Strain Background Research Articles

Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors. Infection with enteroviruses, including poliovirus and coxsackievirus, such as coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALS due to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathological phenotypes closely resemble ALS. However, a causal relationship has not yet been affirmed.MethodsWild-type C57BL/6J and G85R mutant superoxide dismutase 1 (SOD1G85R) ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting at week 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10, week 20 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression.ResultsWe found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted in early onset and progressive motor dysfunction, and shortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeks PI). Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TAR DNA-binding protein 43 (TDP-43) pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, we discovered that early (day 1) but not late (day 15) administration of ribavirin could rescue ALS-like neuropathology and symptoms induced by CVB3 infection.ConclusionsOur study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.

A TLR/CD44 axis regulates T cell trafficking in experimental and human multiple sclerosis

SummaryIn the pathogenesis of autoimmune disorders, the modulation of leukocytes′ trafficking plays a central role, still poorly understood. Here, we focused on the effect of TLR2 ligands in trafficking of T helper cells through reshuffling of CD44 isoforms repertoire. Concurrently, strain background and TLR2 haplotype affected Wnt/β-catenin signaling pathway and expression of splicing factors. During EAE, mCD44v9-v10 was specifically enriched in the forebrain and showed an increased ability to bind stably to osteopontin. Similarly, we observed that hCD44v7 was highly enriched in cells of cerebrospinal fluid from MS patients with active lesions. Moreover, TLRs engagement modulated the composition of CD44 variants also in human T helper cells, supporting the hypothesis that pathogens or commensals, through TLRs, in turn modulate the repertoire of CD44 isoforms, thereby controlling the distribution of lesions in the CNS. The interference with this mechanism(s) represents a potential tool for prevention and treatment of autoimmune relapses and exacerbations.

A Modified Fluctuation Assay with a CAN1 Reporter in Yeast.

Understanding the generation of mutations is fundamental to understanding evolution and genetic disease; however, the rarity of such events makes experimentally identifying them difficult. Mutation accumulation (MA) methods have been widely used. MA lines require serial bottlenecks to fix de novo mutations, followed by whole-genome sequencing. While powerful, this method is not suitable for exploring mutation variation among different genotypes due to its poor scalability with cost and labor. Alternatively, fluctuation assays estimate mutation rate in microorganisms by utilizing a reporter gene, in which Loss-of-function (LOF) mutations can be selected for using drugs toxic to cells containing the WT allele. Traditional fluctuation assays can estimate mutation rates but not their base change compositions. Here, we describe a new protocol that adapts traditional fluctuation assay using CAN1 reporter gene in Saccharomyces cerevisiae , followed by pooled sequencing methods, to identify both the rate and spectra of mutations in different strain backgrounds.

COVID-19 vaccinations and counselling: a mixed-methods survey of Australian general practice in July 2021.

GPs are integral to the COVID-19 vaccination rollout, providing education and administering vaccines. We sought to describe how counselling relating to COVID-19 vaccination was impacting Australian general practice consultations. We conducted an online, mixed-methods, cross-sectional survey of Australian community-based primary care from 7 to 15 July 2021. This survey, number 15 in a series of recurrent cross-sectional surveys conducted over a 14-month period, explored how counselling relating to COVID-19 vaccination was impacting general practice consultations, through multiple selection and open text responses; it also included questions on respondent and practice characteristics, and pandemic-related stress and strain. We calculated descriptive statistics for quantitative variables, and analysed free-text responses using an inductive content analysis approach. We received 73 responses (72 GPs) across all states/territories. Discussions with patients about COVID-19 vaccines and vaccination were common, increasing the duration of routine consultations by 6min on average (s.d. 2.9). Respondents described the impact of the resulting time pressures, and the stress and challenges of participating in COVID-19 vaccine communication and administration. Although our results are illuminating, they are limited by the small sample, with some different characteristics from national estimates, an uncertain response rate and the inability to pilot the survey prior to distribution. The significant impact on general practice consultation from COVID-19 vaccine counselling is on a background of ongoing pandemic-related stress and strain. With a strong track record of population vaccination, GPs are well-placed to deliver COVID-19 immunisations to the Australian population. However, they must be represented in planning and coordination, to reduce the overall burden on primary care.

