response to rapamycin, but offer additional therapeutic opportunities for patients with established LAM.

cell biology↗

bioRxiv2024

mTOR dysregulation induces IL6 and paracrine AT2 cell senescence impeding lung repair in lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare disease which causes lung cysts and respiratory failure. TSC2 deficient LAM cells with dysregulated mTOR signalling form nodules with fibroblasts causing lung injury. We examined if mTOR dysregulation could induce senescence and impair responses to lung injury. Senescence markers p21 and p16 were increased in LAM lungs and co-localised with alveolar type 2 cells. The SenMayo senescence gene panel was upregulated in LAM alveolar type 2 cells with senescence supressed by mTOR inhibition in patients. LAM cell / fibroblast spheroid cultures induced senescence markers in alveolar type 2 cell organoids, altered their growth and delayed epithelial scratch wound repair. Upstream regulator analysis predicted alveolar type 2 cell IL6 receptor activation. IL6 was produced by LAM cells, induced p16 and p21 in alveolar type 2 cells, inhibited epithelial wound resolution and was overexpressed in LAM patient serum where it was related to lung function. Wound repair in the presence of TSC2 null LAM cell / fibroblast spheroids was enhanced by the IL6 receptor antagonist Tocilizumab. Our findings show TSC2 loss induces senescence and IL6 production which are associated with impaired lung repair. Targeting IL6 signalling in parallel with mTOR inhibition, may reduce lung damage in LAM.

cell biology↗

bioRxiv2024

Digital Microbe: A Genome-Informed Data Integration Framework for Collaborative Research on Emerging Model Organisms

The remarkable pace of genomic data generation is rapidly transforming our understanding of life at the micron scale. Yet this data stream also creates challenges for team science. A single microbe can have multiple versions of genome architecture, functional gene annotations, and gene identifiers; additionally, the lack of mechanisms for collating and preserving advances in this knowledge raises barriers to community coalescence around shared datasets. "Digital Microbes" are frameworks for interoperable and reproducible collaborative science through open source, community-curated data packages built on a (pan)genomic foundation. Housed within an integrative software environment, Digital Microbes ensure real-time alignment of research efforts for collaborative teams and facilitate novel scientific insights as new layers of data are added. Here we describe two Digital Microbes: 1) the heterotrophic marine bacterium Ruegeria pomeroyi DSS-3 with >100 transcriptomic datasets from lab and field studies, and 2) the pangenome of the cosmopolitan marine heterotroph Alteromonas containing 339 genomes. Examples demonstrate how an integrated framework collating public (pan)genome-informed data can generate novel and reproducible findings.

bioinformatics↗

bioRxiv2024

A split-GAL4 driver line resource for Drosophila CNS cell types

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in Drosophila melanogaster and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult Drosophila central nervous system and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. Previously published and new lines are included, all validated for driver expression and curated for optimal cell type specificity across diverse cell types. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

neuroscience↗

bioRxiv2023

Employing a honeybee olfactory neural circuit as a novel gas sensor for the detection of human lung cancer biomarkers

Human breath contains biomarkers (odorants) that can be targeted for early disease detection. It is well known that honeybees have a keen sense of smell and can detect a wide variety of odors at low concentrations. Here, for the first time, we employ honeybee olfactory neuronal circuitry to classify human lung cancer volatile biomarkers and their mixtures at concentration ranges relevant to human breath, parts-per-billion to parts-per-trillion. Different lung cancer biomarkers evoked distinct spiking response dynamics in the honeybee antennal lobe neurons indicating that those neurons encoded biomarker-specific information. By investigating lung cancer biomarker-evoked population neuronal responses from the honeybee antennal lobe, we could classify individual human lung cancer biomarkers successfully (88% success rate). When we mixed six lung cancer biomarkers at different concentrations to create synthetic lung cancer vs. synthetic healthy breath, honeybee population neuronal responses were also able to classify those complex breath mixtures successfully (100% success rate with a leave-one-trial-out method). Finally, we used separate training and testing datasets containing responses to the synthetic lung cancer and healthy breath mixtures. We identified a simple metric, the peak response of the neuronal ensemble, with the ability to distinguish synthetic lung cancer breath from the healthy breath with 86.7% success rate. This study provides proof-of-concept results that a powerful biological gas sensor, the honeybee olfactory system, can be used to detect human lung cancer biomarkers and their complex mixtures at biological concentrations.

