title: How does the mode of evolutionary divergence affect reproductive isolation?

abstract: When divergent populations interbreed, the outcome will be affected by the genomic and phenotypic differences that they have accumulated. In this way, the mode of evolutionary divergence between populations may have predictable consequences for the fitness of their hybrids, and so for the progress of speciation. To investigate these connections, we present a new analysis of hybridization under Fishers geometric model. Unlike previous such analyses, we derive results using combinatorics, and so we make few assumptions about the additive and dominance effects that differentiate the hybridizing populations. Results show that the strength and form of postzygotic reproductive isolation (RI) depend on just two properties of the fixed effects, which we call the "total amount" and "net effect" of evolutionary change. It follows that identical patterns of RI can arise in different ways. Nevertheless, the key quantities do contain information about the history of divergence, and the dominance and additive effects contain complementary information. Our results also clarify the roles of large- and small-effect substitutions in generating RI. For example, when hybridizing populations are locally adapted, populations that adapted with a few large-effect mutations will show more intrinsic RI than populations that followed the same phenotypic trajectory, but with many small mutations.


title: Multi-omics profiling, in vitro and in vivo enhancer assays dissect the cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

abstract:
North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disease affecting macular development. With the identification of non-coding single nucleotide variants (SNVs) near PRDM13 and duplications overlapping a DNase I hypersensitive site (DHS) near PRDM13 or IRX1 as its underlying genetic cause, we hypothesize that NCMD is a retinal enhanceropathy. Here we aim to provide insight into the cis-regulatory mechanisms of NCMD by integrating multi-omics profiling of human retina with in vitro and in vivo enhancer assays.

First, we established RegRet (http://genome.ucsc.edu/s/stvdsomp/RegRet), a genome-wide multi-omics retinal database. Next, UMI-4C profiling was performed on adult human retina to fine-map chromatin interactions of cis-regulatory elements (CREs) with the PRDM13 and IRX1 promoters. Multi-omics analysis including the UMI-4C data revealed sixteen candidate CREs (cCREs), seven for the PRDM13 and nine for the IRX1 region. Subsequently, the activity of cCREs was investigated by in vitro luciferase assays and by in vivo enhancer assays in Xenopus laevis and tropicalis. Four cCREs showed in vivo eye- and brain-specific activity in Xenopus. Furthermore, we expanded the genetic architecture of NCMD with two novel non-coding SNVs (V15, V16) with a likely effect on PRDM13 regulation. Luciferase assays showed that the non-coding SNVs that are located in the two hotspot regions of PRDM13 have an effect on cCRE activity. Interestingly, cCRE4 in which V16 is located was shown to interact with the PRDM13 promoter and demonstrated in vivo activity in Xenopus. This cCRE is active at a specific developmental stage (d103) compatible with the timepoint when retinal progenitor cells of the central retina exit mitosis. Mining of single-cell (sc) transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to emerge and begin to synapse with retinal ganglion cells. This supports the hypothesis that altered PRDM13 or IRX1 expression impairs synaptic interactions between amacrine and ganglion cells during retinogenesis. Overall, this study gained insight into the cis-regulatory mechanisms of NCMD and supports that NCMD is a retinal enhanceropathy.

category: genomics


title: Experience does not change the importance of wind support for migratory route selection by a soaring bird

abstract: Understanding how animals develop their ability to perform complex behaviors is fundamental to understanding their fitness and plasticity under global change. Migration can be a costly behavior in terms of time, energy, and mortality, and minimizing these costs is key to survival and reproduction. Thermal soaring birds rely on airflow to offset their energetic costs of flight. Their migratory routes are a record of movement decisions to negotiate the atmospheric environment and achieve efficiency, for example by flying in prevailing winds. We expected that, as individuals gain more experience, these movement decision will also increasingly favor the best thermal uplift conditions, which allow them to fly with minimal energy expenditure. We quantified how route choice during autumn migration of young European honey buzzards (Pernis apivorus) was adjusted to wind support and uplift over up to four years of migration and compared this to the choices of adult birds. We found that wind support was important in all migrations. However, we did not find an increase in the use of thermal uplifts. The young birds varied their responses to airflow, yet they may require more than four years to attain a stable, optimal behavior.

category: animal behavior and cognition


title: A panoramic perspective on human phosphosites

abstract: Protein phosphorylation is the most common post-translational reversible modification of proteins and is key in the regulation of many cellular processes. Due to this importance, phosphorylation is extensively studied, resulting in the availability of a large amount of mass spectrometry based phospho-proteomics data. Here, we leverage the information in these large-scale phospho-proteomics datasets, as contained in Scop3P, to analyze and characterize proteome-wide protein phosphorylation sites (P-sites). First, we set out to differentiate correctly observed P-sites from false positive sites using five complementary site properties. We then describe the context of these P-sites in terms of protein structure, solvent accessibility, structural transitions and disorder, and biophysical properties. We also investigate the relative prevalence of disease-linked mutations on and around P-sites. Moreover, we also assess structural dynamics of P-sites in their phosphorylated and unphosphorylated state. Our study shows that the residues that gets phosphorylated are more flexible than their equivalent non-phosphorylated residues. Our structural and biophysical analyses of P-sites in solvent inaccessible (buried) regions of proteins show that these sites are primarily found in multi-site phospho-proteins, where highly dynamic structural transitions can occur upon binding with another protein. Finally, our analysis of the biophysical properties of P-site mutations shows that P-site mutations that occur in structurally rigid regions are more often involved in disease.

category: bioinformatics


title: Gapless provides combined scaffolding, gap filling and assembly correction with long reads

abstract: Continuity, correctness and completeness of genome assemblies are important for many biological projects. Long reads represent a major driver towards delivering high-quality genomes, but not everybody can achieve the necessary coverage for good long-read-only assemblies. Therefore, improving existing assemblies with low-coverage long reads is a promising alternative. The improvements include correction, scaffolding and gap filling. However, most tools perform only one of these tasks and the useful information of reads that supported the scaffolding is lost when running separate programs successively. Therefore, we propose a new tool for combined execution of all three tasks using PacBio or Oxford Nanopore reads. gapless is available at: https://github.com/schmeing/gapless.


title: mPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations

abstract:
Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies.

Author summaryIn this work we have been able to demonstrate conservation of lipid-interaction sites on proteins from distinct species that deviated from their evolutionary common ancestors a long time ago, as in the case of the membrane-integral pyrophosphatases from a thermophilic bacteria species and a plant species studied here. This retention of a common lipid interaction profile or "fingerprint" and our ability to predict this for other proteins in this family may indicate that they are more integral to protein function than previously thought. By identifying lipid interactions that may act to stabilise the protein structure, these properties could be exploited to gain protein structures, and the interfacial sites potential involvement in inter-subunit communication may be useful for further investigation of the catalytic cycle of this clinically relevant membrane protein family.

category: biophysics


title: Integrative analysis of genomic and transcriptomic alterations of AGR2 and AGR3 in cancer

abstract: The AGR2 and AGR3 genes have been shown by numerous groups to be functionally associated to adenocarcinoma progression and metastasis. We explore in this paper the data available in databases concerning genomic and transcriptomic features concerning these two genes: the NCBI dbSNP database was used to explore the presence and roles of constitutional SNPs, and the NCI, CCLE and TCGA databases were used to explore somatic mutations and copy number variations (CNVs), as well as mRNA expression of these genes in human cancer cell lines and tumours. Relationships of AGR2/3 expression with whole genome mRNA expression and cancer features (i.e. mutations and CNVs of oncogenes and tumour suppressor genes [TSG]) were established using CCLE and TCGA databases. In addition, the CCLE data concerning CRISPR gene extinction screens (Achilles project) were explored concerning these two genes and a panel of oncogenes and TSG. We observed that no functional polymorphism or recurrent mutation could be detected in AGR2 or AGR3. The expression of these genes was positively correlated with the expression of epithelial genes and inversely correlated with that of mesenchymal genes. It was also significantly associated with several cancer features, such as TP53 or SMAD4 mutations, depending on the gene and the cancer type. The CRISPR screens revealed in addition the absence of cell fitness modification upon gene extinction, in contrast to oncogenes (cell fitness decrease) and TSG (cell fitness increase). Overall, these explorations revealed that AGR2 and AGR3 proteins appear as common non-genetic evolutionary factors in the process of human tumorigenesis.

category: cancer biology


title: Probing function in ligand-gated ion channels without measuring ion transport

abstract: Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion-channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are--by no means--new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled -bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric 7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two long-standing questions in the field: whether ligand-binding affinities are sensitive to binding-site occupancy, and whether mutations to amino-acid residues in the transmembrane domain can affect the channels affinities for ligands that bind to the extracellular domain.

category: physiology


title: Ketones facilitate transcriptional resolution of secondary DNA structures in premature aging

abstract: There is currently no established intervention for Cockayne syndrome, a disease characterized by progressive early onset neurodegeneration with features of premature aging. Here, we tested if acetyl-CoA precursors, citrate and beta-hydroxybutyrate, could reduce features of Cockayne syndrome in three model systems. We identified the gene Helicase 89B as a homologue of CSB in drosophila and found that the ketone beta-hydroxybutyrate rescued features of premature aging in Hel89B deficient flies. In mammals, loss of the citrate carrier Indy exacerbated the phenotype of Csbm/m mice which was rescued by a ketogenic diet. The rescue effect appeared to be mediated through ketone stimulated histone acetylation and facilitation of transcriptional readthrough of secondary DNA structures. These findings link a ketogenic diet with transcriptional resolution of secondary structures and DNA repair.