Conserved FimH mutations in the global Escherichia coli ST131 multi-drug resistant lineage weaken interdomain interactions and alter adhesin function

The binding of the type 1 fimbrial adhesin FimH to mannosylated receptors is allosterically regulated to enhance the fitness of uropathogenic Escherichia coli (UPEC) during urinary tract infection (UTI). Mutations in the two FimH domains (pilin and lectin) located outside the mannose binding pocket have been shown to influence mannose binding affinity, yet the details of the allostery mechanism are not fully elucidated. Here we characterised different FimH conformational states (termed low-affinity tense and high-affinity relaxed conformations) of natural FimH variants using molecular dynamics (MD) simulation techniques and report key structural dynamics differences between them. The clinically dominant FimH30 variant from the pandemic multidrug resistant E. coli ST131 lineage contains an R166H mutation that weakens FimH interdomain interactions and allows enhanced mannose interactions with pre-existing high-affinity relaxed conformations. When expressed in an isogenic ST131 strain background, FimH30 mediated high human cell adhesion and invasion, and enhanced biofilm formation over other variants. Collectively, our computational and experimental findings support a model of FimH protein allostery that is mediated by shifts in the pre-existing conformational equilibrium of FimH, additional to the sequential step-wise process of structural perturbations transmitted from one site to another within the protein. Importantly, it is the first study to shed light into how natural mutations in a clinically dominant FimH variant influence the protein’s conformational landscape optimising its function for ST131 fitness at intestinal and extraintestinal niches.

Fructooligosaccharides (FOS) significantly increased the relative abundance of intestinal B. pseudolongum in mice with different genotypes

Fructooligosaccharides (FOS) promote the proliferation of Bifidobacterium, especially Bifidobacterium pseudolongum in C57BL/6J mice. However, the response of intestinal microbes to FOS is influenced by host genotypes. Therefore, we compared the intestinal microbiota of four commonly used mice before and after FOS intervention, including C57BL/6J, BALB/c, Institute Cancer Research (ICR), and Kunming (KM) mice. The intestinal microbiota of the four genotypes exhibited similarities in composition but differences in relative abundance. Bifidobacterium was significantly increased to different degrees in the four genotypes of mice after FOS intervention, and Akkermansia and Bacteroides were also significantly increased in BALB/c and KM mice. Lactobacillus and Alistipes levels were unchanged or decreased. Within the genus Bifidobacterium, B. pseudolongum was the dominant species in the four genotypes of mice and proliferated significantly after FOS intervention, with dramatic proliferation in C57BL/6J mice (9.49%). Furthermore, eight strains of B. pseudolongum were screened from the feces of mice with four genotypes, and there was a great difference in the ability and manner of utilizing FOS among the strains. The strains from C57BL/6J mice exhibited the strongest utilization of 1-kestose (GF2), whereas other strains could utilize both GF2 and nistose (GF3) weakly. The gut microbial analysis of mice with different genotypes complemented our previous studies. The results provided the background strains of the different mouse genotypes and suggested a correlation between the utilization ability and the response of the strains to FOS. Further studies on the utilization ability of strains and competition in the intestine will contribute to the understanding of the mechanisms of the intestinal microbial response to diet.

Identification of Virulence Associated Region during Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus during Attenuation In Vitro: Complex Question with Different Strain Backgrounds.