bioengineering↗

bioRxiv2023

A genome-wide cytotoxicity screen of Cluster F1 mycobacteriophage Girr reveals novel inhibitors of Mycobacterium smegmatis growth

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes ([~]28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function (NKF) and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic NKF proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.

microbiology↗

bioRxiv2023

Tiny Earth CURE improves student persistence in science

Course-based undergraduate research experiences (CUREs) enhance student retention in STEM, particularly among students who belong to historically excluded communities. Yet the mechanisms by which CUREs contribute to student integration and persistence are poorly understood. Utilizing the Tripartite Integration Model of Social Influence (TIMSI), this longitudinal study examines how Tiny Earth, an antibiotic-discovery CURE, impacts students scientific self-efficacy, scientific identity, endorsement of scientific community values, and intentions to persist in science. The study also explores how gains in TIMSI factors vary as a function of student demographics and course characteristics. Results of pre-and post-course measurements show that scientific self-efficacy and identity increased among students in Tiny Earth, and some student demographics and course characteristics moderated these gains. Gains in scientific self-efficacy, identity, and values correlated with gains in persistence intentions, whereas student demographics and course characteristics did not. Results of this study show that the Tiny Earth CURE enhanced students integration into the scientific community, which was linked to intentions of students of both historically underrepresented and majority groups to persist in STEM. We discuss how courses that provide opportunity to learn science skills in the context of a CURE can contribute toward enlarging and diversifying the STEM workforce.

scientific communication and education↗

bioRxiv2022

Fine-scale variability in coral bleaching and mortality during a marine heatwave

Coral bleaching and mortality can show significant spatial and taxonomic heterogeneity at local scales, highlighting the need to understand the fine-scale drivers and impacts of thermal stress. In this study, we used structure-from-motion photogrammetry to track coral bleaching, mortality, and changes in community composition during the 2019 marine heatwave in K[a]ne ohe Bay, Hawai i. We surveyed 30 shallow reef patches every 3 weeks for the duration of the bleaching event (August-December) and one year after, resulting in a total of 210 large-area, high-resolution photomosaics that enabled us to follow the fate of thousands of coral colonies through time. We also measured environmental variables such as temperature, sedimentation, depth, and wave velocity at each of these sites, and extracted estimates of habitat complexity (rugosity R and fractal dimension D) from digital elevation models to better understand their effects on patterns of bleaching and mortality. We found that up to 80% of corals experienced moderate to severe bleaching in this period, with peak bleaching occurring in October when heat stress (DHW) reached its maximum. Mortality continued to accumulate as bleaching levels dropped, driving large declines in more heat-susceptible species (77% loss of Pocillopora cover) and moderate declines in heat-tolerant species (19% and 23% for Porites compressa and Montipora capitata, respectively). Declines in live coral were accompanied by a rapid increase in algal cover across the survey sites. Spatial differences in bleaching were significantly linked to habitat complexity and coral species composition, with reefs that were dominated by Pocillopora experiencing the most severe bleaching. Mortality was also influenced by species composition, fractal dimension, and site-level differences in thermal stress. Our results show that spatial heterogeneity in the impacts of bleaching are driven by a mix of environmental variation, habitat complexity, and differences in assemblage composition.

ecology↗

bioRxiv2022

Umbilical cord blood-derived cell therapy for perinatal brain injury: A systematic review & meta-analysis of preclinical studies - Part B.