title: Lag-optimized BOLD cerebrovascular reactivity derived from breathing task data has a stronger relationship with baseline cerebral blood flow

abstract: Cerebrovascular reactivity (CVR) is an important indicator of cerebrovascular health and is commonly studied with the Blood Oxygenation Level Dependent functional MRI (BOLD-fMRI) response to a vasoactive stimulus. There is theoretical and empirical evidence to suggest that baseline cerebral blood flow (CBF) modulates the BOLD signal amplitude, and that baseline CBF may influence BOLD-CVR estimates. We address how some pertinent data acquisition and modelling choices affect the relationship between baseline CBF and BOLD-CVR: whether BOLD-CVR is modelled with breathing task data or just resting-state data, and whether BOLD-CVR amplitudes are optimized for hemodynamic lag effects. For the relationship between baseline CBF and BOLD-CVR, we assessed both between-subject correlations of average GM values and within-subject spatial correlations across cortical regions. Our results suggest that a simple breathing task addition to a resting-state scan, alongside lag-optimization within BOLD-CVR modelling, can improve BOLD-CVR correlations with baseline CBF, both between- and within-subjects, likely because these CVR estimates are more physiologically accurate. We report positive coupling between baseline CBF and BOLD-CVR, both between and within subjects; the physiological explanation of this positive coupling is unclear, and future research with larger sample sizes and more tightly controlled vasoactive stimuli is needed. Understanding how baseline vascular physiology relates to dynamic cerebrovascular processes will bring further insights into what drives between and within subject participant variability in BOLD-CVR measurements and related measurements of cerebrovascular function. These insights are particularly relevant when interpreting results in populations with altered vascular and/or metabolic baselines or impaired cerebrovascular reserve.

category: neuroscience


title: miR-329 and miR-495-mediated Prr7 downregulation isrequired for homeostatic synaptic depression in rathippocampal neurons

abstract: Homeostatic synaptic depression (HSD) in excitatory neurons is a cell autonomous mechanism which protects excitatory neurons from over-excitation as a consequence of chronic increases in network activity. In this process, excitatory synapses are weakened and eventually eliminated, as evidenced by a reduction in synaptic AMPA receptor expression and dendritic spine loss. Originally considered a global, cell-wide mechanism, local forms of regulation, such as the local control of mRNA translation in dendrites, are being increasingly recognized in HSD. Yet, identification of excitatory proteins whose local regulation is required for HSD is still limited. Here, we show that Proline-rich protein 7/Transmembrane Adapter Protein 3 (Prr7) downregulation in dendrites of rat hippocampal neurons is necessary for HSD induced by chronic increase in network activity resulting from a blockade of inhibitory synaptic transmission by picrotoxin (PTX). We further identify two activity-regulated miRNAs, miR-329-3p and miR-495-3p, which inhibit Prr7 mRNA translation and are required for HSD. Moreover, we found that Prr7 knockdown reduces expression of the synaptic scaffolding protein SPAR, which is rescued by pharmacological inhibition of CDK5, indicating a role of Prr7 protein in the maintenance of excitatory synapses via protection of SPAR from degradation. Together, our findings highlight a novel HSD mechanism in which chronic activity leads to miR-329 and miR-495-mediated local Prr7 reduction upstream of the CDK5-SPAR pathway.


title: Repeat Detector: accurate, efficient, and flexible sizing of expanded CAG/CTG repeats from targeted DNA sequencing

abstract: Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and pre-clinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. In addition, interruptions are sometimes present within repeats and can alter disease manifestation. Despite advances in methodologies, determining repeat size and identifying interruptions in targeted sequencing datasets remains a major challenge. This is because standard alignment tools are ill-suited for the repetitive nature of these sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntingtons disease (HD) and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 HD blood-derived samples and did not require prior knowledge of the flanking sequences or their polymorphisms within the patient population. We demonstrate that RD can be used to identify individuals with repeat interruptions and may provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and the development of novel therapies.


title: Not optimal, just noisy: the geometry of correlated variability leads to highly suboptimal sensory coding

abstract: The brain represents the world through the activity of neural populations. Correlated variability across simultaneously recorded neurons (noise correlations) has been observed across cortical areas and experimental paradigms. Many studies have shown that correlated variability improves stimulus coding compared to a null model with no correlations. However, such results do not shed light on whether neural populations correlated variability achieves optimal coding. Here, we assess optimality of noise correlations in diverse datasets by developing two novel null models each with a unique biological interpretation: a uniform correlations null model and a factor analysis null model. We show that across datasets, the correlated variability in neural populations leads to highly suboptimal coding performance according to these null models. We demonstrate that biological constraints prevent many subsets of the neural populations from achieving optimality according to these null models, and that subselecting based on biological criteria leaves coding performance suboptimal. Finally, we show that the optimal subpopulation is exponentially small as a function of neural dimensionality. Together, these results show that the geometry of correlated variability leads to highly suboptimal sensory coding.


title: Hippocampal gamma and sharp wave/ripples mediate bidirectional interactions with cortical networks during sleep

abstract: Hippocampus-neocortex interactions during sleep are critical for memory processes: hippocampally-initiated replay contributes to memory consolidation in the neocortex and hippocampal sharp wave/ripples are linked to generalized increases in neocortical cell activity and DOWN-UP state transitions. Yet, the spatial and temporal patterns of this exchange are unknown. With voltage imaging, electrocorticography, and laminarly-resolved hippocampal potentials, we characterized cortico-hippocampal interactions during anesthesia and NREM sleep. We observed neocortical activation transients spanning multiple spatial scales hinting at a quasi-critical regime. Transients were organized in a small number of functional networks matching known anatomical connectivity. A network overlapping with the default mode network and centered on retrosplenial cortex was the most associated with the hippocampus. Interestingly, hippocampal slow gamma was the oscillation that best correlated with this neocortical network, outpacing ripples. In fact, neocortical activity predicted hippocampal slow gamma and followed ripples, suggesting that consolidation processes rely on bi-directional exchanges between hippocampus and neocortex.


title: The Olfm4-defined human neutrophil subsets differ in proteomic profile in septic shock

abstract: The specific granule glycoprotein olfactomedin-4 (Olfm4) marks a constitutive subset of neutrophils in humans, where 1-70% of peripheral blood neutrophils produce Olfm4. The proportion of Olfm4-high (Olfm4-H) neutrophils correlates with the severity of paediatric septic shock and could predict mortality in adult septic shock in previous studies, but it is not known whether and how the Olfm4-H neutrophils contribute to sepsis pathogenesis. The aim of this study was to decipher proteomic differences between the Olfm4-H and Olfm4-low (Olfm4-L) human neutrophil subsets at baseline and in the context of septic shock, hypothesizing that Olfm4 marks a neutrophil subset with a distinct proteomic profile, predisposing it for detrimental processes in sepsis. A novel protocol for the preparation of fixed, antibody-stained and sorted neutrophils for LC-MS/MS analysis of proteome was developed. In neutrophil subsets from healthy blood donors, 47 proteins had significantly higher abundance in the Olfm4-H population, and 62 proteins in the Olfm4-L population. Pathway enrichment analysis showed that the differences concerned proteins related to neutrophil degranulation, with e.g. Rab3d and a subunit of the vacuolar ATPase proton pump being more abundant in the Olfm4-H neutrophils, and the alarmin S100-A7, the major neutrophil chemotactic receptor CXCR1 and the antimicrobial peptide defensin alpha-4 being more abundant in the Olfm4-L neutrophils. The data suggest different preparedness to infection in the subsets. In the limited material analysed here, there was no significant correlation between the severity of sepsis and the proportion of Olfm4-H neutrophils, but an increased concentration of Olfm4 in plasma from septic shock patients as compared to healthy blood donors was observed. Furthermore, in neutrophil subsets isolated from septic shock patients, 28 proteins had significantly higher abundance in the Olfm4-H subset and 38 in the Olfm4-L subset, the latter including e.g. Fc receptor proteins and MHC class I molecules, suggesting distinct immunological responses. This is the first report pointing towards differential functions of the Olfm4-defined neutrophil subpopulations in humans and the data are consistent with the idea of distinct responses in the subsets during infection and inflammation.

category: immunology


title: POPEYE directly regulates bHLH Ib genes and its own expression

abstract:
Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency responsive genes. POPEYE (PYE) is a key bHLH transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive. We found that the over-expression of PYE attenuates the expression of Fe deficiency responsive genes. PYE directly represses the transcription of bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101) by associating with their promoters. Although PYE contains an Ethylene response factor-associated Amphiphilic Repression (EAR) motif, it does not interact with the transcriptional corepressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Subcellular localization analysis indicated that PYE localizes in both the cytoplasm and nucleus. PYE contains a Nuclear Export Signal (NES) which is required for the cytoplasmic localization of PYE. The mutation of NES amplifies the repression function of PYE, resulting in downregulation of Fe deficiency responsive genes. Co-expression assays indicated that bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides insights into the complicated Fe deficiency response signaling pathway and enhances the understanding of PYE functions.