Highly pathogenic porcine reproductive and respiratory syndrome virus PRRSV (HP-PRRSV) was one of the most devastating diseases of the pig industry, among various strategies, vaccination was one of the most useful tools for PRRS control. Attenuated live vaccine was used worldwide, however, the genetic basis of HP-PRRSV virulence change during attenuation remain to be determined. Here, to identify virulence associated regions of HP-PRRSV during attenuation in vitro, six full-length infectious cDNA clones with interchanges of 5′UTR + ORF1a, ORF1b, and ORF2-7 + 3′UTR regions between HP-PRRSV strain HuN4-F5 and its attenuated vaccine strain HuN4-F112 were generated, and chimeric viruses were rescued. Piglets were inoculated with chimeric viruses and their parental viruses, and rectal temperature were recorded daily, and serum were collected for future experiments. Our results showed that ORF1a played an important role on virus replication, cytokine response and lung damage, the exchange of ORF1b and ORF2-7 in different backbone led to different exhibition on virus replication in vivo/vitro and cytokine response. Among 9 PRRSV attenuated series, consistent amino acid changes during PRRSV attenuation were found in NSP4, NSP9, GP2, E, GP3 and GP4. Our study provides a fundamental data for the investigation of PRRSV attenuation, the different results of the virulence change among different studies indicated that different mechanisms might be used during PRRSV virulence enhancement in vivo and attenuation in vitro.

Fitness costs associated with chlorantraniliprole resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) strains with different genetic backgrounds.

Spodoptera frugiperda (J.E. Smith) is a difficult pest to manage mainly because of its resistance to insecticides and Bt proteins. We evaluated fitness costs of S. frugiperda resistant strains to diamide insecticides with different genetic backgrounds aiming to highlight the importance of using isogenic strains. We established a near-isogenic strain of S. frugiperda resistant to diamides (Iso-RR), using a chlorantraniliprole resistant strain (RR) selected from a field-collected population and a susceptible reference strain (SS). Fitness costs were assayed using strains with close-related genetic backgrounds (Iso-RR and SS) and strains with distant-related genetic backgrounds (RR and SS). No fitness cost associated with chlorantraniliprole resistance in S. frugiperda was observed using the Iso-RR strain, based on life history traits. The only parameter that differs between Iso-RR and SS strains was the mean length of a generation (T), whereas the Iso-RR strain presented T=35.8 and SS strain showed T=34.6. On the other hand, a significant fitness cost was detected using the RR strain. All population growth parameters differ between RR and SS strains. Based on the intrinsic rate of population increase (rm ) parameter, the relative fitness estimated was 1.02 for the Iso-RR strain and 0.64 for the RR strain. The genetic background of the resistant strains alters fitness cost outcomes. The RR strain showed fitness costs associated with resistance, but the Iso-RR did not. Our work supports the decision-making process of resistance management programs and adds to the growing body of research that enlightens the importance of strain genetics in fitness cost experiments.

A novel CRISPRi platform to study the role of essential genes in antifungal drug-resistant Candida albicans isolates

With the emergence of antifungal resistant Candida albicans strains, the need for new antifungal drugs is critical in combating this fungal pathogen. Investigating essential genes in C. albicans is a vital step in characterizing putative antifungal drug targets. As some of these essential genes are conserved between fungal organisms, developed therapies targeting these genes have the potential to be broad range antifungals. In order to study these essential genes, classical genetic knockout or CRISPR-based approaches cannot be used as disrupting essential genes leads to lethality in the organism. Fortunately, a variation of the CRISPR system (CRISPR interference or CRISPRi) exists that enables precise transcriptional repression of the gene of interest without introducing genetic mutations. CRISPRi utilizes an endonuclease dead Cas9 protein that can be targeted to a precise location but lacks the ability to create a double-stranded break. The binding of the dCas9 protein to DNA prevents the binding of RNA polymerase to the promoter through steric hindrance thereby reducing expression. We recently published the novel use of this technology in C. albicans and are currently working on expanding this technology to large scale repression of essential genes. Through the construction of an essential gene CRISPRi-sgRNA library, we can begin to study the function of essential genes under different conditions and identify genes that are involved in critical processes such as drug tolerance in antifungal resistant background strains. These genes can ultimately be characterized as putative targets for novel antifungal drug development, or targeted as a means to sensitize drug-resistant strains to antifungal treatment.

