title: Species-specific cutaneous protein signatures upon incision injury and correlation with distinct pain-related phenotypes in humans

abstract: Pain after surgery is common, and its management remains a clinical challenge. Severe acute and prolonged post-surgical pain impairs immediate recovery and leads to long-term consequences like chronic pain, opioid dependency, and reduced quality of life. Althought rodent pain incision models exist, translation to patients is still hampered. To bridge this gap, we combined sensory phenotyping with quantitative proteomics and protein networks in humans and mice after skin incision representing an established model for surgical pain. Initially, we revealed, for the first time, similarities and differences of protein-protein interaction (PPI) networks across both species. Next, we comprehensively phenotyped humans for pain-related symptoms and observed phenotypes with incision-induced proteome changes. Remarkably, post-incision PPI-networks differed between volunteers with small incision-related hyperalgesic areas ("Low responder") versus those with large areas ("High responder"). The latter exhibited a pronounced proteolytic environment associated with persistent inflammation, while an anti-inflammatory protein signature was observed in Low responders. Taken together, we provide unprecedented insights into peripheral processes relevant for developing hyperalgesia and pain after incision. This knowledge will immensely facilitate bidirectional translational pain studies and guide future research on the pathophysiology of pain after surgery and the discovery of novel targets for its treatment and prevention.


title: Characterizing Low-Frequency Artifacts During Transcranial Temporal Interference Stimulation (tTIS)

abstract: Transcranial alternating current stimulation (tACS) is a well-established brain stimulation technique to modulate human brain oscillations. However, due to the strong electro-magnetic artifacts induced by the stimulation current, the simultaneous measurement of tACS effects during neurophysiological recordings in humans is challenging. Recently, transcranial temporal interference stimulation (tTIS) has been introduced to stimulate neurons at depth non-invasively. During tTIS, two high-frequency sine waves are applied, that interfere inside the brain, resulting in amplitude modulated waveforms at the target frequency. Given appropriate hardware, we show that neurophysiological data during tTIS may be acquired without stimulation artifacts at low-frequencies. However, data must be inspected carefully for possible low-frequency artifacts. Our results may help to design experimental setups to record brain activity during tTIS, which may foster our understanding of its underlying mechanisms.

category: neuroscience


title: Dentate gyrus mossy cells exhibit sparse coding via adaptive spike threshold dynamics

abstract:
Hilar mossy cells (hMCs) are glutamatergic neurons in the dentate gyrus (DG) that receive inputs primarily from DG granule cells (GCs), CA3 pyramidal cells and local inhibitory interneurons. The hMCs then provide direct excitatory and disynaptic inhibitory feedback input to GCs. Behavioral and in vivo single unit recording experiments have implicated hMCs in pattern separation as well as is in spatial navigation and learning. It has, however, been difficult to mechanistically link the in vivo physiological behavior of hMCs with their intrinsic excitability properties that convert their synaptic inputs into spiking output. Here, we carried out electrophysiological recordings from the main cell types in the DG and found that hMCs displayed a highly adaptive threshold acting over a remarkably protracted time-scale. The hMC spike threshold increased linearly with increasing current stimulation and saturated at high current intensities. This threshold also increased in response to spiking and this effect also decayed over a long timescale, allowing for activity-dependent summation that limited hMC firing rates. This mechanism operates in parallel with a prominent medium after-hyperpolarizing potential (AHP) generated by the small conductance K+ channel. Based on experimentally derived parameters, we developed a phenomenological exponential integrate-and-fire model that closely mimics the hMC adaptive threshold. This lightweight model is amenable to its incorporation into large network models of the DG that will be conducive to deepen our understanding of the neural bases of pattern separation, spatial learning and navigation in the hippocampus.

Statement of significanceRecent studies on hilar mossy cells have revealed that they are implicated in spatial navigation and mnemonic functions. Yet, the basic intrinsic characterization of these hMCs is still too superficial to explain their spiking behavior in vivo. Here, we describe novel biophysical properties of hMCs, including an independent relationship between spike latency and spike threshold as well as a slowly adapting spike threshold. These findings complement several other biophysical and connectivity similarities between hMCs and CA3 pyramidal cells, while emphasizing the contrast with hilar interneurons. Additionally, our results are well captured by a phenomenological model of the hMC which provides a useful framework to study the neural substrate of spatial navigation and learning in the dentate gyrus.


title: Self-navigating the "Island of Reil": a systematic review of real-time fMRI neurofeedback training of insula activity

abstract: Real-time fMRI (rtfMRI) neurofeedback (NF) is a novel noninvasive technique that permits individuals to voluntarily control brain activity or connectivity, with demonstrated feasibility in experimental and therapeutic applications. The crucial role of the insula in emotional and salience processing makes it a popular target in rtfMRI studies although there is no systematic review of their efficacy. To promote our understanding of mechanisms underlying insula regulation and thereby facilitate therapeutic translation, the present review identified 22 preclinical and clinical studies and found that rtfMRI-based NF training is efficient for modulating insula activity (> 86%) and its associated behavioral and neural changes. Based on findings, continuous feedback for multiple training sessions, specific strategies validated before application, inclusion of a practice session, and choosing appropriate localization strategies are suggested as guidelines. We also recommend standardization of training protocol design, statistical analyses and results reports for future studies. In summary, the present review informs both fundamental research and therapeutic translation of NF training as an intervention in mental disorders, particularly those with insula dysfunction.


title: E. coli can eat DNA as excellent nitrogen source to grow quickly

abstract: Is DNA or RNA a good nutrient? Although scientists have raised this question for dozens of years, few textbooks mention the nutritional role of nucleic acids. Paradoxically, mononucleotides are widely added to infant formula milk and animal feed for improving Intestinal health. Here, found that E. coli can grow well in the medium with DNA as carbon and nitrogen sources. More interestingly, in the presence of glucose and DNA, bacteria grew more rapidly, showing that bacteria can use DNA as an excellent nitrogen source. Surprisingly, the amount of DNA in culture media decreased but its length remained unchanged, demonstrating that E. coli ingested long DNA directly. The gene expression study shows that E. coli mainly ingests DNA before digestion and digests it in the periplasm. Bifidobacterium bifidum can also use DNA as the nitrogen source for growth but not efficiently as E. coli. This study is of great significance to study DNA metabolism and utilization in organisms. It also lays a foundation to understand the nutritional function of DNA for intestinal flora and human health.

category: microbiology


title: Myoglobin-derived iron causes phagocyte dysfunction, wound enlargement, and impaired regeneration in pressure injuries of muscle

abstract:
Pressure injuries are classified as chronic wounds, but the reasons for poor healing are not well understood, such as whether impairments are due to comorbidities and bacteria. Chronic vascular ulcers have pathologies caused by extracellular release of hemoglobin, so we questioned whether muscle pressure injuries (mPI) would have intrinsic pathologies due to extracellular myoglobin (Mb), and whether iron chelation therapy could improve them.

Healthy mice in specific-pathogen-free conditions received pressure injuries. Cohorts were Mb-/- versus Mb+/+ in elderly 20-month-olds, or adult Mb+/+ 5-month-olds treated with saline versus iron chelator (subcutaneous deferoxamine, DFO). For comparison, healthy control regeneration was created using cardiotoxin injection.

Unlike acute injuries, the wound bed of mPI displayed failure of phagocytosis at day 3, accompanied by high accumulation of iron, oxidative damage, and a lack of viable immune cells. The necrotic tissue was later expelled as slough (eschar). Mb-knockout mPI had undetectable levels of iron, low oxidative stress (p<0.05), greater immune infiltration (230%, p<0.05), 4-fold decreased marker for extracellular traps (p<0.01), and 50% smaller area of tissue death. Similar to knockout, injecting DFO after wounding caused improvement in oxidative stress (2-fold, p<0.05), extracellular trap marker (4-fold, p<0.01), and diameter of tissue killed (35%, p<0.05). In other words, tissue margins could be protected from dying. At later timepoints, DFO-treated wounds had higher levels of viable immune infiltrate (p<0.05), greater extent of muscle regeneration (46% smaller diameter of gap, p<0.01), and superior myofiber morphology (p<0.0001).

We conclude that myoglobin iron from mPI causes oxidative stress and delayed infiltration, despite a lack of infection. Remarkably, tissue viability could be salvaged by post-injury treatment with subcutaneous injections of an FDA-approved iron chelator. This work also sheds light on how a hostile wound microenvironment impairs myogenesis and morphogenesis.

category: molecular biology


title: Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality

abstract: Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions led to two outcomes -- removal of cytoplasmic material and stem cell division, or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.

category: developmental biology


title: Resource use differences of two coexisting chironomid species at localized scales

abstract: The abundances of competing species may show positive correlations in time and space if they rely on a shared resource. Such positive correlations might obscure resource partitioning that facilitates coexistence of competitors and affects their abundances, spatial distributions, and population dynamics. Here, we examine the potential for resource partitioning between two ecologically similar midge species (Diptera: Chironomidae) in Lake Myvatn, Iceland. Tanytarsus gracilentus and Chironomus islandicus larvae coexist at high abundances in benthic habitats, and they have been previously described as feeding upon diatoms and detritus. Furthermore, both species show large, roughly synchronized population fluctuations, implying potential reliance on a shared fluctuating resource and posing the question of how these species coexist at high abundances. We first considered spatial partitioning of larvae; across multiple sites, abundances of both species were positively correlated. Thus, spatial partitioning across different sites in the lake did not appear to be strong. We then inferred differences in dietary resource use with stable carbon isotopes. T. gracilentus larvae had higher {delta}13C values than C. islandicus (mean difference = 5.39 {+/-} 1.84{per thousand}), suggesting interspecific differences in resource use. Differences in resource selectivity, tube-building behavior, and feeding styles may facilitate resource partitioning between these two species. Relative to surface sediments, T. gracilentus had higher {delta}13C values (1.84 {+/-} 0.96{per thousand}), suggesting that they selectively graze on 13C-enriched resources such as productive algae from the surface of their tubes. In contrast, C. islandicus had lower {delta}13C values than surface sediments (-2.87 {+/-} 1.95{per thousand}), suggesting reliance on isotopically depleted resources, which may include detrital organic matter and associated microbes that larvae selectively consume from the sediment surface or within their tube walls. Overall, our study illustrates that coexisting and ecologically similar species may show positive correlations in space and time while using different resources at fine spatial scales.