IntroductionWe previously described preclinical literature, which supports umbilical cord blood-derived cell (UCBC) therapy use for perinatal brain injury. However, efficacy of UCBCs may be influenced by different patient populations and intervention characteristics. ObjectivesTo systematically review effects of UCBCs on brain outcomes in animal models of perinatal brain injury across subgroups to better understand contribution of model type (preterm versus term), brain injury type, UCB cell type, route of administration, timing of intervention, cell dosage and number of doses. MethodsA systematic search of MEDLINE and Embase databases was performed to identify studies using UCBC therapy in animal models of perinatal brain injury. Subgroup differences were measured by chi2 test where possible. ResultsDifferential benefits of UCBCs were seen in a number of subgroup analyses including intraventricular haemorrhage (IVH) vs. hypoxia ischaemia (HI) model (apoptosis white matter (WM): chi2=4.07; P=0.04, neuroinflammation-TNF-: chi2=5.99; P=0.01), UCB-derived mesenchymal stromal cells (MSCs) vs. UCB-derived mononuclear cells (MNCs) (oligodendrocyte WM: chi2=5.01; P=0.03, neuroinflammation-TNF-: chi2=3.93; P=0.05, apoptosis grey matter (GM), astrogliosis WM) and intraventricular/intrathecal vs. systemic routes of administration (microglial activation GM: chi2=7.51; P=0.02, astrogliosis WM: chi2=12.44; P=0.002). We identified a serious risk of bias and overall low certainty of evidence. ConclusionsPreclinical evidence suggests greater efficacy for UCBCs in IVH compared to HI injury model, use of UCB-MSCs compared to UCB-MNCs, and use of local administrative routes compared to systemic routes in animal models of perinatal brain injury. Further research is needed to improve certainty of evidence found and address knowledge gaps. SIGNIFICANCE STATEMENTIn neonatal medicine there is a clear need for the development of new therapies that can provide neuroregenerative benefits for infants with brain injuries. This review offers a unique and comprehensive resource to inform the development of future preclinical and clinical studies. In part A of this review, we systematically reviewed the preclinical literature surrounding UCBCs as a therapy for perinatal brain injury. In part B of this review, we investigated the effect variables, such as UCB cell type, timing of administration and dosage, have on the efficacy of UCB-derived cell therapy in animal models of perinatal brain injury. We identified UCBCs to show greater efficacy in the brain injury model of IVH compared to HI, the use of UCB-derived MSCs compared to MNCs and the use of local administrative routes compared to systemic routes. In addition to this, we identified knowledge gaps such as the limited preclinical literature surrounding the effect of dose number and sex.

neuroscience↗

bioRxiv2022

Umbilical cord blood-derived cell therapy for perinatal brain injury: A systematic review & meta-analysis of preclinical studies - Part A