Identification of cyclin D1 as a major modulator of 3-nitropropionic acid-induced striatal neurodegeneration

Mitochondria dysfunction occurs in the aging brain as well as in several neurodegenerative disorders and predisposes neuronal cells to enhanced sensitivity to neurotoxins. 3-nitropropionic acid (3-NP) is a naturally occurring plant and fungal neurotoxin that causes neurodegeneration predominantly in the striatum by irreversibly inhibiting the tricarboxylic acid respiratory chain enzyme, succinate dehydrogenase (SDH), the main constituent of the mitochondria respiratory chain complex II. Significantly, although 3-NP-induced inhibition of SDH occurs in all brain regions, neurodegeneration occurs primarily and almost exclusively in the striatum for reasons still not understood.In rodents, 3-NP-induced striatal neurodegeneration depends on the strain background suggesting that genetic differences among genotypes modulate toxicant variability and mechanisms that underlie 3-NP-induced neuronal cell death. Using the large BXD family of recombinant inbred (RI) strains we demonstrate that variants in Ccnd1 - the gene encoding cyclin D1 - of the DBA/2 J parent underlie the resistance to 3-NP-induced striatal neurodegeneration. In contrast, the Ccnd1 variant inherited from the widely used C57BL/6 J parental strain confers sensitivity. Given that cellular stress triggers induction of cyclin D1 expression followed by cell-cycle re-entry and consequent neuronal cell death, we sought to determine if the C57BL/6 J and DBA/2 J Ccnd1 variants are differentially modulated in response to 3-NP. We confirm that 3-NP induces cyclin D1 expression in striatal neuronal cells of C57BL/6 J, but this response is blunted in the DBA/2 J. We further show that striatal-specific alternative processing of a highly conserved 3′UTR negative regulatory region of Ccnd1 co-segregates with the C57BL/6 J parental Ccnd1 allele in BXD strains and that its differential processing accounts for sensitivity or resistance to 3-NP. Our results indicate that naturally occurring Ccnd1 variants may play a role in the variability observed in neurodegenerative disorders involving mitochondria complex II dysfunction and point to cyclin D1 as a possible therapeutic target.

Assessment of late gadolinium enhancement-negative chronic total occlusion by longitudinal strain analysis using cardiac magnetic resonance imaging

Strain analysis has become commonly used in clinical practice in various heart diseases. To explore whether late gadolinium enhancement (LGE)-negative areas with coronary artery chronic total occlusion (CTO) appear normal when analyzed for longitudinal strain using cardiac magnetic resonance (CMR) imaging. A total of 16 patients and 31 healthy controls who underwent 1.5-T MR at our hospital between January 2015 and July 2017 were included in the study. The LGE-CMR of patients with CTO was negative. Left ventricular functional parameters, segmental longitudinal strain/strain rate, and perfusion parameters were measured using CVI42 software. For myocardial segments supplied by CTO vessels, systolic longitudinal strain rate (SLSR)was significantly lower than that of healthy controls, and diastolic longitudinal strain rate (DLSR) was significantly higher (1.19 1/s vs. 1.02 1/s; P = 0.018). Moreover, longitudinal strain (LS), SLSR, and DLSR did not differ between good and poor collateral circulation. Perfusion index of CTO territory segments was lower than non-CTO territory segments (0.20 vs. 0.22; P = 0.027). No correlation was found between longitudinal strain parameters and perfusion parameters. Although LGE-CMR was negative in patients with CTO, the myocardial SLSR of CTO territory segments was significantly lower than that of healthy controls.

APOE variant in the receptor binding domain confers cognitive resilience to familial Alzheimer's mutations and cell-type specific gene expression changes in the hippocampus.