category: ecology


title: Fish microbiomes 101: disentangling the rules governing marine fish mucosal microbiomes across 101 species

abstract: Fish are the most diverse and widely distributed vertebrates, yet little is known about the microbial ecology of fishes nor the biological and environmental factors that influence the fish microbiome. The microbiota from 101 species of Southern California marine fishes, spanning 22 orders, 55 families, and 83 genera representing ~25% of local marine fish diversity, was analyzed to identify patterns that explain microbial diversity patterns in a geographical subset of marine fish biodiversity. We compared fish microbiomes (gill, skin, midgut, and hindgut) using alpha, beta, and gamma diversity along with establishing a novel method to estimate microbial biomass (Qiime2 plugin katharoseq). For oceanic fishes from the neritic zone, host size and distance from shore were negatively associated with microbial biomass densities and diversity in the gills. Body site was the strongest driver for beta diversity with strong evidence of phylosymbiosis observed across the gill, skin, and hindgut, but not midgut. The majority of microbes from all fish body sites were of unknown origin but overall sea water generally contributes more microbes to fish mucus compared to marine sediment. In a meta-analysis of vertebrate hindguts (569 species), mammals had the highest gamma diversity when controlling for host species number while fishes had the highest percent of unique microbial taxa (92%). In fishes, the midgut, gill, and skin contains the majority of microbial diversity which collectively can be 5.5 times higher than the hindgut. The composite dataset will be useful to vertebrate microbiome researchers and fish biologists interested in microbial ecology with applications in aquaculture and fisheries management.


title: Rapid identification of mosquito species, sex and age by mass spectrometric analysis

abstract:
BackgroundA rapid, accurate method to identify and to age grade mosquito populations would be a major advance in predicting the risk of pathogen transmission and evaluating the public health impact of vector control interventions. Whilst other chemometric methods show promise, current approaches rely on challenging morphological techniques or simple binary classifications that cannot identify the subset of the population old enough to be infectious. In this study, the ability of Rapid Evaporative Ionisation Mass Spectrometry (REIMS) to identify the species, sex and age of mosquitoes reared in the laboratory and derived from the wild was investigated.

ResultsThe accuracy of REIMS in identifying morphologically identical species of the Anopheles gambiae complex exceeded 97 % using principal component/linear discriminant analysis (PC-LDA) and 84 % based on random forest analysis. Age separation into 3 different age categories (1 d, 5-6 d, 14-15 d.) was achieved with 99 % (PC-LDA) and 94 % (random forest) accuracy, after including three species and storing samples at room temperature for up to 11 days. When tested on wild mosquitoes from the UK, REIMS data could determine the species and age of the specimens with accuracies of 91 and 90 % respectively.

ConclusionsThe accuracy of REIMS to resolve the species and age of Anopheles mosquitoes is comparable to that achieved by chemometric approaches. The processing time and ease of use represent significant advantages over current, dissection-based methods. Importantly the accuracy was maintained when using wild mosquitoes reared under differing environmental conditions, and when mosquitoes were stored frozen or desiccated. This high throughput approach thus has potential to conduct rapid, real- time monitoring of vector populations, providing entomological evidence of the impact of alternative interventions.


title: Host phylogeny, but not diet diversity, influences gut microbiome composition in breeding wood-warblers

abstract: Understanding the factors that shape microbiomes can provide insight on the importance of host-symbiont interactions and on co-evolutionary dynamics. Unlike for mammals, previous studies have found little or no support for an influence of host evolutionary history on avian gut microbiome diversity and instead have suggested a greater influence of the environment or diet. Because effects of different factors may be conflated by captivity and sampling design, examining natural variation using large sample sizes is important. Our goal was to overcome these limitations by sampling wild birds to compare environmental, dietary, and evolutionary influences on gut microbiome structure. We performed fecal metabarcoding to characterize both the gut microbiome and diet of fifteen wood-warbler species across a four-year period and from two geographic localities. We find host taxonomy generally explained ~10% of the variation between individuals, which is ~6-fold more variation of any other factor considered, including diet diversity. Further, gut microbiome similarity was more congruent with the host phylogeny than with host diet similarity, and diet diversity was not associated with microbiome diversity. Together, our results suggest evolutionary history is the strongest predictor of gut microbiome differentiation among wood-warblers. Although the phylogenetic signal of the warbler gut microbiome is not very strong, our data suggest a stronger influence of diet (as measured by diet diversity) does not account for this pattern. The mechanism underlying this phylogenetic signal is not clear, but we argue host traits may filter colonization by microbes despite small host body size (and fast gut turnover) which is characteristic of wood-warblers.

category: evolutionary biology


title: Yeast galactokinase in closed conformation can switch between catalytic and signal transducer states.

abstract:
S.cerevisiae galactokinase (ScGal1p), in closed conformation catalyzes the phosphorylation of galactose to galactose 1-phopshate using ATP as the phosphate donor as well as allosterically activates the GAL genetic switch in response to galactose and ATP as ligands. How both kinase and signaling activities of ScGal1p are associated with closed conformation of the protein is not understood. Conformational sampling of ScGal1p indicated that this protein samples closed kinase and closed non-kinase conformers. Closed non-kinase conformers are catalytically incompetent to phosphorylate galactose and act as a bonafide signal transducer. It was observed that toggling of side chain of highly conserved K266 of ScGal1p between S171and catalytic base D217 is responsible for transitioning of ScGal1p between signal transducer and kinase states. Interestingly in ScGal3p, the paralog of ScGal1p, which has only signal transduction activity and lacks kinase activity, a H bond between a non-conserved Y433, and a highly conserved Y57, gets broken during MD simulation. The corresponding H-bond present in ScGal1p between residues Y441 and Y63 respectively, remains intact throughout the simulations of ScGal1p.Therefore, we predicted that K266 and Y441 have a role in bifunctionality of ScGal1p. To test the above predictions, we monitored the signaling and kinase activity of ScGal1K266Rp and ScGal1Y441Ap variants. Signaling activity increased in both ScGal1Y441Ap and ScGal1K266Rp variants as compared to ScGal1wtp, whereas the kinase activity increased in ScGal1Y441Ap, but decreased in ScGal1K266Rp Based on the above, we propose that K266 and Y441 are crucial for conferring bifunctionality to ScGal1p.

Author summaryGalactokinase of S.cerevisiae(ScGal1p), the first enzyme of Leloir pathway of galactose metabolism, phosphorylates galactose using ATP as the phosphate donor. ScGal1p also functions as a signal transducer of GAL regulon wherein galactose and ATP allosterically activate galactokinase. The active form of galactokinase, then sequesters the repressor ScGal80p, to activate the GAL switch. ScGal1p has a single site each for binding to galactose and ATP. How ScGal1p, a monomeric protein, performs the above two mutually exclusive activities using the same set of substrates/ligands, with the same site acting as the active site for enzymatic activity as well as allosteric site for signal transduction activity is unclear. Our findings are that this protein has a distinct conformational state for functioning as a signal transducer and a distinct conformational state for functioning as a kinase. A highly conserved lysine residue (K266) present only in fungal galactokinases, triggers the interconversion between catalysis and signal transduction states. This interconversion is subdued by H bond between Y441 and Y63. These studies suggest that the two activities of ScGal1p are fine tuned by evolution to regulate metabolism through transcriptional control.

category: genetics


title: Calcium homeostasis disruption initiates rapid growth after micro-fragmentation in the scleractinian coral Porites lobata

abstract: Coral reefs are ecosystems under increasing threat from global climate change. Coral restoration is a tool for preserving biological and ecological function of coral reefs by mitigating coral loss and maintaining the structural integrity and complexity of reefs. To generate the necessary stock for coral restoration, larger coral colonies are usually fragmented to generate smaller specimens for outplanting, taking advantage of the high regenerative ability of corals. In this study, we utilized RNA-seq technology to understand the physiological responses of Porites lobata colonies to physical fragmentation and outplanting, which have thus far not been characterized. Our results demonstrate that P. lobata fragments undergoing physical injury recover through two distinct phases: rapid wound regeneration of the cut margins, followed by a slower growth phase that cements the colony to the substrate. Our study found rapid physiological responses to acute physical injury and outplanting in the coral host that involved significantly increased energy production, calcium homeostasis disruption, and ER stress leading to increased antioxidant expression and rates of protein turnover. Our results suggest that phosphoinositide-mediated acute calcium homeostasis disruption stimulates wound recovery processes in response to physical injury. Contrary to other coral transcriptomic experiments, symbiont gene expression revealed extremely low gene differences in response to fragmentation, growth, and outplanting. These results provide insight into the physiological mechanisms that allow for rapid wound healing and stabilization in response to physical injury in corals.