Short summaryPYE is a negative regulator of Fe homeostasis; however, it was still unclear how PYE integrates the Fe deficiency response signaling. Our study shows that conditional nuclear localization of PYE is crucial for Fe homeostasis. PYE not only negatively regulates FIT-dependent Fe uptake genes by directly targeting bHLH Ib genes, but also negatively regulates its own expression.

category: molecular biology


title: Single-cell atlas of human liver development reveals pathways directing hepatic cell fates

abstract: The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances, especially in regenerative medicine, are currently hampered by the lack of knowledge concerning human hepatic cell development. Here, we addressed this limitation by describing the developmental trajectories of different cell types comprising the human fetal liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing critical, supportive roles during organogenesis. We utilised this information to derive bipotential hepatoblast organoids and then exploited this novel model system to validate the importance of key signalling pathways and developmental cues. Furthermore, these insights into hepatic maturation enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a new platform to investigate the basic mechanisms of human liver development and to produce cell types for clinical applications.

category: developmental biology


title: Comparative host-pathogen dynamics of Snake Fungal Disease in sympatric species of water snakes (Nerodia)

abstract: The ascomycete fungus Ophidiomyces ophiodiicola (Oo) is the causative agent of ophidiomycosis (Snake Fungal Disease), which has been detected globally. However, surveillance efforts in the central U.S., specifically Texas, have been minimal. The threatened and rare Brazos water snake (Nerodia harteri harteri) is one of the most range restricted snakes in the U.S. and is sympatric with two wide-ranging congeners, N. erythrogaster transversa and N. rhombifer, in north central Texas; thus, providing an opportunity to test comparative host-pathogen dynamics in this system. To accomplish this, we surveyed a portion of the Brazos river drainage (~400 river km) over 29 months and tested 150 Nerodia spp. for the presence of Oo via quantitative PCR and recorded any potential signs of Oo infection. We found Oo was distributed across the entire range of N. h. harteri, Oo prevalence was 46 % overall, and there was a significant association between Oo occurrence and signs of infection in our sample. Models indicated adults had a higher probability of Oo infection than juveniles and subadults, and adult N. h. harteri had a higher probability of infection than adult N. rhombifer but not higher than adult N. e. transversa. High Oo prevalence estimates (94.4%) in adult N. h. harteri has implications for their conservation and management owing to their patchy distribution, comparatively low genetic diversity, and threats from anthropogenic habitat modification.

category: ecology


title: A thermal radiator underlies plasticity to extreme temperatures in the world's largest bird

abstract: Organisms inhabiting extreme thermal environments, such as desert birds, have evolved various adaptations to thermoregulate during hot days and cold nights. However, our knowledge of selection acting on thermoregulatory traits and their evolutionary potential is limited, particularly for large organisms experiencing extreme temperature fluctuations. Here we show, using thermal imaging that the featherless neck of the ostrich (Struthio camelus) acts as a  thermal radiator, protecting the head from overheating during hot conditions and conserving heat during cool conditions. We found substantial individual variation in thermal plasticity of the neck to dissipate heat away from the head that was associated with increased egg-laying rates during high ambient temperatures. Combined with low, but significant, heritability estimates of individual thermal profiles, these findings suggest that the ostrich neck functions as an adaptive thermal radiator with evolutionary potential. There were also signatures of past selection, since ostriches originating from more volatile climatic regions and females that incubate during hot daytime conditions exhibited especially high thermal plasticity. Taken together our results indicate that morphological adaptations involved in ostrich thermoregulation, such as the neck, are experiencing ongoing selection and are crucial for successfully reproducing under fluctuating climatic conditions.

category: evolutionary biology


title: G matrix stability of clinally diverging populations of an annual weed.

abstract: How phenotypic and genetic divergence among populations is influenced by the genetic architecture of those traits, and how microevolutionary changes in turn affect the within-population patterns of genetic variation, are of major interest to evolutionary biology. Work on Ipomoea hederacea, an annual vine, has found genetic clines in the means of a suite of ecologically important traits, including flowering time, growth rate, seed mass, and corolla width. Here we investigate the genetic (co)variances of these clinally varying traits in two northern range-edge and two central populations of Ipomoea hederacea to evaluate the influence of the genetic architecture on divergence across the range. We find 1) limited evidence for clear differentiation between northern and southern populations in the structure of G, suggesting overall stability of G across the range despite mean trait divergence and 2) that the axes of greatest variation (gmax) were unaligned with the axis of greatest multivariate divergence. Together these results indicate the role of constraint on the divergence among populations across the range.


title: ezQTL: A Web Platform for Interactive Visualization and Colocalization of Quantitative Trait Loci and GWAS

abstract: Genome-wide association studies (GWASs) have identified thousands of genomic loci associated with complex diseases and traits, including cancer. The vast majority of common trait-associated variants identified via GWAS fall in non-coding regions of the genome, posing a challenge in elucidating the causal variants, genes, and mechanisms involved. Expression quantitative trait locus (eQTL) and other molecular QTL studies have been valuable resources in identifying candidate causal genes from GWAS loci through statistical colocalization methods. While QTL colocalization is becoming a standard analysis in post-GWAS investigation, an easy web tool for users to perform formal colocalization analyses with either user-provided or public GWAS and eQTL datasets has been lacking. Here, we present ezQTL, a web-based bioinformatic application to interactively visualize and analyze genetic association data such as GWAS and molecular QTLs under different linkage disequilibrium (LD) patterns (1000 Genomes Project, UK Biobank, or user-provided data). This application allows users to perform data quality control for variants matched between different datasets, LD visualization, and two-trait colocalization analyses using two state-of-the-art methodologies (eCAVIAR and HyPrColoc), including batch processing. ezQTL is a free and publicly available cross-platform web tool, which can be accessed online at https://analysistools.cancer.gov/ezqtl.

category: genetics


title: Nanobody-tethered transposition allows for multifactorial chromatin profiling at single-cell resolution

abstract: Chromatin states are functionally defined by a complex combination of histone modifications, transcription factor binding, DNA accessibility, and other factors. However, most current single-cell-resolution methods are unable to measure more than one aspect of chromatin state in a single experiment, limiting our ability to accurately measure chromatin states. Here, we introduce nanobody-tethered transposition followed by sequencing (NTT-seq), a new assay capable of measuring the genome-wide presence of multiple histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq utilizes recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. We apply bulk- and single-cell NTT-seq to generate high-resolution multimodal maps of chromatin states in cell culture and in cells of the human immune system, demonstrating the high accuracy and sensitivity of the method. We further extend NTT-seq to enable simultaneous profiling of cell-surface protein expression alongside multimodal chromatin states to study cells of the immune system.


title: Identifying commonalities between cell lines and tumors at the single cell level using Sobolev Alignment of deep generative models

abstract: Preclinical models are essential to cancer research, however, key biological differences with patient tumors result in reduced translatability to the clinic and high attrition rates in drug development. Variability among and between patients, preclinical models, and individual cells obscures commonalities which could otherwise be exploited therapeutically. To discover the shared biological processes between cell line models and clinical tumors we developed Sobolev Alignment, a computational framework which uses deep generative models to capture non-linear processes in single-cell RNA sequencing data and kernel methods to align and interpret these processes. We show that our approach faithfully captures shared processes on a set of three synthetic datasets. Exploiting two large panels of untreated non-small cell lung cancer cell lines and patients, we identify the similarities between cell lines and tumors and show the conservation of key mitotic and immune-related pathways. Employing our approach on a large in-vitro perturbation screen, we show that processes captured by our method faithfully recapitulate the known modes of action of clinically approved drugs and allow investigation into the mode of action of an uncharacterized drug.


title: Myosin essential light chain 1sa decelerates actin and thin filament gliding on β-myosin molecules

abstract:
The {beta}-myosin heavy chain expressed in ventricular myocardium and the myosin heavy chain (MyHC) in slow-twitch skeletal soleus muscle type-I fibers are both encoded by MYH7. Thus, these myosin molecules are deemed equivalent. However, some reports suggested variations in the light chain composition between soleus and ventricular myosin, which could influence functional parameters such as maximum velocity of shortening. To test for functional differences of the actin gliding velocity on immobilized myosin molecules we made use of the in vitro motility assay.

We found that ventricular myosin moved actin filaments with approx. 0.9 m/s significantly faster than soleus myosin (0.3 m/s). Unregulated actin filaments are not the native interaction partner of myosin and are believed to slow down movement. Yet, using native thin filaments purified from soleus or ventricular tissue, the gliding velocity of soleus and ventricular myosin remained significantly different. When comparing the light chain composition of ventricular and soleus {beta}-myosin a difference became evident. Soleus myosin contains not only the "ventricular" essential light chain (ELC) MLC1sb/v, but also an additional longer and more positively charged MLC1sa. Moreover, we revealed that on a single muscle fiber level, a higher relative content of MLC1sa was associated with significantly slower actin gliding.