Cognitive resilience to familial Alzheimer's Disease (FAD) is a phenomenon whereby cognitive functioning is better than predicted based on highly penetrant mutations in APP and PS1. This resilience is likely mediated by unidentified genetic factors, and we hypothesize that these factors provide key targets for treatment and prevention of AD. We previously discovered that the most commonly used background strain in AD model research (C57BL/6J, B6) is resilient to AD-related cognitive decline. Using genetic mapping, we determined that cognitive resilience is associated with a variant in the Apoe receptor binding domain and utilized CRISPR to generate a novel Apoe knockin mouse (Apoe KIB6/E163D ). Contextual fear conditioning (CFC), tau pathology, and single-nuclear RNAseq hippocampi analyses were performed in male and female Apoe KIB6/E163D x 5XFAD progeny that differed by only a single variant in Apoe leading to the E163D base pair change in the protein. We found that, indeed, cognitive resilience to the 5XFAD transgene was conferred in ApoeB6/B6 mice compared to Apoe KIB6/E163D mice tested on CFC. Although minimal differences in hyperphosphorylated tau abundance were observed between 5XFAD-ApoeWT/WT mice compared to 5XFAD-Apoe KIB6/E163D , we uncovered robust differences in the transcriptomes of hippocampal excitatory and inhibitory neurons and microglia using single-nuclear sequencing. Gene enrichment and pathway analyses of differentially expressed genes suggest that the 5XFAD-ApoeB6/B6 genotype promotes cation transport and suppression of amyloid fibril formation in excitatory neurons, promotes genes involved in ribosome assembly and protein translation in inhibitory neurons, and enhances gene expression associated with stress response in microglia. Our results suggest that 5XFAD-ApoeB6/B6 homozygotes exhibit cognitive resilience to causal FAD mutations that involve dynamic regulation of gene networks across multiple cell types, emphasizing the need for well-aligned cell culture models of cognitive resilience to validate targets and screen resilience-based interventions. Future work leveraging in vivo electrophysiology and imaging will establish mechanisms whereby Apoe variants alter neural and synaptic function. Understanding the relationship between protective genetic factors and cognitive resilience are crucial for determining mechanisms of resilience and uncovering novel targets for intervention.

Establishment of a Reproducible Ischemic Stroke Model in Nestin-GFP Mice with High Survival Rates.

An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP+ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP+ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP+ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.

C57BL/6 Background Attenuates mHTT Toxicity in the Striatum of YAC128 Mice.

Mouse models are frequently used to study Huntington’s disease (HD). The onset and severity of neuronal and behavioral pathologies vary greatly between HD mouse models, which results from different huntingtin expression levels and different CAG repeat length. HD pathology appears to depend also on the strain background of mouse models. Thus, behavioral deficits of HD mice are more severe in the FVB than in the C57BL/6 background. Alterations in medium spiny neuron (MSN) morphology and function have been well documented in young YAC128 mice in the FVB background. Here, we tested the relevance of strain background for mutant huntingtin (mHTT) toxicity on the cellular level by investigating HD pathologies in YAC128 mice in the C57BL/6 background (YAC128/BL6). Morphology, spine density, synapse function and membrane properties were not or only subtly altered in MSNs of 12-month-old YAC128/BL6 mice. Despite the mild cellular phenotype, YAC128/BL6 mice showed deficits in motor performance. More pronounced alterations in MSN function were found in the HdhQ150 mouse model in the C57BL/6 background (HdhQ150/BL6). Consistent with the differences in HD pathology, the number of inclusion bodies was considerably lower in YAC128/BL6 mice than HdhQ150/BL6 mice. This study highlights the relevance of strain background for mHTT toxicity in HD mouse models.

Interplay between IncF plasmids and topoisomerase mutations conferring quinolone resistance in the Escherichia coli ST131 clone: stability and resistance evolution.