title: Decreased spliceosome fidelity inhibits mTOR signalling and promotes longevity via an intron retention event

abstract: Changes in splicing fidelity are associated with loss of homeostasis and ageing1-3, yet only a handful of splicing factors have been shown to be causally required to promote longevity 1-3, and the underlying mechanisms and downstream targets in these paradigms remain elusive. Surprisingly, we found a hypomorphic mutation within RNP-6/PUF60, a spliceosome component promoting weak 3 splice site recognition, which causes aberrant splicing, elevated stress responses, and enhances longevity in Caenorhabditis elegans. Through genetic suppressor screens, we identify a gain-of-function mutation within rbm-39, an RNP-6 interacting splicing factor, which increases nuclear speckle formation, alleviates splicing defects and curtails longevity caused by rnp-6 mutation. By leveraging the splicing changes induced by RNP-6/RBM-39 activities, we uncover a single intron retention event in egl-8/phospholipase C B4 as a key splicing target prolonging life. Genetic and biochemical evidence show that neuronal RNP-6/EGL-8 downregulate mTORC1 signaling to control organismal life span. In mammalian cells, PUF60 downregulation also potently and specifically inhibits mTORC1 signaling. Altogether, our results reveal that splicing fidelity modulates mTOR signaling and suggest a potential therapeutic strategy to delay ageing.


title: Dissecting the Pyrenophora tritici-repentis (tan spot of wheat) pangenome

abstract:
We sequenced the genome of a global collection (40 isolates) of the fungus Pyrenophora tritici-repentis (Ptr), a major foliar pathogen of wheat and model for the evolution of necrotrophic pathogens. Ptr exhibited an open-pangenome, with 43% of genes in the core set and 57% defined as accessory (present in only a subset of isolates), of which 56% were singleton genes (present in only one isolate). A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around the effector coding genes, were explored further using the annotated genomes of two isolates sequenced by PacBio RS II and Illumina HiSeq. The Ptr genome exhibited major chromosomal rearrangements, including chromosomal fusion, translocation, and segment duplications. An intraspecies translocation of ToxA, the necrosis-inducing effector-coding gene, was facilitated within Ptr via a 143 kb  Starship transposon (dubbed  Horizon). Additionally, ToxB, the gene encoding the chlorosis-inducing effector, was clustered as three copies on a 294 kb transposable element in a ToxB-producing isolate. ToxB and its carrying transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and the transposon were both present in another non-coding isolate. The Ptr genome also appears to exhibit a  one-compartment organization, but may still possess a  two-speed genome that is facilitated by copy-number variation as reported in other fungal pathosystems.

IMPORTANCEPtr is one of the most destructive wheat pathogens worldwide. Its genome is a mosaic of present and absent effectors, and serves as a model for examining the evolutionary processes behind the acquisition of virulence in necrotrophs and disease emergence. In this work, we took advantage of a diverse collection of pathogenic Ptr isolates with different global origins and applied short- and long-read sequencing technologies to dissect the Ptr genome. This study provides comprehensive insights into the Ptr genome and highlights its structural organization as an open pangenome with  one-compartment. In addition, we identified the potential involvement of transposable elements in genome expansion and the movement of virulence factors. The ability of effector-coding genes to shuffle across chromosomes on large transposons was illustrated by the intraspecies translocation of ToxA and the multi-copy ToxB. In terms of gene contents, the Ptr genome exhibits a large percentage of orphan genes, particularly in non-pathogenic or weakly-virulent isolates.

category: genomics


title: A global pangenome for the wheat fungal pathogen Pyrenophora tritici-repentis and prediction of effector protein structural homology

abstract:
The adaptive potential of plant fungal pathogens is largely governed by the gene content of a species, comprised of core and ancillary genes across the pathogen isolate repertoire. To approximate the complete gene repertoire of a globally significant crop fungal pathogen, a pan genomic analysis was undertaken for Pyrenophora tritici-repentis (Ptr), the causal agent of tan (or yellow) spot disease in wheat.

In this study, fifteen new Ptr genomes were sequenced, assembled and annotated, including isolates from three races not previously sequenced. Together with eleven previously published Ptr genomes, a pangenome for twenty-six Ptr isolates from Australia, Europe, North Africa and America, representing nearly all known races, revealed a conserved core-gene content of 56% and presents a new Ptr resource for searching natural homologues using remote protein structural homology. Here, we identify for the first time a nonsynonymous mutation in the Ptr effector gene ToxB, multiple copies of toxb, a distant natural Pyrenophora homologue of a known Parastagonopora nodorum effector, and clear genomic break points for the ToxA effector horizontal transfer region.

This comprehensive genomic analysis of Ptr races includes nine isolates sequenced via long read technologies. Accordingly, these resources provide a more complete representation of the species, and serve as a resource to monitor variations potentially involved in pathogenicity.

Author NotesAll supporting data, code and protocols have been provided within the article or through supplementary data files. Five supplementary data files and fifteen supplementary figures are available with the online version of this article.

Impact StatementOur Pyrenophora tritici-repentis (Ptr) pangenome study provides resources and analyses for the identification of pathogen virulence factors, of high importance to microbial research. Key findings include: 1) Analysis of eleven new sequenced (with three new races not previously available) and previously published isolates, 26 genomes in total, representing the near complete Ptr race set for known effector production collected from Australia, Europe, North Africa and the Americas. 2) We show that although Ptr has low core gene conservation, the whole genome divergence of other wheat pathogens was greater. 3) The new PacBio sequenced genomes provide unambiguous genomic break points for the large ToxA effector horizontal transfer region, which is only present in ToxA producing races. 4) A new web-based Ptr resource for searching in silico remote protein structural homology is presented, and a distant natural Pyrenophora protein homologue of a known effector from another wheat pathogen is identified for the first time.

Data SummaryThe sources and genomic sequences used throughout this study have been deposited in the National Centre for Biotechnology Information (NCBI), under the assembly accession numbers provided in Tables 1 and 2 (available in the online version of this article). The new M4 resource for protein structural homology is freely available through the BackPhyre web-portal URL, http://www.sbg.bio.ic.ac.uk/phyre2/.

O_TBL View this table:
org.highwire.dtl.DTLVardef@b709bdorg.highwire.dtl.DTLVardef@e089ecorg.highwire.dtl.DTLVardef@17f159dorg.highwire.dtl.DTLVardef@642b80org.highwire.dtl.DTLVardef@6405a1_HPS_FORMAT_FIGEXP  M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary statistics for our four PacBio sequenced Ptr genome assemblies, compared with those of two previously published Ptr assemblies.

C_TABLECAPTION C_TBL O_TBL View this table:
org.highwire.dtl.DTLVardef@ada1beorg.highwire.dtl.DTLVardef@155e117org.highwire.dtl.DTLVardef@12ceb3corg.highwire.dtl.DTLVardef@7ec3dorg.highwire.dtl.DTLVardef@26c5f1_HPS_FORMAT_FIGEXP  M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONIllumina sequenced genome assemblies of 11 new Ptr isolates. Table shows isolate source, race and de novo assembly statistics.

C_TABLECAPTION C_TBL


title: Differential DNA methylation in Pacific oyster reproductive tissue associated with ocean acidification

abstract:
BackgroundThere is a need to investigate mechanisms of phenotypic plasticity in marine invertebrates as negative effects of climate change are experienced by coastal ecosystems. Environmentally-induced changes to the methylome can regulate gene expression, but methylome responses may be tissue- and species-specific. To assess how ocean acidification affects the Pacific oyster (Crassostrea gigas) epigenome, we exposed oysters to either low pH (7.31 {+/-} 0.02) or ambient pH (7.82 {+/-} 0.02) conditions for seven weeks. Whole genome bisulfite sequencing was used to identify methylated regions in female oyster gonad samples. C->T single nucleotide polymorphisms were identified and removed to ensure accurate methylation characterization.

ResultsAnalysis of gonad methylomes revealed a total of 1,284 differentially methylated loci (DML) found primarily in genes, with several genes containing multiple DML. Gene ontologies for genes containing DML were involved in development and stress response, suggesting methylation may promote gonad growth homeostasis in low pH conditions. Additionally, several of these genes were associated with cytoskeletal structure regulation, metabolism, and protein ubiquitination -- commonly-observed responses to ocean acidification. Comparison of these DML with other Crassostrea spp. exposed to ocean acidification demonstrates that similar pathways, but not identical genes, are impacted by methylation.

ConclusionsOur work suggests DNA methylation may have a regulatory role in gonad and larval development. Combined with existing molluscan methylome research, our work further supports the need for tissue- and species-specific studies to understand the potential regulatory role of DNA methylation.


title: Comparative analysis of the Illumina Mouse Methylation BeadChip and Reduced Representation Bisulphite Sequencing for routine DNA methylation analysis of murine samples

abstract:
BackgroundResearching the murine epigenome in disease models has been hampered by the lack of an appropriate and cost-effective DNA methylation array. Until recently, investigators have been limited to the relatively expensive and analysis intensive bisulphite sequencing methods. Here, we performed a comprehensive, comparative analysis between the new Mouse Methylation BeadChip (MMB) and reduced representation bisulphite sequencing (RRBS) in two murine models of colorectal carcinogenesis, providing insight into the utility to each platforms in a real world environment.

ResultsWe captured 1.47x106 CpGs by RRBS and 2.64x105 CpGs by MMB, mapping to 13,778 and 13,365 CpG islands, respectively. RRBS captured significantly more CpGs per island (median 41 for RRBS versus 2 for MMB). We found that 64.4% of intra-island CpG methylation variability can be captured by measuring approximately one quarter of CpG island (CGI) CpGs. MMB was more precise in measuring DNA methylation, especially at sites that had low RRBS coverage. This impacted differential methylation analysis, with more statistically significantly differentially methylated CpG sites identified by MMB in all experimental conditions, however the difference was minute when appropriate thresholding for the magnitude of methylation change (0.2 beta value difference) was applied, providing confidence that both techniques can identify similar differential DNA methylation. Gene ontology enrichment analysis of differentially hypermethylated gene promoters identified similar biological processes and pathways by both RRBS and MMB across two murine model systems.