We conclude that the ELC MLC1sa decelerates gliding velocity presumably by a decreased dissociation rate from actin associated with a higher actin affinity compared to MLC1sb/v. Such ELC/actin interactions might also be relevant in vivo as differences between soleus and ventricular myosin persisted when native thin filaments were used.

SummaryCompared to the "ventricular" essential myosin light chain MLC1sb/v, the longer and more positively charged MLC1sa present in slow-twitch soleus muscle fibers decelerates actin filament gliding on {beta}-myosin molecules presumably by a decreased dissociation rate from actin filaments.


title: Identification of genetic loci in lettuce mediating quantitative resistance to fungal pathogens

abstract:
Lactuca sativa L. (lettuce) is an important leafy vegetable crop grown and consumed globally. Chemicals are routinely used to control major pathogens, including the causal agents of grey mould (Botrytis cinerea) and lettuce drop (Sclerotinia sclerotiorum). With increasing prevalence of pathogen resistance to fungicides and environmental concerns, there is an urgent need to identify sources of genetic resistance to B. cinerea and S. sclerotiorum in lettuce. We demonstrated genetic variation for quantitative resistance to B. cinerea and S. sclerotiorum in a set of 97 diverse lettuce and wild relative accessions, and between the parents of lettuce mapping populations. Transcriptome profiling across multiple lettuce accessions enabled us to identify genes with expression correlated with resistance, predicting the importance of post-transcriptional gene regulation in the lettuce defence response. We identified five genetic loci influencing quantitative resistance in a F10 mapping population derived from a Lactuca serriola (wild relative) x lettuce cross, which each explained 5-10% of the variation. Differential gene expression analysis between the parent lines, and integration of data on correlation of gene expression and resistance in the diversity set, highlighted potential causal genes underlying the quantitative trait loci.

Key MessageWe demonstrate genetic variation for quantitative resistance against important fungal pathogens in lettuce and its wild relatives, map loci conferring resistance and predict key molecular mechanisms using transcriptome profiling.

category: plant biology


title: Kaempferol over-accumulation in the flavonoid 3' hydroxylase tt7 mutant disrupts seed coat outer integument differentiation and compromises seed longevity

abstract:
O_LISeeds slowly accumulate damage during storage, which ultimately results in germination failure. The seed coat is the barrier between the embryo and the external environment, and its composition is critical for seed longevity. Flavonols accumulates in the outer integument, but the effect of altering flavonol composition on outer integument development has not been explored.
C_LIO_LIGenetic, biochemical, ultrastructural and transcriptomics assays on a battery of loss-of-function mutants in the flavonoid biosynthesis pathway were used to study the effect of altered flavonoid composition in seed development and seed longevity.
C_LIO_LIControlled deterioration assays indicate that loss-of-function of the flavonoid 3 hydroxylase TT7 gene dramatically affects seed longevity and seed development. Seed outer integument differentiation is compromised from nine days after pollination in tt7 seeds, with a defective suberine layer and incomplete degradation of seed-coat starch. These distinctive phenotypes are not shared by other mutants showing also altered flavonoid composition. Double-mutant analysis indicate that over-accumulation of kaempferol is the primary cause of the observed phenotypes. Expression analysis suggest that the tt7 flavonoid pattern affects transcriptional and non-transcriptional levels of regulation.
C_LIO_LIThe increase of kaempferols in the seed coat influences seed development. This positions TT7 as an essential player modulating seed coat development and seed longevity.
C_LI


title: Mental Workload Classification of Oceanauts from EEG Data Using Multiclass Support Vector Machines

abstract:
BackgroundIn the actual operation task, the workload of the oceanaut is mainly mental workload. For the oceanaut, too high or too low mental workload will significantly reduce work efficiency and even lead to major safety accidents. Classification of mental workload of the oceanaut in operational task research is one of the key problems in operational task research. However, the traditional mechanism modeling method is complex, computational complexity and low accuracy.

MethodIn this paper, machine learning method got used to the model. Based-on Electroencephalograph (EEG) data collected from the simulation experiment of the operating manipulator, multiclass support vector machine (MSVM) was used to train the samples, and the optimal training sample size was selected. The parameters of the model were optimized by grid search mixed with particle-swarm optimization algorithm (GSPSO) to obtain the optimal classification of oceanauts mental workload.

ResultsAs the research results showed that the method could accurately classify the mental workload of oceanauts. Furthermore, GSPSO-MSVM in the classification of oceanauts mental workload had the advantage over K-nearest neighbors (KNN), BP neural network (BP), random forest (RF) and support vector machines (SVM).

ConclusionGSPSO-MSVM in the classification of oceanauts mental workload had the advantage over K-nearest neighbors (KNN), BP neural network (BP), random forest (RF) and support vector machines (SVM).

Key points: The significance of the studyO_LIClassification of mental workload of the oceanaut in operational task research is one of the key problems in operational task research.
C_LIO_LIBased-on Electroencephalograph (EEG) data collected from the simulation experiment of the operating manipulator, multiclass support vector machine (MSVM) was used to train the samples, and the optimal training sample size was selected.
C_LIO_LIThe parameters of the model were optimized by grid search mixed with particle-swarm optimization algorithm (GSPSO) to obtain the optimal classification of oceanauts mental workload.
C_LIO_LIGSPSO-MSVM in the classification of oceanauts mental workload had the advantage over K-nearest neighbors (KNN), BP neural network (BP), random forest (RF) and support vector machines (SVM).
C_LI

category: scientific communication and education


title: Co-expression analysis reveals gene cluster associated with methylation of enhancers and chromosomal instability under TP63 and TRIM29 regulation

abstract:
Prostate adenocarcinoma (PRAD) is the second most common cause of cancer-related deaths in men. In PRAD, high variability in DNA methylation and a high rate of large genomic rearrangements is often observed. To elucidate the reasons behind such high variance, we integrated DNA methylation, RNA-seq, and copy number alterations datasets from The Cancer Genome Atlas (TCGA) focusing on PRAD and subsequently employed weighted gene co-expression network analysis (WGCNA). Results from the studies show that only a single cluster of co-expressed genes is associated with genomic and epigenomic instability. Within this cluster, TP63 and TRIM29 are regulators and were both downregulated in PRAD. We show that TP63 regulates the level of enhancer methylation in prostate basal epithelium cells. TRIM29 forms a complex with TP63 and together regulate the expression of genes specific to the prostate basal epithelium. Moreover, TRIM29 binds DNA repair proteins and prevents the formation of the TMPRSS2:ERG gene fusion typically seen in PRAD. Therefore, the study shows that TRIM29 and TP63 are important regulators maintaining the identity of the basal epithelium. Finally, we uncover the role of TRIM29 in genome protection functions in PRAD.

HIGHLIGHTSO_LIIdentification of clusters of co-expressed genes associated with epigenomic variability and chromosomal instability in prostate adenocarcinoma
C_LIO_LITP63 and TRIM29 together regulate the expression of the cluster genes
C_LIO_LITP63 regulates methylation of enhancers associated with the cluster
C_LIO_LITRIM29 decreases chromosomal instability in prostate adenocarcinoma
C_LI

category: systems biology


title: The choice of the objective function in flux balance analysis is crucial for predicting replicative lifespans in yeast

abstract: Flux balance analysis (FBA) is a powerful tool to study genome-scale models of the cellular metabolism, based on finding the optimal flux distributions over the network. While the objective function is crucial for the outcome, its choice, even though motivated by evolutionary arguments, has not been directly connected to related measures. Here, we used an available multi-scale mathematical model of yeast replicative ageing, integrating cellular metabolism, nutrient sensing and damage accumulation, to systematically test the effect of commonly used objective functions on features of replicative ageing in budding yeast, such as the number of cell divisions and the corresponding time between divisions. The simulations confirmed that assuming maximal growth is essential for reaching realistic lifespans. The usage of the parsimonious solution or the additional maximisation of a growth-independent energy cost can improve lifespan predictions, explained by either increased respiratory activity using resources otherwise allocated to cellular growth or by enhancing antioxidative activity, specifically in early life. Our work provides a new perspective on choosing the objective function in FBA by connecting it to replicative ageing.


title: Differential Activity of Repurposed Drugs as Receptor Binding Domain Antagonists for Omicron and Native Strains of SarsCov2

abstract:
Omicron strain is the latest variant of concern of SarsCov2 virus. The mutations in this strain in the S protein Receptor Binding domain (RBD) enable it to be more transmissible as well as escape neutralizing activity by antibodies in response to vaccine. Thus, Omicron specific strategies are need to counter infection by this strain.

We investigated a collection of approved drugs shown to antagonize the binding of native strain RBD to human ACE2, for their ability to antagonize binding to Omicron strain RBD.