IntroductionPerinatal brain injury is a major contributor to long-term adverse neurodevelopment. There is mounting preclinical evidence for use of umbilical cord blood (UCB)-derived cell therapy as potential treatment. ObjectivesTo systematically review and analyse effects of UCB-derived cell therapy on brain outcomes in preclinical models of perinatal brain injury. MethodsMEDLINE and Embase databases were searched for relevant studies. Brain injury outcomes were extracted for meta-analysis to calculate standard mean difference (SMD) with 95% confidence interval (CI), using an inverse variance, random effects model. Outcomes were separated based on grey matter (GM) and white matter (WM) regions where applicable. Risk of bias was assessed using SYRCLE, and GRADE was used to summarise certainty of evidence. ResultsFifty-five eligible studies were included (7 large, 48 small animal models). UCB-derived cell therapy significantly improved outcomes across multiple domains, including decreased infarct size (SMD 0.53; 95%CI (0.32, 0.74), P<0.00001), apoptosis (WM, SMD 1.59; 95%CI (0.86, 2.32), P<0.0001), astrogliosis (GM, SMD 0.56; 95%CI (0.12, 1.01), P=0.01), microglial activation (WM, SMD 1.03; 95%CI (0.40, 1.66), P=0.001), neuroinflammation (TNF-, SMD 0.84; 95%CI (0.44, 1.25), P<0.0001); as well as improved neuron number (SMD 0.86; 95%CI (0.39, 1.33), P=0.0003), oligodendrocyte number (GM, SMD 3.35; 95%CI (1.00, 5.69), P=0.005) and motor function (cylinder test, SMD 0.49; 95%CI (0.23, 0.76), P=0.0003). Risk of bias was determined as serious, and overall certainty of evidence was low. ConclusionsUCB-derived cell therapy is an efficacious treatment in pre-clinical models of perinatal brain injury, however findings are limited by low certainty of evidence. SIGNIFICANCE STATEMENTPerinatal brain injury can lead to significant long-term neurodevelopmental deficits. There are limited treatment options available, and new interventions are urgently required. Through assessment of preclinical studies, this systematic review and meta-analysis shows that umbilical cord blood-derived cell therapy is an efficacious treatment for perinatal brain injury across a wide range of neuropathological and functional domains, albeit with low certainty of evidence. It also identified knowledge gaps, including that future studies should focus on non-hypoxic ischemic models, preterm models, large animal models and should explore the heterogeneity that exists in treatment protocols.

neuroscience↗

bioRxiv2022

History of previous midlife estradiol treatment permanently alters interactions of brain insulin-like growth factor-1 signaling and hippocampal estrogen synthesis to enhance cognitive aging in a rat model of menopause

Across species, including humans, elevated levels of brain estrogen receptor (ER) are associated with enhanced cognitive aging even in the absence of circulating estrogens. In rodents, short-term estrogen treatment--such as that commonly used in the menopausal transition--results in long-term increases in levels of ER in the hippocampus, leading to enhanced memory long after termination of estrogen treatment. However, mechanisms by which increased levels of brain ER enhances cognitive aging remain unclear. Here we show that in the hippocampus, insulin-like growth factor-1 (IGF-1)--which can activate ER via ligand-independent mechanisms--requires concomitant synthesis of brain-derived neuroestrogens to phosphorylate ER via MAPK signaling, ultimately resulting in enhanced memory. In a rat model of menopause involving long-term ovarian hormone deprivation, hippocampal neuroestrogen activity decreases, altering IGF-1 activity and resulting in impaired memory. However, this process is reversed by short-term estradiol treatment. Forty-days of estradiol exposure following ovariectomy results in maintenance of neuroestrogen levels that persist beyond the period of hormone treatment, allowing for continued interactions between IGF-1 and neuroestrogen signaling, elevated levels of hippocampal ER, and ultimately enhanced memory. Collectively, results demonstrate that short-term estradiol use following loss of ovarian function has long-lasting effects on hippocampal function and memory by dynamically regulating cellular mechanisms that promote activity of ER in the absence of circulating estrogens. Translational impacts of these findings suggest lasting cognitive benefits of short-term estrogen use near menopause and highlight the importance of hippocampal ER--independent from the role of circulating estrogens--in regulating memory in aging females. Significance statementDeclines in ovarian hormones following menopause coincide with increased risk of cognitive decline. Due to potential health risks, current recommendations are that menopausal estrogen therapy be limited to a few years. Long-term consequences for the brain and memory of this short-term midlife estrogen therapy are unclear. Here, in a rodent model of menopause, we determined mechanisms by which short-term midlife estrogen exposure can enhance hippocampal function and memory with cognitive benefits and molecular changes enduring long after termination of estrogen exposure. Our model indicates long-lasting benefits of maintaining hippocampal estrogen receptor function in the absence of ongoing estrogen exposure and suggests potential strategies for combating age-related cognitive decline.

neuroscience↗

bioRxiv2021

Preclinical studies for plant-based oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice with transgenic tobacco seeds expressing human GAA (tobrhGAA)