ConclusionMMB is an effective tool for profiling the murine methylome that performs comparably to RRBS, identifying similar differentially methylated pathways. Although MMB captures a similar proportion of CpG islands, it does so with fewer CpGs per island. We show that subsampling informative CpGs from CpG islands is an appropriate strategy to capture whole island variation. Choice of technology is experiment dependent and will be predicated on the underlying biology being probed.


title: Enzyme-Responsive Nanoparticles for the Targeted Delivery of an MMP Inhibitor to the Heart post Myocardial Infarction

abstract: In this paper, we describe block copolymer amphiphiles consisting of a hydrophilic matrix metalloproteinase (MMP) peptide substrate, and a hydrophobic small molecule MMP inhibitor PD166793 for the treatment of acute myocardial infarction. These resulting drug loaded peptide-polymer amphiphiles (PPAs) assemble in aqueous solution to yield drug loaded micellar nanoparticles. Following minimally invasive intravenous injection, these nanoparticles preferentially exit the vasculature and are physically trapped at the infarcted region of the heart due to MMP-induced peptide cleavage and aggregation. This MMP directed active assembly prevents the material from leaking out into the blood stream, enabling long-term retention. Further, we show that the conjugated MMP inhibitor (PD166793) is inactivated in the core of the micelles and can be released upon the action of proteases and esterases, leading to MMP inhibition. This work establishes a promising targeted nanoparticle platform for delivering small molecule therapeutics to the heart.

category: bioengineering


title: KMCP: accurate metagenomic profiling of both prokaryotic and viral populations by pseudo-mapping

abstract:
MotivationThe growing number of microbial reference genomes enables the improvement of metagenomic profiling accuracy but also imposes greater requirements on the indexing efficiency, database size, and runtime of taxonomic profilers. Additionally, most profilers focus mainly on bacterial, archaeal, and fungal populations, while less attention is paid to viral communities.

ResultsWe present KMCP, a novel k-mer-based metagenomic profiling tool that introduces genomic positions to nucleotide k-mers by splitting the reference genomes into chunks and then stores k-mers in a modified and optimized COBS index for fast alignment-free sequence searching. The index size of KMCP is smaller than that of COBS, and the batch searching speed is increased by 10 times. KMCP combines k-mer similarity and genome coverage information to reduce the false positive rate of k-mer-based taxonomic classification and profiling methods. Benchmarking results based on simulated and real data demonstrate that KMCP not only allows the accurate taxonomic profiling of prokaryotic and viral populations but also provides confident pathogen detection in clinical samples of low coverage.

Availability and ImplementationThe software is open-source under the MIT license and available at https://github.com/shenwei356/kmcp.

Contactshenwei356@cqmu.edu.cn (W., Shen); hp_cq@163.com, hupengcq@hospital.cqmu.edu.cn (P., Hu); renhong0531@cqmu.edu.cn (H., Ren)

category: bioinformatics


title: Protease Activity Analysis: A Toolkit for Analyzing Enzyme Activity Data

abstract: Analyzing the activity of proteases and their substrates is critical to defining the biological functions of these enzymes and to designing new diagnostics and therapeutics that target protease dysregulation in disease. While a wide range of databases and algorithms have been created to better predict protease cleavage sites, there is a dearth of computational tools to automate analysis of in vitro and in vivo protease assays. This necessitates individual researchers to develop their own analytical pipelines, resulting in a lack of standardization across the field. To facilitate protease research, here we present Protease Activity Analysis (PAA), a toolkit for the preprocessing, visualization, machine learning analysis, and querying of protease activity datasets. PAA leverages a Python-based object-oriented implementation that provides a modular framework for streamlined analysis across three major components. First, PAA provides a facile framework to query datasets of synthetic peptide substrates and their cleavage susceptibilities across a diverse set of proteases. To complement the database functionality, PAA also includes tools for the automated analysis and visualization of user-input enzyme-substrate activity measurements generated through in vitro screens against synthetic peptide substrates. Finally, PAA can supports a set of modular machine learning functions to analyze in vivo protease activity signatures that are generated by activity-based sensors. Overall, PAA offers the protease community a breadth of computational tools to streamline research, taking a step towards standardizing data analysis across the field and in chemical biology and biochemistry at large.


title: The MIF SNP rs755622 is a germline determinant of tumor immune activation in Glioblastoma

abstract: While immunotherapies have shown durable responses for multiple tumors, their efficacy remains limited in some advanced cancers, including glioblastoma. This may be due to differences in the immune landscape, as the glioblastoma microenvironment strongly favors immunosuppressive myeloid cells, which are linked to an elevation in immune-suppressive cytokines, including macrophage migration inhibitory factor (MIF). We now find that a single-nucleotide polymorphism (SNP) rs755622 in the MIF promoter associates with increased leukocyte infiltration in glioblastoma. Furthermore, we identified lactotransferrin expression as being associated with the rs755622 SNP, which could also be used as a biomarker for immune infiltrated tumors. These findings provide the first example in glioblastoma of a germline SNP that underlies differences in the immune microenvironment and identifies high lactotransferrin as a potential factor promoting immune activation.

category: cancer biology


title: Mechanisms underlying the efficacy of a rodent model of vertical sleeve gastrectomy - a focus on energy expenditure

abstract:
Background and aimsBariatric surgery remains the only effective and durable treatment option for morbid obesity. Vertical Sleeve Gastrectomy (VSG) is currently the most widely performed of these surgeries primarily because of its proven efficacy in generating rapid onset weight loss, improved glucose regulation and reduced mortality compared with other invasive procedures. VSG is associated with reduced appetite, however, the relative importance of energy expenditure to VSG-induced weight loss and changes in glucose regulation, particularly that in brown adipose tissue (BAT), remains unclear. The aim of this study is to investigate the role of BAT thermogenesis in the efficacy of VSG in a rodent model.

MethodsDiet-induced obese male Sprague-Dawley rats were either sham-operated, underwent VSG surgery or were pairfed to the food consumed by the VSG group. Rats were also implanted with biotelemetry devices between the interscapular lobes of BAT to assess local changes in BAT temperature as a surrogate measure of thermogenic activity. Metabolic parameters including food intake, body weight and changes in body composition were assessed. To further elucidate the contribution of energy expenditure via BAT thermogenesis to VSG-induced weight loss, a separate cohort of lean rats underwent complete excision of the interscapular BAT (iBAT lipectomy) or chemical denervation using 6-hydroxydopamine (6-OHDA). To localize glucose uptake in specific tissues, an oral glucose tolerance test was combined with an intraperitoneal injection of 2 deoxy-D-glucose (2DG)-14C, administered intraperitoneally. Transneuronal viral tracing was used to identify 1) sensory neurons directed to the stomach or small intestine (H129-RFP) or 2) chains of polysynaptically linked neurons directed to BAT (PRV-GFP) in the same animals.

ResultsFollowing VSG, there was a rapid reduction in body weight that was associated with reduced food intake, elevated BAT temperature and improved glucose regulation. Rats that underwent VSG had elevated glucose uptake into BAT compared to sham operated animals as well as elevated gene markers related to increased BAT activity (Ucp1, Dio2, Cpt1b, Cox8b, Ppargc) and markers of increased browning of white fat (Ucp1, Dio2, Cited1, Tbx1, Tnfrs9). Both iBAT lipectomy and 6-OHDA treatment significantly attenuated the impact of VSG on changes in body weight and adiposity in lean animals. In addition, surgical excision of iBAT following VSG significantly reversed VSG-mediated improvements in glucose tolerance, an effect that was independent of circulating insulin levels. Viral tracing studies highlight a patent neural link between the gut and BAT that include groups of premotor BAT-directed neurons in the dorsal raphe and raphe pallidus.

ConclusionCollectively, these data support a role for BAT in mediating the metabolic sequelae, particularly the improvement in glucose regulation following VSG surgery and highlight the need to better understand the contribution from this tissue in human patients.

category: physiology


title: TOR mediates cytokinin-driven development and defense cues

abstract: Target of Rapamycin (TOR) kinase is a conserved energy sensor that regulates plant growth and development in response to nutritional and environmental inputs. TOR is also involved in the regulation of plant immunity. TOR downregulation, for example, results in enhanced plant defense responses and plant resistance to several pathogens in Arabidopsis, rice, and tomato. Similarly, the plant hormone cytokinin (CK) has also been demonstrated to mediate both development and defense processes. Although TOR is positioned at the interface between development and defense, little is known about the mechanisms in which TOR may potentially regulate the switch between these two modalities. Here, we investigated the interplay between TOR-mediated development and TOR-mediated defense. We show that TOR-silencing or inhibition led to enhanced defense responses and disease resistance in WT or CK-deficient backgrounds. However, in a high-CK background, which is already immuno-activated and disease resistant, TOR-inhibition reduced defense responses and disease resistance. TOR-silencing resulted in the normalization of developmental phenotypes associated with high or low CK levels, as well as of other classical developmental mutants, demonstrating that TOR is required for the execution of developmental cues. We found that CK represses TOR activity, suggesting the existence of a cross talk mechanism between the two pathways. Our results demonstrate that TOR likely acts downstream to CK signaling and hormonal signaling in general, executing signaling cues resulting from both high and low CK, in both defense and development. Thus, differential regulation of TOR or TOR-mediated processes could underlie a development-defense switch.