While most of the drugs the drugs that antagonize binding to native RBD are also active for Omicron RBD but some were inactive, namely drugs that contain iodine are completely inactive against Omicron RBD. Our data strongly indicate that presence of a single iodine molecule in the drug renders it inactive against Omicron strain. Thus, there is molecular specificity of drugs for antagonizing Omicron strain RBD versus native strain RBD of this virus. Such information will pave way for specific drugs for Omicron. A pragmatic message from our data is that the often-used iodine containing mouth wash and rises may be ineffective in antagonizing receptor binding of Omicron strain.

category: pharmacology and toxicology


title: Nucleocapsid-specific humoral responses improve the control of SARS-CoV-2

abstract:
The spike protein of SARS-CoV-2 is a critical antigen present in all approved SARS-CoV-2 vaccines. This surface viral protein is also the target for all monoclonal antibody therapies, but it is unclear whether antibodies targeting other viral proteins can also improve protection against COVID-19. Here, we interrogate whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine, and then transferred sera from these mice into naive mice. On the next day, the recipient mice were challenged intranasally with SARS-CoV-2 to evaluate whether nucleocapsid-specific humoral responses affect viral control. Interestingly, mice that received nucleocapsid-specific sera exhibited enhanced control of a SARS-CoV-2 infection. These findings provide the first demonstration that humoral responses specific to an internal coronavirus protein can help clear infection, warranting the inclusion of other viral antigens in next-generation SARS-CoV-2 vaccines and providing a rationale for the clinical evaluation of nucleocapsid-specific monoclonals to treat COVID-19.

HighlightsA SARS-CoV-2 nucleocapsid vaccine elicits robust nucleocapsid-specific antibody responses.

This nucleocapsid vaccine generates memory B cells (MBC).

Nucleocapsid-specific humoral responses do not prevent SARS-CoV-2 infection.

Nucleocapsid-specific humoral responses help control a SARS-CoV-2 infection.


title: Modeling within-host and aerosol dynamics of SARS-CoV-2: the relationship with infectiousness

abstract:
The relationship between transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the amount of virus present in the proximity of a susceptible host is not understood. Here, we developed a within-host and aerosol mathematical model and used it to determine the relationship between viral kinetics in the upper respiratory track, viral kinetics in the aerosols, and new transmissions in golden hamsters challenged with SARS-CoV-2. We determined that infectious virus shedding early in infection correlates with transmission events, shedding of infectious virus diminishes late in the infection, and high viral RNA levels late in the infection is a poor indicator of transmission. We further showed that viral infectiousness increases in a density dependent manner with viral RNA and that their relative ratio is time-dependent. Such information is useful for designing interventions.

Author summaryQuantifying the relationship between SARS-CoV-2 dynamics in upper respiratory tract and in aerosols is key to understanding SARS-CoV-2 transmission and evaluating intervention strategies. Of particular interest is the link between the viral RNA measured by PCR and a subjects infectiousness. Here, we developed a mechanistic model of viral transmission in golden hamsters and used data in upper respiratory tract and aerosols to evaluate key within-host and environment based viral parameters. The significance of our research is in identifying the timing and duration of viral shedding, how long it stays infectious, and the link between infectious virus and total viral RNA. Such knowledge enhances our understanding of the SARS-CoV-2 transmission window.


title: DiSCERN - Deep Single Cell Expression ReconstructioN for improved cell clustering and cell subtype and state detection

abstract:
Single cell sequencing provides detailed insights into biological processes including cell differentiation and identity. While providing deep cell-specific information, the method suffers from technical constraints, most notably a limited number of expressed genes per cell, which leads to suboptimal clustering and cell type identification. Here we present DISCERN, a novel deep generative network that reconstructs missing single cell gene expression using a reference dataset. DISCERN outperforms competing algorithms in expression inference resulting in greatly improved cell clustering, cell type and activity detection, and insights into the cellular regulation of disease. We used DISCERN to detect two novel COVID-19-associated T cell types, cytotoxic CD4+ and CD8+ Tc2 T helper cells, with a potential role in adverse disease outcome. We utilized T cell fraction information of patient blood to classify mild or severe COVID-19 with an AUROC of 81 % that can serve as a biomarker of disease stage. DISCERN can be easily integrated into existing single cell sequencing workflows and readily adapted to enhance various other biomedical data types.

2010 MSC: 00-01, 99-00


title: Comparative thermophysiology of marine Synechococcus CRD1 strains isolated from different thermal niches in iron-depleted areas

abstract:
Marine Synechococcus cyanobacteria are ubiquitous in the ocean, a feature likely related to their extensive genetic diversity. Amongst the major lineages, clades I and IV preferentially thrive in temperate and cold, nutrient-rich waters, whilst clades II and III prefer warm, nitrogen or phosphorus-depleted waters. The existence of such cold (I/IV) and warm (II/III) thermotypes is corroborated by physiological characterization of representative strains. A fifth clade, CRD1, was recently shown to dominate the Synechococcus community in iron-depleted areas of the world ocean and to encompass three distinct ecologically significant taxonomic units (ESTUs CRD1A-C) occupying different thermal niches, suggesting that distinct thermotypes could also occur within this clade.

Here, using comparative thermophysiology of strains representative of these three CRD1 ESTUs we show that the CRD1A strain MITS9220 is a warm thermotype, the CRD1B strain BIOS-U3-1 a cold temperate thermotype, and the CRD1C strain BIOS-E4-1 a warm temperate stenotherm. Curiously, the CRD1B thermotype lacks traits and/or genomic features typical of cold thermotypes. In contrast, we found specific physiological traits of the CRD1 strains compared to their clade I, II, III and IV counterparts, including a lower growth rate and photosystem II maximal quantum yield at most temperatures and a higher turnover rate of the D1 protein. Together, our data suggests that the CRD1 clade prioritizes adaptation to low-iron conditions over temperature adaptation, even though the occurrence of several CRD1 thermotypes likely explains why the CRD1 clade as a whole occupies most iron-limited waters.

category: microbiology


title: Performance of the cobas(R) HBV RNA Automated Investigational Assay for the Detection and Quantification of Circulating HBV RNA in Chronic HBV Patients

abstract:
BackgroundThe amount of HBV RNA in peripheral blood may reflect HBV covalently closed circular DNA (cccDNA) transcriptional activity within infected hepatocytes. Quantification of circulating HBV RNA (cirB-RNA) is thus a promising biomarker for monitoring antiviral treatment.

ObjectivesWe evaluated the performance of an automated, prototype quantitative HBV RNA assay for use on the Roche cobas(R) 6800/8800 systems.

Study DesignThe sensitivity, specificity, linearity, and potential interference by HBV DNA of the cobas(R) HBV RNA assay were assessed using synthetic HBV armored RNA and clinical specimens.

Resultscobas(R) HBV RNA results were linear between 10 and 107 copies/mL in clinical samples of several HBV genotypes, and up to 109 copies/mL with synthetic RNA. Precision and reproducibility were excellent, with standard deviation below 0.15 log10 copies/mL and coefficients of variation below 5% throughout the linear range. The presence of HBV DNA had minimal (<0.3 log10 copies/mL) impact on HBV RNA quantification at DNA:RNA ratios of up to approximately one million. In a panel of 36 untreated patient samples, cirB-RNA concentrations were approximately 200-fold lower than HBV DNA. cirB-RNA was detected in all 13 HBeAg-positive patients (mean 6.0 log10 copies/mL), and in 20 of 23 HBeAg-negative patients (mean of quantifiable samples 2.2 log10 copies/mL). Finally, cirB-RNA was detected in 12 of 20 nucleoside analog-treated patients (mean of quantifiable samples 3.4 log10 copies/mL).

ConclusionsThe cobas(R) 6800/8800 investigational HBV RNA assay is a high throughput, sensitive and inclusive assay to evaluate the clinical relevance of cirB-RNA quantification in patients with chronic hepatitis B.


title: Do lanthanide-dependent microbial metabolisms drive the release of REEs from weathered granites?

abstract: Prior to soil formation, phosphate liberated by rock weathering is often sequestered into highly insoluble lanthanide phosphate minerals. Dissolution of these minerals is critical for the release of phosphate to the biosphere, yet the microorganisms involved, and the genes required for lanthanide metabolism, are poorly understood. Here, we sampled weathered granite and associated soil to identify the zones of lanthanide phosphate mineral solubilization and genomically define the organisms implicated in lanthanide utilisation. We reconstructed 136 genomes from 11 bacterial phyla and found gene clusters implicated in lanthanide-based metabolism of methanol (primarily XoxF3 and XoxF5) are surprisingly common in microbial communities in moderately weathered granite where lanthanide phosphate minerals are dissolving. Notably, XoxF3 systems were found in Verrucomicrobia for the first time, and in Acidobacteria, Gemmatimonadetes, and Alphaproteobacteria. The XoxF-containing gene clusters are shared by diverse Acidobacteria and Gemmatimonadetes, and include conserved hypothetical proteins and transporters not associated with the few well studied XoxF systems. Given that siderophore-like molecules that strongly bind lanthanides may be required to solubilize lanthanide phosphates, it is notable that candidate siderophore biosynthesis systems were most prevalent in bacteria in moderately weathered rock, especially in Acidobacteria with lanthanide-based systems. We conclude that the confluence in the zone of moderate weathering of phosphate mineral dissolution, lanthanide utilisation, and methanol oxidation (thus carbonic acid production) may be important during the conversion of granitic rock to soil.


title: Insights into β-manno-oligosaccharide uptake and metabolism in Bifidobacterium adolescentis DSMZ 20083 from whole-genome microarray analysis

abstract:
AimsTo determine the ability of adult-associated B. adolescentis DSMZ 20083 to utilize dietary {beta}-manno-oligosaccharides and understand the underlying molecular mechanism.