Genetic deficiency of acid -glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe disease (PD) encompassing at least four clinical subtypes of varying severity (infantile; childhood, juvenile and late onset). Our objective is to develop an innovative and affordable approach for enzyme replacement therapy (ERT) via oral administration (Oral-ERT) to maintain a sustained, therapeutic level of enzyme on a daily basis to improve efficacy of treatment and quality of life for people living with Pompe disease. A consensus at a 2019 US Acid Maltase Deficiency (AMDA) conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment of Pompe disease. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. Previously, we have shown that a lysate from transgenic tobacco seeds expressing human GAA (tobrhGAA) was enzymatically active and can correct enzyme deficiency in cultured PD cells and in adult lymphocytes of Pompe patients and in vivo in disease-relevant tissues in GAA knockout (KO) mice when administered IP. We have extended these pre-clinical studies in PD knockout (KO) mice with ground tobrhGAA seeds that supports proof-of-concept for Oral-ERT for future clinical trials. Briefly in GAA KO mice, Oral-ERT with ground tobrhGAA seeds showed significant reversal of fore-limb and hind-limb muscle weakness, increased motor coordination/balance/strength and mobility, improved spontaneous learning, increased GAA baseline activity in tissues, reduced glycogen in tissues and negible serum titers to GAA. Pharmacokinetics showed maximum serum GAA concentration (Cs) at 8-10 hr and peak urine excretion at 10-12 hr. The tobrhGAA was taken up in PD fibroblast, lymphoid and myoblast cells. Enzyme kinetics compared favorably or superior to placental hGAA, plus alglucosidase alfa or other rhGAAs for Km, Vmax, pH optima, thermal heat stability and IC50 for inhibitors. The tobrhGAA in seeds was extremely stable stored for 15 years at room temperature. NGS-genome sequencing of the tobrhGAA and wild-type plants and RNA expression profiles was performed and will be posted on our website. Thus, Oral-ERT with ground tobrhGAA seeds is an innovative approach that overcomes some of the challenges of alglucosidase alfa-ERT and provides a more effective, safe and significantly less expensive treatment.

genetics↗

bioRxiv2021

Manual curation and phylogenetic analysis of chitinase family genes in the Asian citrus psyllid, Diaphorina citri

Chitinases are enzymes that digest the polysaccharide polymer chitin. Chitin is a major component of insect exoskeletons, which must be replaced multiple times during growth and development in a process known as molting. Insect genomes usually have multiple chitinase genes, some of which are required for molting and some whose functions are still unknown. Since knockdown of the chitinases required for molting causes high levels of lethality, chitinase genes have drawn interest as targets for RNAi-based pest control methods. The Asian citrus psyllid, Diaphorina citri, carries the bacterium that causes Huanglongbing, also known as citrus greening disease, which is devastating the citrus industry worldwide. We have identified and annotated 12 chitinase family genes from D. citri as part of a community effort to create high quality gene models to facilitate the design of interdictory molecules for pest control. Using predicted protein domain content and phylogenetic analysis, we categorized the D. citri chitinases according to a previously established classification scheme and re-evaluated the classification of chitinases in other hemipterans. In addition to chitinases belonging to known groups, we identified a novel class of chitinases present in D. citri and several related hemipterans that appears to be the result of horizontal gene transfer.

bioinformatics↗

bioRxiv2021

Optimizing Polymerase Chain Reaction (PCR) using Machine Learning

Despite substantial standardization, polymerase chain reaction (PCR) experiments frequently fail. Troubleshooting failed PCRs can be costly in both time and money. Using a crowdsourced data set spanning 290 real PCRs from six active research laboratories, we investigate the degree to which PCR success rates can be improved by machine learning. While human designed PCRs succeed at a rate of 55-63%, we find that a machine learning model can accurately predict reaction outcome 81% of the time. We validate this level of improvement by then using the model to guide the design and predict the outcome of 39 new PCR experiments. In addition to improving outcomes, the model identifies 15 features of PCRs that researchers did not optimize well compared to the learned model. These results suggest that PCR success rates can easily be improved by 17-26%, potentially saving millions of dollars and thousands of hours of researcher time each year across the scientific community. Other common laboratory methods may benefit from similar data-driven optimization effort.