category: plant biology


title: Plastid-specific RsmD methyltransferase and ribosome maturation factor RimM are crucial for 16S rRNA maturation and proteostasis

abstract: Chloroplast pre-ribosomal RNA (rRNA) undergoes maturation, which is critical for ribosome assembly. While the central and auxiliary factors in rRNA maturation have been elucidated in bacteria, their mode of action remains largely unexplored in chloroplasts. We now reveal chloroplast-specific factors involved in 16S rRNA maturation, RsmD methyltransferase (AtRsmD) and ribosome maturation factor RimM-like protein (AtRimM) in Arabidopsis thaliana. A forward genetic screen aimed to find suppressors of the Arabidopsis yellow variegated 2 (var2) mutant defective in photosystem II (PSII) quality control found a causal nonsense mutation in AtRsmD. The substantially impaired 16S rRNA maturation and translation due to the mutation rescued the leaf variegation phenotype by lowering the levels of PSII core proteins in var2. The subsequent co-immunoprecipitation coupled with mass spectrometry analyses and bimolecular fluorescence complementation assay found that AtRsmD interacts with AtRimM. Consistent with their interaction, loss of AtRimM also considerably impairs 16S rRNA maturation, with less methylation in m2G915 in 16S rRNA catalyzed by AtRsmD. The atrimM mutation also rescued var2 mutant phenotypes, corroborating the functional interplay between AtRsmD and AtRimM towards 16S rRNA maturation and chloroplast proteostasis.


title: Genetic context effects can override canonical cis regulatory elements in Escherichia coli

abstract: Recent experiments have shown that in addition to control by cis regulatory elements, the local chromosomal context of a gene also has a profound impact on its transcription. Although this chromosome-position dependent expression variation has been empirically mapped at high-resolution, the underlying causes of the variation have not been elucidated. Here, we demonstrate that 1 kb of flanking, non-coding synthetic sequences with a low frequency of guanosine and cytosine (GC) can dramatically reduce reporter expression compared to neutral and high GC-content flanks in E. coli. Despite the strong reduction in the maximal expression level from the fully-induced reporter, low GC synthetic flanks do not affect the time required to reach the maximal expression level after induction. Expression of the reporter construct is also affected by proximity to highly expressed ribosomal RNA operons depending on the relative orientation of transcription despite being insulated by strong transcriptional terminators, in a manner consistent with supercoiling competition. Overall, we demonstrate key determinants of transcriptional propensity that appear to act as tunable modulators of transcription, independent of regulatory sequences such as the promoter. These findings provide insight into the regulation of naturally occurring genes and specific rules for optimizing control of synthetic biology constructs.

category: systems biology


title: Comparative metaproteomics demonstrates different views on the complex granular sludge microbiome

abstract:
The tremendous progress in sequencing technologies has made 16S amplicon and whole metagenome sequencing routine in microbiome studies. Furthermore, advances in mass spectrometric techniques has expanded conventional proteomics into the field of microbial ecology. Commonly referred to as metaproteomics, this approach measures the gene products (i.e., proteins) to subsequently identify the actively-expressed metabolic pathways and the protein-biomass composition of complete microbial communities.

However, more systematic studies on metaproteomic and genomic approaches are urgently needed, to determine the orthogonal character of these approaches. Here we describe a deep, comparative metaproteomic study on the complex aerobic granular sludge microbiome obtained from different wastewater treatment plants. Thereby, we demonstrate the different views that can be obtained on the central nutrient-removing organisms depending on the  omic approach and reference sequence databases. Furthermore, we demonstrate a  homogenized Genome Taxonomy Database (GTDB) that subsequently enables a more accurate interpretation of data from different omics approaches. Ultimately, our systematic study underscores the importance of metaproteomics in the characterization of complex microbiomes; and the necessity of accurate reference sequence databases to improve the comparison between approaches and accuracy in scientific reporting.


title: The ZuCo Benchmark on Cross-Subject Reading Task Classification with EEG and Eye-Tracking Data

abstract:
We present a new machine learning benchmark for reading task classification with the goal of advancing EEG and eye-tracking research at the intersection between computational language processing and cognitive neuroscience. The benchmark task consists of a cross-subject classification to distinguish between two reading paradigms: normal reading and task-specific reading. The data for the benchmark is based on the Zurich Cognitive Language Processing Corpus (ZuCo 2.0), which provides simultaneous eye-tracking and EEG signals from natural reading. The training dataset is publicly available, and we present a newly recorded hidden testset. We provide multiple solid baseline methods for this task and discuss future improvements. We release our code and provide an easy-to-use interface to evaluate new approaches with an accompanying public leaderboard: www.zuco-benchmark.com.

HighlightsO_LIWe present a new machine learning benchmark for reading task classification with the goal of advancing EEG and eye-tracking research.
C_LIO_LIWe provide an interface to evaluate new approaches with an accompanying public leaderboard.
C_LIO_LIThe benchmark task consists of a cross-subject classification to distinguish between two reading paradigms: normal reading and task-specific reading.
C_LIO_LIThe data is based on the Zurich Cognitive Language Processing Corpus of simultaneous eye-tracking and EEG signals from natural reading.
C_LI


title: Distinguishing shadows from surface boundaries using local achromatic cues

abstract:
In order to accurately parse the visual scene into distinct surfaces, it is essential to determine whether a local luminance edge is caused by a boundary between two surfaces or a shadow cast across a single surface. Previous studies have demonstrated that local chromatic cues may help to distinguish edges caused by shadows from those caused by surface boundaries, but the information potentially available in local achromatic cues like contrast, texture, and penumbral blur remains poorly understood. In this study, we develop and analyze a large database of hand-labeled achromatic shadow edges to better understand what image properties distinguish them from occlusion edges. We find that both the highest contrast as well as the lowest contrast edges are more likely to be occlusions than shadows, extending previous observations based on a more limited image set. We also find that contrast cues alone can reliably distinguish the two edge categories with nearly 70% accuracy at 40x40 resolution. Logistic regression on a Gabor Filter bank (GFB) modeling a population of V1 simple cells separates the categories with nearly 80% accuracy, and furthermore exhibits tuning to penumbral blur. A Filter-Rectify Filter (FRF) style neural network extending the GFB model performed at better than 80% accuracy, and exhibited greater sensitivity to texture differences. Comparing the models with humans performing the same occlusion/shadow classification task using the same stimuli reveals better agreement on an image-by-image basis between human performance and the FRF model than the GFB model. Taken as a whole, the present results suggest that local achromatic cues like contrast, penumbral blur, and texture play an important role in distinguishing edges caused by shadows from those caused by surface boundaries.

AUTHOR SUMMARYDistinguishing edges caused by changes in illumination from edges caused by surface boundaries is an essential computation for accurately parsing the visual scene. Previous psychophysical investigations examining the utility of various locally available cues to classify edges as shadows or surface boundaries have primarily focused on color, as surface boundaries often give rise to a change in color whereas shadows will not. However, even in grayscale images we can readily distinguish shadows from surface boundaries, suggesting an important role for achromatic cues in addition to color. We demonstrate using statistical analysis of natural shadow and occlusion edges that locally available achromatic cues can be exploited by machine classifiers to reliably distinguish these two edge categories. These classifiers exhibit sensitivity to blur and local texture differences, and exhibit reasonably good agreement with humans classifying edges as shadows or occlusion boundaries. As trichromatic vision is relatively rare in the animal kingdom, our work suggests how organisms lacking rich color vision can still exploit other cues to avoid mistaking illumination changes for surface changes.


title: Emergence of integrated information at macro timescales in real neural recordings

abstract: How a system generates conscious experience remains an elusive question. One approach towards answering this is to consider the information available in the system from the perspective of the system itself. Integrated information theory (IIT) proposes a measure to capture this, integrated information ({Phi}). While {Phi} can be computed at any spatiotemporal scale, IIT posits that it be applied at the scale at which the measure is maximised. Importantly, {Phi} should emerge to be maximal not at the smallest spatiotemporal scale, but at some macro scale where system elements or timesteps are grouped into larger elements or timesteps. Emergence in this sense has been demonstrated in artificial binary systems, but it remains unclear whether it occurs in real neural recordings which are generally continuous and noisy. Here we first utilise a computational model to confirm that {Phi} becomes maximal at the temporal scales underlying its generative mechanisms. Second, we search for emergence in local field potentials from the fly brain recorded during wakefulness and anaesthesia, finding that normalised {Phi} (wake/anaesthesia), but not raw {Phi} values, peaks at 5 ms. Lastly, we extend our model to investigate why raw {Phi} values themselves did not peak. This work extends the application of {Phi} to simple artificial systems consisting of logic gates towards searching for emergence of a macro spatiotemporal scale in real neural systems.


title: Induced pluripotent stem cell-derived astrocytes from patients with schizophrenia exhibit an inflammatory phenotype that affects vascularization

abstract: Molecular and functional abnormalities of astrocytes have been implicated in the etiology and pathogenesis of schizophrenia (SCZ). In this study, we examined the proteome, inflammatory responses, and secretome effects on vascularization of human induced pluripotent stem cell (hiPSC)-derived astrocytes from patients with SCZ. Proteomic analysis revealed alterations in proteins related to immune function and vascularization. Reduced expression of the nuclear factor kappa B (NF-{kappa}B) p65 subunit was observed in these astrocytes, with no incremental secretion of cytokines after tumor necrosis factor alpha (TNF-) stimulation. Among inflammatory cytokines, secretion of interleukin (IL)-8 was found particularly elevated in SCZ-patient-derived- astrocyte conditioned medium (ASCZCM). In a chicken chorioallantoic membrane (CAM) assay, ASCZCM reduced the diameter of newly grown vessels, and this effect could be mimicked with exogenous addition of IL-8. Taken together, our results suggest that SCZ astrocytes are immunologically dysfunctional and may consequently affect vascularization through secreted factors.