Methods and ResultsIn-vitro fermentation and TLC were used to determine the ability of B. adolescentis DSMZ 20083 to utilize {beta}-manno-oligosaccharides from guar gum, locust bean gum, konjac and copra meal. Further, Whole-genome transcriptome analysis, q-RT-PCR and molecular docking were employed to reconstruct copra meal {beta}-manno-oligosaccharides (CM-{beta}-MOS) utilization pathway in B. adolescentis DSMZ 20083.

B. adolescentis DSMZ 20083 grew appreciably (A600nm up to 0.8) on all tested {beta}-manno-oligosaccharides but maximally on CM-{beta}-MOS. Transcriptome analysis revealed differential up-regulation of three distinct gene clusters encoding, ABC transporter cassette (ESBPs and permeases), MFS transporter, GH1 {beta}-glucosidase and, GH32 {beta}-fructofuranosidases. ABC and MFS possibly transported majorly DP[≥]2 and DP[≥]3 CM-{beta}-MOS, respectively. Sugar absorption and utilization pathways; ABC transport system, pyruvate metabolism, glycolysis/gluconeogenesis, pentose, and glucouronate inter-conversions were also up-regulated.

ConclusionsB. adolescentis DSMZ 20083 possibly utilizes ABC & MFS transporters to internalise and GH1 {beta}-glucosidase, and GH32 {beta}-fructofuranosidase to deconstruct CM-{beta}-MOS.

Significance and Impact of the StudyFirst study reporting possible molecular determinants used by B. adolescentis DSMZ 20083 to utilize {beta}-manno-oligosaccharides and thus, can prove resourceful in precision microbiome modulation.


title: The ADP-heptose biosynthesis enzyme GmhB is a conserved Gram-negative bacteremia fitness factor

abstract:
Klebsiella pneumoniae is a leading cause of Gram-negative bacteremia, which is a major source of morbidity and mortality worldwide. Gram-negative bacteremia requires three major steps: primary site infection, dissemination to the blood, and bloodstream survival. Since K. pneumoniae is a leading cause of healthcare-associated pneumonia, the lung is a common primary infection site leading to secondary bacteremia. K. pneumoniae factors essential for lung fitness have been characterized, but those required for subsequent bloodstream infection are unclear. To identify K. pneumoniae genes associated with dissemination and bloodstream survival, we performed insertion site sequencing (InSeq) using a pool of >25,000 transposon mutants in a murine model of bacteremic pneumonia. This analysis revealed the gene gmhB as important for either dissemination from the lung or bloodstream survival. In Escherichia coli, GmhB is a partially redundant enzyme in the synthesis of ADP-heptose for the lipopolysaccharide (LPS) core. To characterize its function in K. pneumoniae, an isogenic knockout strain ({Delta}gmhB) and complemented mutant were generated. During pneumonia, GmhB did not contribute to lung fitness and did not alter normal immune responses. However, GmhB enhanced bloodstream survival in a manner independent of serum susceptibility, specifically conveying resistance to spleen-mediated killing. In a tail-vein injection of murine bacteremia, GmhB was also required by K. pneumoniae, E. coli and Citrobacter freundii for optimal bloodstream survival. Together, this study identifies GmhB as a conserved Gram-negative bacteremia fitness factor that acts through LPS-mediated mechanisms to enhance bloodstream survival.

IMPORTANCEKlebsiella pneumoniae frequently causes healthcare-associated infections including pneumonia and bacteremia. This is particularly concerning due to emerging antimicrobial resistance and the propensity for bacteremia to initiate sepsis, which has high mortality and is the most expensive hospital-treated condition. Defining mechanisms of bloodstream survival is critical to understanding the pathology of bacteremia and identifying novel targets for future therapies. In this study, we identified the K. pneumoniae enzyme GmhB as a bloodstream-specific fitness factor that enables the bacteria to survive in the spleen but is dispensable in the lung. Furthermore, GmhB is also needed by the related bacterial pathogens Escherichia coli and Citrobacter freundii to cause bacteremia. Conserved bacteremia fitness factors such a GmhB could be the basis for future therapeutics that would alleviate significant disease caused by from multiple diverse pathogens.


title: Cell-surface PD-L1 expression identifies a sub-population of distal epithelial cells enriched in idiopathic pulmonary fibrosis

abstract: Idiopathic lung fibrosis (IPF) is a fatal lung disease characterized by chronic epithelial injury and exhausted repair capacity of the alveolar compartment, associated with the expansion of cells with intermediate alveolar epithelial cell (AT2) characteristics. Using SftpcCreERT2/+: tdTomatoflox/flox mice, we previously identified a lung population of quiescent injury activated alveolar epithelial progenitors (IAAPs) marked by low expression of the AT2 lineage trace marker tdTomato (Tomlow), and characterized by high levels of Pd-l1 (Cd274) expression. This led us to hypothesize that a population with similar properties exists in the human lung. To that end, we used flow cytometry to characterize the CD274 cell surface expression in lung epithelial cells isolated from donor and end-stage IPF lungs. The identity and functional behavior of these cells were further characterized by qPCR analysis, in vitro organoid formation, and ex vivo precision-cut lung slices (PCLS). Our analysis led to the identification of a population of CD274pos cells expressing intermediate levels of SFTPC, which was expanded in IPF lungs. While donor CD274pos cells initiated clone formation, they did not expand significantly in 3D organoids in AT2-supportive conditions. However, an increased number of CD274pos cells was found in cultured PCLS. In conclusion, we demonstrate that, similar to the IAAPs in the mouse lung, a population of CD274 expressing cells exists in the normal human lung and this population is expanded in the IPF lung and in an ex vivo PCLS assay, suggestive of progenitor cell behavior.

category: cell biology


title: Complex regulatory role of DNA methylation in caste- and age-specific expression of a termite.

abstract: The reproductive castes of eusocial insects are often characterised by extreme lifespans and reproductive output, indicating an absence of the fecundity/longevity trade-off. The role of DNA methylation in the regulation of caste- and age-specific gene expression in eusocial insects is controversial. While some studies find a clear link to caste formation in honeybees and ants, others find no correlation when replication is increased across independent colonies. Although recent studies have identified transcription patterns involved in the maintenance of high reproduction throughout the long lives of queens, the role of DNA methylation in the regulation of these genes is unknown. We carried out a comparative analysis of DNA methylation in the regulation of caste-specific transcription and its importance for the regulation of fertility and longevity in queens of the higher termite, Macrotermes natalensis. We found evidence for significant, well-regulated changes in DNA methylation in mature compared to young queens, especially in several genes related to ageing and fecundity in mature queens. We also found a strong link between methylation and caste-specific alternative splicing. This study reveals a complex regulatory role of fat body DNA methylation both in the division of labour in termites, and during the reproductive maturation of queens.


title: Tissue-Restricted Inhibition of mTOR Using Chemical Genetics

abstract: mTOR is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product Rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system that restricts the activity of a Rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this Rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in S. cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a Rapamycin-induced developmental delay in Drosophila.

category: biochemistry


title: Temporin B forms hetero-oligomers with Temporin L, modifies its membrane activity and increases the cooperativity of its antibacterial pharmacodynamic profile

abstract: The pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A-L, as an effective chemical dermal defence. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly, when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.


title: Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors

abstract: Bacteriophages operate via pathogen-specific mechanisms of action distinct from conventional, broad-spectrum antibiotics and are emerging as promising alternatives. However, phage-mediated killing is often limited by bacterial resistance development (1,2). Here, we engineer phages for target-specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Using urinary tract infection (UTI) as a model, we show how heterologous effector phage therapeutics (HEPTs) suppress resistance and improve uropathogen killing by dual phage- and effector-mediated targeting. Moreover, we designed HEPTs to control polymicrobial uropathogen communities through production of effectors with cross-genus activity. Using a phage-based companion diagnostic (3), we identified potential HEPT responder patients and treated their urine ex vivo. Compared to wildtype phage, a colicin E7-producing HEPT demonstrated superior control of patient E. coli bacteriuria. Arming phages with heterologous effectors paves the way for successful UTI treatment and represents a versatile tool to enhance and adapt phage-based precision antimicrobials.

category: bioengineering


title: Activation of targetable inflammatory immune signaling is seen in Myelodysplastic Syndromes with SF3B1 mutations

abstract:
BackgroundMutations in the SF3B1 splicing factor are commonly seen in Myelodysplastic syndromes (MDS) and Acute Myeloid Leukemia (AML), yet the specific oncogenic pathways activated by missplicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood.

MethodsRNA-seq data from SF3B1 mutant samples was analyzed and functional roles of IRAK4 isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in pre-clinical models of MDS/AML

ResultsRNA-seq splicing analysis of innate immune mediators in SF3B1 mutant MDS samples revealed retention of full-length exon 6 of interleukin-1 receptor-associated kinase 4 (IRAK4), a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-Long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteosomal degradation. Expression of IRAK4-Long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models.