bioinformatics↗

bioRxiv2021

Genetic compensation of triacylglycerol biosynthesis in the green microalga Chlamydomonas reinhardtii

Genetic compensation through transcriptional adaptation has been proposed to explain phenotypic differences between gene knockouts and knockdowns. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in Chlamydomonas reinhardtii, it was discovered that knockout of certain genes catalyzing rate-limiting steps of TAG biosynthesis, type-2 diacylglycerol acyltransferase genes (DGTTs), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, e.g., CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a {Delta}vtc1 mutant, in which PDAT was up-regulated under stress. Knockout of PDAT in the {Delta}vtc1 background induced a 12.8-fold upregulation of DGTT1 and a 272.3% increase in TAG content in {Delta}vtc1/pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress with a mechanism distinct from that found in plants. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis, and provides guidance for future genetic engineering and mutant characterization efforts.

plant biology↗

bioRxiv2021

An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces sterile immunity and prevents transmission of SARS-CoV-2 between cats

Early in the global SARS-CoV-2 pandemic concerns were raised regarding infection of other animal hosts and whether these could play a significant role in the viral epidemiology. Infection of animals could be detrimental by causing clinical disease but also of concern if they become a viral reservoir allowing further mutations, plus having the potential to infect other animals or humans. The first reported animals to be infected both under experimental conditions and from anecdotal field evidence were cats described in China early in 2020. Given the concerns this finding raised and the close contacts between humans and cats, we aimed to determine whether a vaccine candidate could be developed that was suitable for use in multiple susceptible animal species and whether this vaccine could reduce infection of cats in addition to preventing spread to other cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus (VEEV), known to be safe and efficacious for use in a variety of animals, expressing a stabilised Spike antigen, could induce neutralising antibody titers in guinea pigs and cats. After two intramuscular vaccinations, virus neutralising antibodies were detected in the respiratory tract of the guinea pigs and a cell mediated immune response was induced. The design of the SARS-CoV-2 antigen was shown to be critical in developing a strong neutralising antibody response. Vaccination of cats was able to induce a serum neutralising antibody response which lasted for the course of the experiment. Interestingly, in contrast to control animals, infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after challenge. Moreover, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit virus. The results show that the RP vaccine induces sterile immunity preventing SARS-CoV-2 infection and transmission. This data suggests that this RP vaccine could be a multi-species vaccine useful for preventing spread to and between other animals should that approach be required.

microbiology↗

bioRxiv2020

Segmentation pathway genes in the Asian citrus psyllid, Diaphorina citri

Insects have a segmented body plan that is established during embryogenesis when the anterior-posterior (A-P) axis is divided into repeated units by a cascade of gene expression. The cascade is initiated by protein gradients created by translation of maternally provided mRNAs, localized at the anterior and posterior poles of the embryo. Particular combinations of these proteins activate specific gap genes to divide the embryo into distinct regions along the A-P axis. Gap genes then activate pair-rule genes, which are usually expressed in part of every other segment. The pair-rule genes, in turn, activate expression of segment polarity genes in a portion of each segment. The segmentation genes are generally conserved among insects, although there is considerable variation in how they are deployed. We annotated 24 segmentation gene homologs in the Asian citrus psyllid, Diaphorina citri. We identified most of the genes that were expected to be present based on known phylogenetic distribution. Two exceptions were eagle and invected, which are present in at least some hemipterans, but were not identified in D. citri. Many of these genes are likely to be essential for D. citri development and thus may be useful targets for pest control methods.

genomics↗