title: Locus coeruleus neuromelanin predicts ease of attaining and maintaining neural states of arousal

abstract: The locus coeruleus (LC), a small subcortical structure in the brainstem, is the brains principal source of norepinephrine. It plays a primary role in regulating stress, the sleep-wake cycle, and attention, and its degradation is associated with aging and neurodegenerative diseases associated with cognitive deficits (e.g., Parkinsons, Alzheimers). Yet precisely how norepinephrine drives brain networks to support healthy cognitive function remains poorly understood - partly because LCs small size makes it difficult to study noninvasively in humans. Here, we characterized LCs influence on brain dynamics using a hidden Markov model fitted to functional neuroimaging data from healthy young adults across four attention-related brain networks and LC. We modulated LC activity using a behavioral paradigm, and also measured individual differences in LC neuromelanin. The model revealed five hidden states, including a stable  arousal state that occurred when subjects actively engaged with the task. LC neuromelanin correlated with this states stability across experimental manipulations, and with subjects propensity to enter into and remain in this state. These results provide new insight into LCs role in driving spatiotemporal neural patterns associated with attention and arousal, and demonstrate that neuromelanin variation can explain individual differences in these patterns even in healthy young adults.


title: Thyroid hormone induces B cells abnormal differentiation via overexpression of B cell-activating factor

abstract: Breakdown of tolerance and abnormal activation in B cells is an important mechanism in Graves disease (GD) pathogenesis. However, the mechanism by which B cells are abnormal differentiated and activated in GD remains elusive. Here, we show that elevated BAFF expression is positively correlated with serum thyroid hormone (TH) levels in GD patients and high TH levels can induce BAFF overexpression and lead to the abnormal differentiation of B cells in mice. This BAFF overexpression can be seen in many tissues. In the spleens of mice, high TH levels induce M1 macrophages polarization, which generates BAFF overexpression. Our findings open a new perspective on the interactions between endocrine and immune system and provide insight into the involvement of thyroid hormones in the development and progression of GD.

category: immunology


title: Helix 8 in chemotactic receptors of the complement system.

abstract: Host response to infection involves activation of the complement system leading to producing of anaphylotoxins C3a and C5a. A complement factor C5a exerts its effect through activation of C5aR1, chemotactic receptor 1, and triggers the G protein-coupled signaling cascade. Orthosteric and allosteric antagonists of C5aR1 are a novel strategy for anti-inflammatory therapies. Here, we discuss recent crystal structures of inactive C5aR1 in terms of an inverted orientation of helix H8, unobserved in other GPCR structures. Analysis of mutual interactions of subunits in the C5aR1 - G protein complex has provided new insights into the activation mechanism of this distinct receptor. By comparison of C5aR1 and its homolog C5aR2 we explained differences between their signaling pathways on the molecular level. A comparison of microsecond MD trajectories started from active and inactive receptor conformations also enabled to elucidate details of local and global changes in the transmembrane domain induced by interactions with the G subunit and to explain the impact of inverted H8 on the receptor activation.


title: Distinct denitrifying phenotypes of predominant bacteria mould nitrous oxide metabolism in upland agricultural soils

abstract: Denitrifying nitrous oxide (N2O) emissions in agroecosystems result from variations in microbial composition and soil properties. However, the microbial mechanisms of differential N2O emissions in agricultural soils are less understood. Microcosm experiments of two types of Chinese farmland soil were conducted with nitrate (250 mg/kg) and a combination of glucose (1000 mg/kg) and nitrate, and a case with no addition was used as the control. The results show that N2O accumulation in black soil (BF) was significantly higher than that in fluvo-aquic soil (FF) independent of carbon and nitrogen supply. The abundance of denitrifying genes was significantly higher in FF, but the ratios of genes responsible for N2O production (narG, nirS, and nirK) to the gene for N2O reduction (nosZ) did not significantly differ between the two soils. However, the soils showed obvious discrepancies in denitrifying bacterial communities. High accumulation of N2O was verified by the isolates of Rhodanobacter, which is predominant in BF due to its truncated denitrifying genes and lack of N2O reduction capacity. The dominance of complete denitrifiers such as Castellaniella in FF led to a rapid reduction in N2O and reduced N2O accumulation, as demonstrated when its corresponding isolate was inoculated into both studied soils. Therefore, the different phenotypes of N2O metabolism of the distinct denitrifiers maintained in the two soils caused their differing N2O accumulation. This knowledge could guide the regulation of the denitrifying bacterial community and the phenotypes of N2O metabolism in agricultural soils to reduce N2O emissions.


title: Composition and functionality of bacterioplankton communities in marine coastal zones adjacent to finfish aquaculture

abstract: Finfish aquaculture is one of the fastest-growing primary industries globally and is increasingly common in coastal ecosystems demanding effective impact monitoring tools. Bacterioplankton is ubiquitous in marine environment and its rapid response to environmental change makes them a potential bioindicator for environmental impacts. This study aims to verify the use of the taxonomic and functional profiles of bacterioplankton using high-throughput amplicon sequencing. Water was sampled along a distance gradient in three different depths from two lease areas and three control sites in a low-oxygen, highly stratified marine embayment. Our results revealed a vertical variation in bacterioplankton community strongly associated to NOx, conductivity, salinity, temperature and PO4. The overall shift in the taxonomic profile (ASVs) were more noticeable than in the functional profile. Despite depth stratification, ASVs and functional annotations were detected as potential bioindicators for aquaculture activity. Differentially abundant bacteria found in lease areas were associated with nutrient enrichment (e.g., Desulfovibrionaceae, Psychromonadaceae, and Fusibacter) or are known as potential pathogens (e.g., Pseudomonas, Vibrio and Aliivibrio) and members of the fish gut microbiome (e.g., Leptotrichiaceae, Enterobacterales and Pseudomonadales). Differences in the predicted functional profile were strongest in the bottom waters near leases, where pathways assigned to organic compounds and vitamin degradation, fermentation, methanogenesis and antibiotic resistance were detected. Overall, our findings indicated that the use of 16S rRNA sequencing of bacterioplankton communities are a promising and alternative method for detecting variations along an aquaculture gradient in the water column.


title: Identification of genes required for swarming motility in Bacillus subtilis using transposon mutagenesis and high-throughput sequencing (TnSeq)

abstract:
Bacillus subtilis exhibits swarming motility, a flagellar-mediated form of surface motility. Here we use high-throughput transposon mutagenesis and sequencing (TnSeq) to identify candidate genes required for swarming. The TnSeq approach identified all of the known genes required for flagellar biosynthesis as well as nearly all of the previously reported regulators that promote swarming. Moreover, we identified an additional 36 genes that improve swarming and validated them individually. Among these, two mutants with severe defects were recovered including fliT, required for flagellar biosynthesis, and a gene of unknown function yolB, whose defect could not be attributed to a lack of flagella. Our work indicates that TnSeq is a powerful approach for the identification of genes required for motility that drives outward expansion of a colony on plates.

IMPORTANCEIn TnSeq, transposons are randomly inserted throughout the chromosome at a population level, but insertions that disrupt genes of essential function cause strains that carry them to fall out of the population and appear under-represented at the sequence level. Here we apply TnSeq to the non-essential phenotype of motility in B. subtilis and spatially select for cells proficient in swarming. We find that insertions in nearly all genes previously identified as required for swarming are under-represented in TnSeq analysis, and we identify 36 additional genes that enhance swarming. We suggest that TnSeq is a powerful tool for the genetic analysis of motility and likely other non-lethal screens for which enrichment is strong.


title: E. coli Leucine-responsive regulatory protein bridges DNA in vivo and tunably dissociates in the presence of exogenous leucine

abstract: Feast-Famine Response Proteins are a widely conserved class of global regulators in prokaryotes, the most highly studied of which is the E. coli leucine-responsive regulatory protein (Lrp). Lrp senses environmental nutrition status and subsequently regulates up to one-third of the genes in E. coli, either directly or indirectly. Lrp exists predominantly as octamers and hexadecamers (16mers), where leucine is believed to shift the equilibrium towards the octameric state. In this study, we analyzed the effects of three oligomerization state mutants of Lrp in terms of their ability to bind to DNA and regulate gene expression in response to exogenous leucine. We find that oligomerization beyond dimers is required for Lrps regulatory activity, and that contrary to prior speculation, exogenous leucine modulates Lrp activity at its target promoters exclusively by inhibiting Lrp binding to DNA. We also find evidence that Lrp binding bridges DNA over length scales of multiple kilobases, revealing a new range of mechanisms for Lrp-mediated transcriptional regulation.


title: Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

abstract: Probing epigenomic marks such as histone modifications at a single cell level in thousands of cells has been recently enabled by technologies such as scCUT&Tag. Here we developed a multimodal and optimized iteration of scCUT&Tag called nano-CT (for nano-CUT&Tag) that allows simultaneous probing of three epigenomic modalities at single-cell resolution, using nanobody-Tn5 fusion proteins. nano-CT is compatible with starting materials as low as 25 000 cells and has significantly higher resolution than scCUT&Tag, with a 16-fold increase in the number of fragments per cells. We used nano-CT to simultaneously profile chromatin accessibility, H3K27ac and H3K27me3 in a complex tissue - juvenile mouse brain. The obtained multimodal dataset allowed for discrimination of more cell types/states that scCUT&Tag, and inference of chromatin velocity between ATAC and H3K27ac in the oligodendrocyte (OL) lineage. In addition, we used nano-CT to deconvolute H3K27me3 repressive states and infer two sequential waves of H3K27me3 repression at distinct gene modules during OL lineage progression. Thus, given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in different biological systems at single cell level.


title: A versatile pattern-forming cortical circuit based on Rho, F-actin, Ect2 and RGA-3/4

abstract:
Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner and, when co-expressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces a range of cortical behaviors from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho and F-actin form the core of a circuit that drives a diverse range of cortical behaviors, and demonstrate that the immature oocyte is a powerful model for characterizing these dynamics.