ConclusionsSF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models.

category: cancer biology


title: High-throughput molecular recording can determine the identity and biological activity of sequences within single cells

abstract:
Methods of measuring the activities of biomolecules and pathways are fundamental to the study and engineering of biological systems. Traditional marker genes that generate light or color are limited in their dynamic range and often require expensive equipment. Here we describe a way to simultaneously detect the identity and measure the activity of each molecular variant within a mixed population of cells using nanopore sequencing. By linking the desired activity to expression of a base editor that induces mutations in a "canvas" adjacent to the DNA encoding the molecule or pathway of interest, we show that a single long-read sequencing step can retrieve identity and activity at numerous timepoints in bacteria, potentially greatly increasing the available dynamic range. Our technique can replace flow cytometry in directed evolution and may be capable of directly mapping biomolecular fitness landscapes.

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category: synthetic biology


title: Tradeoffs in bacterial physiology determine the efficiency of antibiotic killing

abstract: Antibiotics can kill or stop the growth of bacteria, and their effectiveness depends on many factors. It is important to understand the relation between bacterial physiology, the environment and antibiotic action. While many of the mechanistic details of antibiotic action are known, the connection between death rate and bacterial physiology is poorly understood. Death rate in antibiotics has often been shown to rise linearly with growth rate; however, it remains unclear how environmental factors, in concert with whole-cell physiological properties, affect bactericidal activity. To address this, we developed a high-throughput assay to precisely measure antibiotic-mediated bacterial death. We found that death rate is linear in growth rate only under certain conditions and deviates significantly from linearity in stressful growth environments: stressors lower the death rate compared to a non-stressed environment with the same growth rate. To understand the role of stress, we developed a mathematical model of bacterial death based on resource allocation that takes into account a newly defined stress-response sector; we identify this sector using RNAseq. Our model accurately predicts the death rate and minimal inhibitory concentration of antibiotics across a wide range of conditions, including a previously unknown increase in the stress response and protection from death at very low levels of cAMP. The present death-growth model suggests conditions that may improve antibiotic efficacy.


title: Neonatal exposures to sevoflurane in rhesus monkeys alter synaptic ultrastructure in later life

abstract:
Early-life exposure to anesthesia in infant humans and monkeys increases the risk for cognitive and socioemotional impairments. However, the long-term effects of neonatal anesthesia on synaptic ultrastructure have not been thoroughly investigated in primates. We used electron microscopy with unbiased stereological sampling to assess synaptic ultrastructure in the CA1 of the hippocampus and the dorsolateral prefrontal cortex (dlPFC) of female and male rhesus macaques four years after three 4-hour exposures to sevoflurane during the first five postnatal weeks. We counted synapses and measured synapse areas for all synapses and those classified as perforated or nonperforated with spine or dendritic shaft targets. We measured numbers and shapes of mitochondria within presynaptic boutons and calculated vesicle docking rates. In monkeys exposed to anesthesia as infants, synapse areas were reduced in the largest 20% of synapses in CA1 and the largest 5% of synapses in dlPFC, with differential sex effects for the largest 10% of synapses in CA1. Synapse areas were reduced by 7.6% for perforated spinous synapses in CA1, and by 10.4% for nonperforated spinous synapses in dlPFC. Perforated and nonperforated dendritic synapse numbers in CA1 increased by 180% and 63% respectively. Curved mitochondria decreased 25% in CA1 after anesthesia exposure, and dlPFC boutons with 0 mitochondria showed an interaction of anesthesia and sex. These results demonstrate that exposure to anesthesia in infancy can cause long-term structural deficits in primates. These structural changes may be substrates for long-term alterations in the strength and efficiency of synaptic transmission in hippocampus and prefrontal cortex.

Key pointsO_LIExposure to anesthesia in early life causes lasting cognitive and socioemotional impairments in human and nonhuman primates, but the extent to which early-life exposure to anesthesia alters synaptic ultrastructure in later life has not been known.
C_LIO_LIFour years after rhesus monkeys were given multiple exposures to anesthesia in infancy, the area of spinous synapses was reduced in CA1 and dlPFC, dendritic synapse numbers were elevated in CA1, there were fewer curved presynaptic mitochondria in CA1, and numbers of presynaptic boutons without mitochondria were altered in dlPFC.
C_LIO_LIThe long-term ultrastructural changes to synapses and presynaptic mitochondria of rhesus monkeys that were exposed to anesthesia as infants could help explain their behavioral deficits later in life.
C_LI

category: neuroscience


title: Three functionally distinct classes of cGAS proteins in nature revealed by self-DNA-induced interferon responses

abstract:
Innate immune pattern recognition receptors (PRRs) emerged early in evolution. It is generally assumed that structurally homologous proteins in distinct species will operate via similar mechanisms. We tested this prediction through the study of interferon responses to self-DNA by the enzymatic PRR cyclic GMP-AMP synthase (cGAS). Contrary to expectations, we identified three functional classes of this PRR in mammals. Class 1 proteins (including human) contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream N-terminal domain. Class 2 and 3 proteins were either not self-DNA reactive (including chimpanzee) or included proteins whose N-terminal domain promoted self-DNA reactivity (mouse). While self-DNA reactivity of Class 1 cGAS was linked to an ability to access intra-mitochondrial DNA, mitochondrial localization was not associated with other classes. These studies reveal unexpected diversity in the mechanisms of self-DNA reactivity of a PRR.

One Sentence SummaryThe regulation of self-DNA reactivity of cGAS is evolutionarily diverse in mammals.

category: immunology


title: Identification of a new Inovirus in the absence of bacterial infection in the human virome

abstract: Viruses infecting bacteria, known as bacteriophages, represent the most abundant viral particles in the human body. They participate in the control of the human associated bacterial communities and play an important role in the dissemination of virulence genes. Here we present the identification of a new genetic element, named RIP1, in the human virome. RIP1 shares conserved structural genes with the single-stranded DNA viruses of the Inoviridae family. Furthermore structure-function studies identified nuclear subcellular localization of the RIP1 DNA replication/recombination machinery. Additional metagenomics analysis and polymerase chain reaction detected RIP1 in multiple body sites including blood, cerebrospinal pericardial and amniotic fluids, nasal swabs and feces in the absence of concomitant bacterial infection, uncovering inovirus phage persistence in the human virome.


title: Unblocking genome editing of major animal mycoplasmas using CRISPR/Cas9 base editor systems

abstract:
Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically important livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is still a bottleneck for many mycoplasma species. Here, we demonstrate the efficacy of a CRISPR-derived genetic tool to introduce targeted mutations in three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible dCas9-cytidine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was sufficiently powerful to disrupt 54 of 55 insertion sequence transposases in a single step. Whole genome sequencing showed that off-target mutations were limited and suggest that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and, most likely, the entire class Mollicutes.

SignificanceMycoplasmas are minimal wall-less pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies for many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. We developed a CRISPR-derived base editor for three major pathogenic species, Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies in these economically important bacteria.


title: The therapeutic potential of D-Serine in reducing expression of the cytopathic genotoxin colibactin

abstract: Some Escherichia coli strains belonging mainly to the B2 phylogroup harbour the pks island, a 54 kb genomic island encoding the biosynthesis genes for a genotoxic compound named colibactin. In eukaryotic cells, colibactin can induce DNA damage, cell cycle arrest and chromosomal instability. Moreover, production of colibactin has been implicated in the development of colorectal cancer. In this study, we demonstrate the inhibitory effect of D-Serine on the expression of the pks island in two colibactin-producing strains, CFT073 and Nissle 1917, and determine the implications for cytopathic effects on host cells. To investigate the specificity of the inhibitory effect of D-Serine, we also tested a comprehensive panel of proteinogenic L-amino acids and corresponding D-enantiomers for their ability to modulate clbB transcription using RT-qPCR. Several D-amino acids exhibited the ability to inhibit expression of clbB, with D-Serine exerting the strongest repressing activity (3.81-fold in CFT073; 3.80-fold in Nissle 1917) and thus, we focussed additional experiments on D-Serine. To investigate the cellular effect, we investigated if repression of colibactin by D-Serine could reduce the cytopathic responses normally observed during infection of HeLa cells with pks+ strains. Levels of {gamma}-H2AX (a marker of DNA double strand breaks) were reduced 2.75-fold in cells infected with D-Serine treatment. Moreover, exposure of pks+ E. coli to D-Serine during infection caused a reduction in cellular senescence that was observable at 72 h post infection. The recent finding of an association between pks-carrying commensal E. coli and CRC, highlights the necessity for the development of colibactin targeting therapeutics. Here we show that D-Serine can reduce expression of colibactin, and inhibit downstream cellular cytopathy, illuminating its therapeutic potential to prevent colibactin-associated disease.


title: The Anti-Burkholderia Lasso Peptide Ubonodin Co-Opts the Siderophore Receptor PupB for Cellular Entry

abstract: New antibiotics are needed as bacterial infections continue to be a leading cause of death. Notorious among antibiotic-resistant bacteria is the Burkholderia cepacia complex (Bcc), which infects cystic fibrosis patients, causing lung function decline. We recently discovered a novel ribosomally synthesized and post-translationally modified peptide (RiPP), ubonodin, with potent activity against several Burkholderia pathogens. Ubonodin inhibits RNA polymerase, but only select Bcc strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we speculate that cellular uptake of ubonodin determines susceptibility. Here, we report a new outer membrane siderophore receptor, PupB, that is required for ubonodin uptake in B. cepacia. Loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. Thus, outer membrane transport is the major determinant of ubonodins spectrum of activity. We also show that PupB is activated by a TonB protein and examine a transcriptional pathway that further regulates PupB. Finally, we elucidate the complete cellular uptake pathway for ubonodin by also identifying its inner membrane transporter in B. cepacia. Our work unravels central steps in the mechanism of action of ubonodin and establishes a general framework for dissecting RiPP function.