The Escherichia coli ST131 H30-Rx subclone vehicles CTX-M-15 plasmids and mutations in gyrA and parC conferring multidrug resistance successfully in the clinical setting. The aim of this study was (1) to investigate the relationship of specific topoisomerase mutations on the stability of IncF (CTX-M producing) plasmids using isogenic E. coli mutants and (2) to investigate the impact of the IncF-type plasmids present in the E. coli clone ST131 on the evolution of quinolone resistance. E. coli ATCC 25922 (background strain) and derived mutants encoding specific QRDR substitutions were used. Also, NGS-characterized IncFIA and IncFIB plasmids (encoding CTX-M genes) were included. Plasmid stability was evaluated by sequential dilutions into Luria broth medium without antibiotics for 7days. Mutant frequency to ciprofloxacin was also evaluated. Moderate differences in the IncF plasmids stability were observed among E. coli ATCC 25922 and isogenic mutants. Under our experimental conditions, the fluctuation of bacteria harboring plasmids was less than 0.5-log(10) in all cases. In the mutant frequency tests, it was observed that the presence of these IncF plasmids increased this value significantly (10-1000-fold). Quinolone resistance substitutions in gyrA or parC genes, frequently found associated with E. coli clone ST131, do not modify the stability of ST131-associated IncFIA and IncFIB plasmids under in vitro conditions. IncF-type plasmids present in E. coli clone ST131 facilitate the selection of resistance to quinolones. These results are consistent with the clinical scenario in which the combination of resistance to quinolones and beta-lactams is highly frequent in the E. coli clone ST131.

Introduction of NADH-dependent nitrate assimilation in Synechococcus sp. PCC 7002 improves photosynthetic production of 2-methyl-1-butanol and isobutanol

Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. In this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products.

UBB+1 reduces amyloid-β cytotoxicity by activation of autophagy in yeast.

UBB+1 is a mutated version of ubiquitin B peptide caused by a transcriptional frameshift due to the RNA polymerase II “slippage”. The accumulation of UBB+1 has been linked to ubiquitin-proteasome system (UPS) dysfunction and neurodegeneration. Alzheimer’s disease (AD) is defined as a progressive neurodegeneration and aggregation of amyloid-β peptides (Aβ) is a prominent neuropathological feature of AD. In our previous study, we found that yeast cells expressing UBB+1 at lower level display an increased resistance to cellular stresses under conditions of chronological aging. In order to examine the molecular mechanisms behind, here we performed genome-wide transcriptional analyses and molecular/cellular biology assays. We found that low UBB+1 expression activated the autophagy pathway, increased vacuolar activity, and promoted transport of autophagic marker ATG8p into vacuole. Furthermore, we introduced low UBB+1 expression to our humanized yeast AD models, that constitutively express Aβ42 and Aβ40 peptide, respectively. The co-expression of UBB+1 with Aβ42 or Aβ40 peptide led to reduced intracellular Aβ levels, ameliorated viability, and increased chronological life span. In an autophagy deficient background strain (atg1Δ), intracellular Aβ levels were not affected by UBB+1 expression. Our findings offer insights for reducing intracellular Aβ toxicity via autophagy-dependent cellular pathways under low level of UBB+1 expression.

Waving Through the Window: A Model of Volitional Social Interaction in Female Mice

BackgroundMouse models of social behavior fail to account for volitional aspects of social interaction, and current neurobiological investigation of social behavior is performed almost exclusively using C57BL/6J mice, the background strain of most transgenic mice. Here, we introduce a mouse model of operant social self-administration and choice, using a custom-made apparatus. MethodsFirst, we trained adolescent and adult female C57BL/6J and CD1 mice to self-administer palatable food pellets and then to lever press under increasing fixed-ratio response requirements for access to an age-matched female social partner. Next, we tested their motivation to seek social interaction using a progressive ratio reinforcement schedule, relapse to social seeking after social isolation, and choice between palatable food versus social interaction. We also tested social conditioned place preference in adult female CD1 and C57BL/6J mice. ResultsAdolescent and adult female mice of both strains showed similar rates of food self-administration. In contrast, CD1 mice demonstrated significantly stronger social self-administration than C57BL/6J mice under both reinforcement schedules. CD1 but not C57BL/6J mice demonstrated robust social seeking after social isolation. In the choice task, CD1 mice preferred social interaction, whereas C57BL/6J mice preferred food. CD1 but not C57BL/6J mice demonstrated robust social conditioned place preference. The strain differences were age independent. ConclusionsOur data show that CD1 mice are a better strain for studying female social reward learning. Our mouse operant social model provides a tool for research on neurobiological substrates of female social reward and disruption of social reward in psychiatric disorders using mouse-specific genetic tools.