SummaryMichaud et al. identify Ect2 and RGA-3/4 as core components of the cortical excitability circuit associated with cytokinesis. Additionally, they demonstrate that the immature Xenopus oocyte is a powerful model for characterizing excitable dynamics.

category: cell biology


title: Cell Surface-Bound La Protein Regulates The Cell Fusion Stage Of Osteoclastogenesis

abstract:
Multinucleated osteoclasts, essential for skeletal remodeling in health and diseases, are formed by fusion of osteoclast precursors with each fusion event raising their bone-resorbing activity. Here we report that nuclear RNA chaperone, La protein moonlights as an osteoclast fusion regulator. Monocyte-to-osteoclast differentiation starts with a drastic decrease in La levels. Then La reappears as a proteasecleaved species at the cell surface where it promotes fusion by mechanisms independent of La-RNA interactions. Appearance-and-disappearance of cell-surface La act as an on-and-off switch of the fusion activity with fusion slowing down when surface La is replaced in mature osteoclasts by full-length nuclear La. Inhibiting surface La in a novel explant model of fibrous dysplasia inhibits excessive osteoclast formation characteristic of this disease, highlighting Las potential as a therapeutic target.

One-Sentence SummaryA nuclear RNA chaperon moves to the surface of osteoclasts to control their formation and function in bone metabolism.


title: Small molecule-mediated insulin hypersecretion induces transient unfolded protein response and loss of function in pancreatic islet beta cells

abstract:
Aims/hypothesisInsulin production and its regulated exocytosis by pancreatic islet beta cells is the major physiological mechanism controlling blood glucose concentrations. Beta cells are susceptible to failure due to genetic and environmental influences, leading to type 1 and 2 diabetes. In this work we aimed to advance understanding of the regulation of beta-cell function by identifying small molecule modulators of secretion.

MethodsFor high-throughput screening and routine medium-throughput beta cell function assays we used mouse MIN6 beta cells stably expressing a Gaussia luciferase-linked insulin secretion reporter (InsGLuc-MIN6). We validated hit small molecules using cadaveric human islets in static culture insulin secretion assays. We measured effects on calcium influx and cAMP in MIN6 cells using Fura-2 and a bioluminescence resonance energy transfer reporter assay, respectively. Metabolism was analyzed by targeted metabolomics and Seahorse oxygen consumption assays. We measured relative gene expression and protein amounts by quantitative RT-PCR and SDS-PAGE immunoblotting, respectively.

ResultsThrough our high-throughput screen we discovered 2-(3-benzyl-2-iminobenzimidazol-1-yl)-1-thiophen-2-ylethanol (SW016789) which acutely potentiated nutrient-induced calcium influx and insulin secretion. More than 2 hours of exposure to SW016789 transiently induced the unfolded protein response of the endoplasmic reticulum and shut down of insulin secretory function. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or death as determined by multiplexed cytotoxicity assays and a lack of induction of cleaved PARP. This may be due in part to a more rapid induction of Hspa5, and a lesser induction of signalling through the eIF2 kinase PERK and lack of expression of oxidative stress genes like Txnip. We determined that SW016789 acted upstream of the voltage-dependent calcium channel (VDCC) and potentiated nutrient-stimulated, but not KCl-stimulated, calcium influx. The potentiating effects of SW016789 were not due to altered metabolic pathways, mitochondrial function, or actions on G protein-coupled receptors. In chemical co-treatment experiments we discovered synergy between SW016789 and small molecule activators of protein kinase C and VDCCs, suggesting potential involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as co-treatment with dihydropyridine-class VDCC inhibitors protected MIN6 beta cells and human islets from loss of function.

Conclusions/interpretationThese data suggest the major mechanism of action of SW016789 in beta cells is to potentiate opening of VDCCs. This activity may partially depend on protein kinase C. Beta cells under pharmacological hypersecretory conditions have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. Further study of the mechanisms underlying these processes will increase understanding of beta cell function in normal and pathophysiological states.

Research in contextWhat is already known about this subject? O_LIPharmacologic and environmental chemicals can stimulate aberrant beta cell activity.
C_LIO_LIHypersecretion of insulin from pancreatic beta cells can cause ER stress and lead to beta cell dysfunction.
C_LIO_LIDefects in beta cell ER stress handling contribute to diabetes pathogenesis.
C_LI

What is the key question? O_LICan the chemical tool SW016789 help uncover novel insulin secretion regulatory pathways and ER proteostasis factors?
C_LI

What are the new findings? O_LISW016789 caused hypersecretion of insulin via enhanced nutrient-stimulated Ca2+ influx followed by transient ER stress and shutdown of beta cell function without apoptosis.
C_LIO_LIBlockade of voltage-dependent Ca2+ channels protected human islets and beta cells from hypersecretion-induced dysfunction.
C_LI

How might this impact on clinical practice in the foreseeable future? O_LIThese results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.
C_LI


title: The push-pull intercrop Desmodium does not repel, but intercepts and kills pests

abstract: Over two decades ago, scientists developed a push-pull intercropping strategy that received critical acclaim for synergizing food security with ecosystem resilience in smallholder farming. The strategy suppresses Lepidopteran pests in maize through a combination of a repellent intercrop (push), commonly Desmodium spp., and an attractive, dead-end border crop (pull). Key is the intercrops constitutive release of volatiles that repel herbivores. Surprisingly, however, we found that Desmodium does not constitutively release volatiles, and only minimally upon herbivory. Further, in oviposition choice settings, Spodoptera frugiperda, a devastating invasive pest, was not repelled by Desmodium volatiles. In search of an alternative mechanism, we found that neonate larvae strongly preferred Desmodium over maize. However, their development stagnated and none survived. In addition, larvae were frequently seen impaled and immobilized by the dense network of silica-fortified, non-glandular trichomes. Thus, entirely different from repelling adult moths, Desmodium intercepts and decimates dispersing offspring. As a hallmark of sustainable pest control, maize-Desmodium intercropping has inspired countless efforts trying to emulate a stimulo-deterrent diversion in other cropping systems. However, detailed knowledge of the actual mechanisms is required to rationally improve the strategy, and translate the concept into other cropping systems.


title: Parasite and pathogen prevalence in our closest animal companions is determined by accessibility of sanitation services

abstract:
Despite the critical role parasites play in ecosystem functioning and their considerable influence on human society, little is known about their variations in abundance on a global scale. This gap in knowledge is amplified by a lack of holistic understanding on how the abundance of parasites of wildlife and humans varies across environmental and socioeconomic gradients, despite a need to integrate study of parasites across social and environmental spheres. Free-roaming companion animals (e.g., domestic cats (Felis catus) and dogs (Canis lupus familiaris)) share pathogens and have frequent contact with humans and wildlife. Thus, they are an effective model to understand how parasite and pathogen prevalence of humans and wildlife varies across environmental and socioeconomic gradients. Through a global systematic review and analysis of socioeconomic and environmental variables, including per capita GDP, income disparity, sanitation, biodiversity, island habitation, and latitude, we find that sanitation and island habitation best explained free-roaming companion animal parasite and pathogen prevalence. Sanitation was significantly associated with parasite and pathogen prevalence in free-roaming companion animals, such that for every 10% increase in the proportion of the human population with improved sanitation access, parasite and pathogen prevalence in free-roaming companion animals decreased by 12% (5-19%, 95% C.L.; p = 0.0023). Since companion animals share many parasites with humans and wildlife, these results suggest that actionable interventions to improve sanitation access could reduce parasite and pathogen exposure risks from companion animals to humans and wildlife.

Significance StatementIn addition to playing a critical role in ecosystem functioning, parasites also influence human health, behavior, and society. Further, parasites are also impacted by human activities, as much as by ecological phenomena in natural environments. Despite these dualities, little is known about their variations in abundance on a global scale across environmental and socioeconomic gradients. Using free-roaming companion animals (e.g., domestic cats (Felis catus) and dogs (Canis lupus familiaris)) as a model system, we find that access to safely managed sanitation services is strongly associated with parasite and pathogen prevalence. This finding underscores improvements to sanitation as an actionable One Health intervention that could reduce parasite and pathogen exposure risks from companion animals to humans and wildlife.