title: Antibiotic-induced recombination in Gram-negative bacteria requires the formation of double-strand breaks

abstract: Recombination is an essential process in bacterial drug resistance evolution. Fluoroquinolones, an important class of antibiotics, are known to stimulate recombination in Escherichia coli. On the other hand, when bacteria are exposed to sublethal concentrations of different antibiotic families, including strong inducers of the SOS system, which upregulates recombination pathways, there is no detectable change in the recombination rate. Here we explore why fluoroquinolones, but not any other tested antimicrobials, increase recombination rates. One feature that makes the fluoroquinolones different from other antibiotics is the generation of DNA double-strand breaks (DSBs). We tested whether other antimicrobials known for their ability to cause double-strand breaks, such as mitomycin C and bleomycin, are also affecting bacterial recombination rate. We found that both drugs indeed affected recombination rates in E. coli. Furthermore, there was a positive correlation between the number of DNA double-strand breaks and the recombination frequency. The manipulation of the level of DSBs directly impacted the recombination frequency. These results imply that recombination, at least in Gram-negative bacteria, is conservative, and even if multiple copies of a DNA sequence are available, the basal recombination rate is low. Our results highlight that homologous recombination is intended more for DNA damage repair than generating diversity, or at least that they occur at a very different scale. We also show that only antibiotics that cause DNA double-strand breaks can increase genetic diversity via recombination. The stimulation of recombination by DSBs-causing antimicrobials is an additional factor leading to the risk of antibiotic resistance evolution.


category: molecular biology


title: Genomic landscape of the DHA1 family in the newly emerged pathogen Candida auris and mapping substrate repertoire of the prominent member CauMdr1

abstract: The last decade has witnessed the rise of extremely threatening healthcare-associated multidrug resistant non-albicans Candida (NAC) species, Candida auris. Thus, understanding the molecular basis of antifungal resistance has emerged as the single most important goal amongst the research community. Since besides target alterations, efflux mechanisms contribute maximally to antifungal resistance, it is imperative to investigate their contributions in this pathogen. Of note, within the Major Facilitator Superfamily (MFS) of efflux pumps, Drug/H+ antiporter family 1 (DHA1) has been established as a predominant contributor towards xenobiotic efflux. Our study provides a complete landscape of DHA1 transporters encoded in the genome of C. auris. This study identifies 14 DHA1 transporters encoded in the genome of the pathogen. We also construct deletion and heterologous overexpression strains for the most important DHA1 drug transporter, viz., CauMdr1 to map the spectrum of its substrates. While the knockout strain did not show any significant changes in the resistance patterns against the majority of the tested substrates, the ortholog when overexpressed in a minimal background S. cerevisiae strain, AD1-8u-, showed significant enhancement in the Minimum inhibitory concentrations (MICs) against a large panel of antifungal molecules. Altogether, the present study provides a comprehensive template for investigating the role of DHA1 members of C. auris in antifungal resistance mechanisms.


title: The Fate of DIET Consortium exposed to continuous and intermittent electrochemical stimulation

abstract: Electromethanogenesis is the electrochemical conversion of carbon dioxide (CO2) to methane (CH4) with microorganisms as catalysts, and it is a promising approach for biogas upgrading. Many studies have shown increased methane production by electrochemical stimulation, allegedly due to the enhancement of direct interspecies electron transfer (DIET) between electrogenic bacteria and methanogens. However, these studies were done with mixed cultures where the interspecies interactions were undefined, so the impact of an electrochemical stimulus on these interactions remains unresolved. Here we report that an electrochemical stimulus diminished the survival and activity of a defined DIET consortium. We investigated the impact of electrochemical stimulation on a co-culture of Geobacter metallireducens and Methanosarcina barkeri, where the two partners interact syntrophically via DIET. The electrochemical stimulus was a cathode poised at - 700 mV (versus the standard hydrogen electrode, SHE). Electrochemical stimulation was provided either continuously or intermittently, and so was the food substrate. Compared to the untreated DIET co-culture, all treatment strategies relying on cathodic current additions resulted in acetate accumulation, lower methane production and lower cell numbers for both partners, indicating a detrimental impact of electrochemical stimulation to a DIET consortium. These findings suggest that future bioelectrochemical technologies must guarantee the survival of the syntrophic partners during current additions.


title: Profibrotic priming of airway cell types and drug responses in early-stage idiopathic pulmonary fibrosis

abstract: Early genetic studies hinted the role of airway epithelial cells in the development of idiopathic pulmonary fibrosis (IPF), while recent single-cell RNA sequencing (scRNA-seq) atlases utilized explant IPF lungs and therefore represent late-stage disease. Here, we used air liquid interface (ALI) cultures of primary cells taken from the subsegmental bronchi of newly diagnosed IPF patients, reflecting early-stage fibrosis, to interrogate the transcriptional landscape of the airway mucosa. Profiling of 129,986 cells identified a shared proinflammatory state in epithelial cells and an early activation state of fibroblasts. Moreover, IPF basal cells initiated awry repair mechanisms and primed the airway mucosa for TGF-{beta} activation. Treatment with nintedanib, pirfenidone, both established antifibrotic drugs, and saracatinib, an Src kinase inhibitor that can limit IPF progression, only significantly affected certain IPF signatures. This study provides insight into the early disease mechanisms of IPF and may serve as a resource to further investigate pharmacological inhibition effects.


title: Lrig3 restricts the size of the colon stem cell compartment

abstract:
The cellular census of the colonic crypt is tightly regulated, yet the molecular mechanisms that regulate this census are not fully understood. Lrig3, a transmembrane protein, is expressed in colonic crypt epithelial cells, including the stem, progenitor, and differentiated cell types. Mice missing Lrig3 have a disruption in their cellular census: using a novel Lrig3-/- mouse we demonstrate that Lrig3-/- mice have more cells per crypt, a greater mucosal area, and longer colons compared to wildtype mice, suggesting the expression of Lrig3 is required for both the total number of epithelial cells in the mouse colon, as well as colon length. In addition, we show Lrig3-/- mice have significantly more stem, progenitor, and deep crypt secretory cells, yet harbor a normal complement of enteroendocrine, Tuft, and absorptive cells. Lrig3-/- mice also have a concomitant decrease in phosphorylated Extracellular signal-related kinases, indicating the loss of Lrig3 leads to an expansion of the colonic stem cell compartment, in an Erk-dependent manner. Our study describes the expression of Lrig3 within the colon, defines perturbations in mice lacking Lrig3, and supports a role for Lrig3 in the establishment of both colonic crypt structure and cellular census, defined as the epithelial cell type and number in colon crypts.

Graphical Abstract

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title: Genome-wide base editor screen identifies regulators of protein abundance in yeast

abstract: Abundance of proteins is extensively regulated both transcriptionally and post-transcriptionally. To systematically characterize how regulation of protein abundance is encoded in the genome and identify protein regulators on a genome-wide scale, we developed a genetic screen that uses a CRISPR base editor. We examined the effects of 16,452 genetic perturbations on the abundance of eleven yeast proteins representing a variety of cellular functions. Thereby, we uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Our results provide unprecedented insights into the components, scale and connectedness of the protein regulatory network.


title: Molecular profiling of XPO1 inhibitor and gemcitabine-nab-paclitaxel combination in cellular and LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) pancreatic cancer model

abstract:
The majority of pancreatic ductal adenocarcinoma (PDAC) patients experience disease progression while on treatment with gemcitabine and nab-Paclitaxel (GemPac) treatment indicating the need for more effective combinations for this recalcitrant disease.

Earlier we showed that nuclear exporter protein exportin 1 (XPO1) is a valid therapeutic target in PDAC and the selective inhibitor of nuclear export (SINE) selinexor (Sel), synergistically enhances the efficacy of GemPac in pancreatic cancer cells, spheroids, patient derived tumors and had promising activity in a phase I study in patients with PDAC. Here we investigated the mechanisms of synergy by molecular profiling of Sel or Sel-GemPac treated PDAC cells, in vitro and by utilizing genetically modified LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) mouse model.

In KPC model, Sel given with GemPac at a sub-MTD dose enhanced the survival compared to controls (p < 0.05). Molecular analysis of residual KPC tumors showed re-organization of tumor stromal architecture, suppression of proliferation and nuclear retention of tumor suppressors. Single cell nuclear RNA sequencing (snRNAseq) revealed significant loss of cellular clusters in the Sel-GemPac treated mice including CD44 stem cell population. RNA-seq, Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) analysis showed inhibition of several tumor promoting molecules.

Prioritized RNA-seq identified molecules were validated in in vitro or in the PDAC patient samples through siRNA mediated silencing, quantitative gene expression, cytotoxicity assays and confirmed their role in observed synergy. Sel or Sel-GemPac caused broad penetration in PDAC supporting signaling networks.