Aurora kinase B inhibits aurora kinase A to control maternal mRNA translation in mouse oocytes.

Mammalian oocytes are transcriptionally quiescent, and meiosis and early embryonic divisions rely on translation of stored maternal mRNAs. Activation of these mRNAs is mediated by polyadenylation. Cytoplasmic polyadenylation binding element 1 (CPEB1) regulates mRNA polyadenylation. One message is aurora kinase C (Aurkc), encoding a protein that regulates chromosome segregation. We previously demonstrated that AURKC levels are upregulated in oocytes lacking aurora kinase B (AURKB), and this upregulation caused increased aneuploidy rates, a role we investigate here. Using genetic and pharmacologic approaches, we found that AURKB negatively regulates CPEB1-dependent translation of many messages. To determine why translation is increased, we evaluated aurora kinase A (AURKA), a kinase that activates CPEB1 in other organisms. We find that AURKA activity is increased in Aurkb knockout mouse oocytes and demonstrate that this increase drives the excess translation. Importantly, removal of one copy of Aurka from the Aurkb knockout strain background reduces aneuploidy rates. This study demonstrates that AURKA is required for CPEB1-dependent translation, and it describes a new AURKB requirement to maintain translation levels through AURKA, a function crucial to generating euploid eggs.

Metabolic engineering Yarrowia lipolytica for a dual biocatalytic system to produce fatty acid ethyl esters from renewable feedstock in situ and in one pot.

Given the grave concerns over increasing consumption of petroleum resources and dramatic environmental changes arising from carbon dioxide emissions worldwide, microbial biosynthesis of fatty acid ethyl ester (FAEE) biofuels as renewable and sustainable replacements for petroleum-based fuels has attracted much attention. As one of the most important microbial chassis, the nonconventional oleaginous yeast Yarrowia lipolytica has emerged as a paradigm organism for the production of several advanced biofuels and chemicals. Here, we report the engineering of Y. lipolytica for use as an efficient dual biocatalytic system for in situ and one-pot production of FAEEs from renewable feedstock. Compared to glucose with 5.7% (w/w) conversion rate to FAEEs, sunflower seed oil in the culture medium was efficiently used to generate FAEEs with 84% (w/w) conversion rate to FAEEs by the engineered Y. lipolytica strain GQY20 that demonstrates an optimized intercellular heterologous FAEE synthesis pathway. In particular, the titer of extracellular FAEEs from sunflower seed oil reached 9.9 g/L, 10.9-fold higher than that with glucose as a carbon source. An efficient dual biocatalytic system combining ex vivo and strengthened in vitro FAEE production routes was constructed by overexpression of a lipase (Lip2) variant in the background strain GQY20, which further increased FAEEs levels to 13.5 g/L. Notably, deleting the ethanol metabolism pathway had minimal impact on FAEE production. Finally, waste cooking oil, a low-cost oil-based substance, was used as a carbon source for FAEE production in the Y. lipolytica dual biocatalytic system, resulting in production of 12.5 g/L FAEEs. Thus, the developed system represents a promising green and sustainable process for efficient biodiesel production. KEY POINTS: • FAEEs were produced by engineered Yarrowia lipolytica. • A Lip2 variant was overexpressed in the yeast to create a dual biocatalytic system. • Waste cooking oil as a substrate resulted in a high titer of 12.5 g/L FAEEs.