title: Flexible estimation of biodiversity with short-range multispectral imaging in a temperate grassland

abstract:
O_LIImage sensing technologies are rapidly increasing the cost-effectiveness of biodiversity monitoring efforts. Species differences in the reflectance of electromagnetic radiation have recently been highlighted as a promising target to estimate plant biodiversity using multispectral image data.
C_LIO_LIHowever, these efforts are currently hampered by logistical difficulties in broad-scale implementation and their use in characterizing biodiversity at different spatial scales.
C_LIO_LIHere, we investigate the utility of multispectral imaging technology from commercially available unmanned aerial vehicles (UAVs, or drones) in estimating biodiversity metrics at short-range (<10 m image recording height) in a temperate calcareous grassland ecosystem in Oxfordshire, UK. We calculate a suite of moments (coefficient of variation, standard deviation, skew, kurtosis) for the distribution of radiance from multispectral images at five wavelength bands (Blue 450{+/-}16 nm; Green 560{+/-}16 nm; Red 650{+/-}16 nm; Red Edge 730{+/-}16 nm; Near Infrared 840{+/-}16 nm) and test their effectiveness at estimating ground-truthed biodiversity metrics from in-situ botanical surveys for 37 - 1 m x 1 m quadrats.
C_LIO_LIWe find positive associations between the average coefficient of variation in spectral radiance and both the Shannon-Weiner and Simpsons biodiversity indices. Furthermore, we find that the average coefficient of variation in spectral radiance is consistent and highly repeatable, across sampling days and recording heights. Positive associations with biodiversity indices hold irrespective of the image recording height (2-8 m), but we report reductions in estimates of spectral diversity with increases to UAV recording height. UAV imaging reduced sampling time by 16-fold relative to in-situ botanical surveys.
C_LIO_LISynthesis - We demonstrate the utility of multispectral radiance moments as an indicator of grassland biodiversity metrics at high spatial resolution using a widely available UAV monitoring system at a coarse spectral resolution. The use of UAV technology with multispectral sensors has far-reaching potential to provide cost-effective and high-resolution monitoring of biodiversity in complex environments.
C_LI


title: Impacts of recreational angling on fish population recovery after a commercial fishing ban

abstract: It is often assumed that recreational fishing has negligible impact on fish stocks compared to commercial fishing. Yet, for inland water bodies in densely populated areas, this is unlikely to be true. In this study we demonstrate remarkably variable stock recovery rates among different fish species with similar life histories in a large productive inland freshwater ecosystem (Kaunas Reservoir, Lithuania), where all commercial fishing has been banned since 2013. We conducted over 900 surveys of recreational anglers during a period of four years (2016 to 2021) to assess recreational fishing catches. These surveys are combined with drone and fishfinder device-based assessment of recreational fishing effort. Fish population recovery rates were assessed using standardised catch per unit effort time series. We show that recreational fishing is having a major impact in retarding the recovery of predatory species, such as pikeperch and perch. In contrast, recovery of roach, rarely caught by anglers, has been remarkably rapid and the species is now dominating the ecosystem. Our study demonstrates that recreational fishing can have strong impacts on some fish species, alter relative species composition and potentially change ecosystem state and dynamics.


title: Urbanization enhances multiple carotenoid-based signals despite poor body condition in male and female Northern Cardinals

abstract:
If humans aim to sustainably coexist with wildlife, we must understand how our activity impacts the communication systems of urban animal populations. We know much about the effects of anthropogenic noise on bird song, but relatively little about how avian visual signals are affected by urbanization. Such an effect may occur if urbanization alters the food available to species with color based on carotenoids, which they must obtain from their diet. Over three years, we compared a comprehensive suite of visual signals in male and female Northern Cardinals (Cardinalis cardinals) in a rural and an urban population. We predicted that urban birds would have enhanced carotenoid-based signals as they likely have access to more carotenoids from invasive plants, especially honeysuckle (Lonicera spp.), that thrive in cities. We used reflectance spectrometry, digital image analyses, and avian visual models to quantify hue, saturation, and brightness of chest (male), underwing (female), and bill (male and female) signals. Compared to rural males, urban males had redder chest feathers in one year and redder bills in every year. Urban females had more saturated underwing color than rural females in every year. These color differences were sufficient to be distinguished by the avian visual system. Urbanization did not affect female bill color. Interestingly, urban birds had significantly reduced mass-related body condition compared to rural birds. These results show that both male and female urban birds can display enhanced carotenoid-based signals despite being in relatively poor condition. The consequences of this color enhancement are unknown, but it could affect the information content of the signals and the dynamics of the social and mating systems. These results stand in stark contrast to the predominant trend in birds of decreased color in urban areas and highlight the complex and varied potential effects of urbanization on animal communication.

LAY SUMMARYO_LIUrbanization typically dulls bird color, but carotenoid-based signals may be enhanced if urban birds have increased access to carotenoids from invasive plants.
C_LIO_LIUrban Northern Cardinals of both sexes had enhanced signal color compared to rural birds, but were in worse body condition.
C_LIO_LIUrban birds likely had greater access to carotenoids from invasive plants, allowing them to produce enhanced color despite poor condition.
C_LIO_LIUrbanization can disrupt animal communication and possibly alter evolutionary dynamics.
C_LI


title: Demuxafy: Improvement in droplet assignment by integrating multiple single-cell demultiplexing and doublet detection methods

abstract:
Recent innovations in droplet-based single-cell RNA-sequencing (scRNA-seq) have provided the technology necessary to investigate biological questions at cellular resolution. With the ability to assay thousands of cells in a single capture, pooling cells from multiple individuals has become a common strategy. Droplets can subsequently be assigned to a specific individual by leveraging their inherent genetic differences, and numerous computational methods have been developed to address this problem. However, another challenge implicit with droplet-based scRNA-seq is the occurrence of doublets - droplets containing two or more cells. The inaccurate assignment of cells to individuals or failure to remove doublets contribute unwanted noise to the data and result in erroneous scientific conclusions. Therefore, it is essential to assign cells to individuals and remove doublets accurately. We present a new framework to improve individual singlet classification and doublet removal through a multi-method intersectional approach.

We developed a framework to evaluate the enhancement in donor assignment and doublet removal through the consensus intersection of multiple demultiplexing and doublet detecting methods. The accuracy was assessed using scRNA-seq data of [~]1.4 million peripheral blood mononucleated cells from 1,034 unrelated individuals and [~]90,000 fibroblast cells from 81 unrelated individuals. We show that our approach significantly improves droplet assignment by separating singlets from doublets and classifying the correct individual compared to any single method. We show that the best combination of techniques varies under different biological and experimental conditions, and we present a framework to optimise cell assignment for a given experiment. We offer Demuxafy (https://demultiplexing-doublet-detecting-docs.readthedocs.io/en/latest/index.html) - a framework built-in Singularity to provide clear, consistent documentation of each method and additional tools to simplify and improve demultiplexing and doublet removal. Our results indicate that leveraging multiple demultiplexing and doublet detecting methods improves accuracy and, consequently, downstream analyses in multiplexed scRNA-seq experiments.


title: Analyses of Mutational Patterns Induced by Formaldehyde and Acetaldehyde Reveal Similarity to a Common Mutational Signature in Cancers

abstract: Formaldehyde (CH2O) and acetaldehyde (C2H4O) are reactive small molecules produced endogenously in cells as well as being environmental contaminants. Both of these small aldehydes are classified as human carcinogens, as they are known to damage DNA and cause mutations. However, the mutagenic properties of formaldehyde and acetaldehyde remain incompletely understood, at least in part because they are relatively weak mutagens. Here, we use a highly sensitive yeast genetic reporter system featuring controlled generation of long single-stranded DNA regions to show that both small aldehydes induced mutational patterns characterized by mostly C/G [->] A/T, C/G [->] T/A, and T/A [->] C/G substitutions, each in similar proportions. We also observed an excess of C/G [->] A/T transversions when compared to mock-treated controls. Many of these C/G [->] A/T transversions occurred at TC/GA motifs. Interestingly, the formaldehyde mutational pattern resembles single base substitution (SBS) signature 40 from the Catalog of Somatic Mutations in Cancer (COSMIC). SBS40 is a cancer mutational signature of unknown etiology. We also noted that acetaldehyde treatment caused an excess number of deletion events longer than four bases while formaldehyde did not. This latter result could be a distinguishing feature between the mutational patterns of these two simple aldehydes. These findings shed new light on the mutagenic characteristics of two important carcinogens.


title: Oil accumulation in leaves driven by a native promoter-gene fusion created using CRISPR/Cas9 mediated genomic deletion

abstract: Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual-guide CRISPR/Cas9. We fuse the promoter of a non-essential photosynthesis-related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) to drive oil biosynthesis in leaves of a lipase deficient sugar-dependent 1 mutant. DGAT2 expression is enhanced more than twenty-fold and the triacylglycerol content increases by around thirty-fold. This strategy offers a transgene-free route to engineering gain-of-function traits such as high lipid forage to increase the productivity and sustainability of ruminant farming.


title: Molecular Dynamic Investigation of H5N1 Influenza Virus Dual H274Y-I222K Mutation Resistance to Peramivir

abstract: In light of the rapid rise of an influenza pandemic, the constant genetic mutations of H5N1 influenza viruses pose a threat. Mutations at the sialic site are often responsible for multiple drug resistance. To design effective new inhibitors, it is necessary to undertake research into the mechanism of resistance of influenza viruses and their rapid mutations. The molecular dynamic simulation technique has been an instrumental tool in understanding how proteins function from an atomic perspective. A thorough investigation has not been conducted using molecular dynamics to examine the impact of these mutations (I222K, H274Y, and H274Y-I222K) on Peramivir. This study investigates the effects of I222K, H274Y, H274Y-I222K substitution on the neuraminidase-Peramivir complex and identifies responsible residues for complex conformations. The mutations caused distorted Peramivir orientation in the enzyme active site, which affected the inhibitors binding. In the presence of various mutations, interaction between protein and ligand became less thermodynamically favorable. We observed the following trend in binding free energy difference: WT<I222<H274Y<H274Y-I222K. As a result of the thermodynamic instability of the mutant complexes, Peramivirs potency is reduced due to impaired binding interactions. Wild type complex displays thermodynamic stability and strong protein-ligand interactions due to their high total energy contributions and low residue flexibility. Based on the energy decomposition analysis, Arg117, Arg224, and Arg292 contributed the largest residual energy for the binding of Peramivir to wild type and mutants. These residues are thought to play a key role in the formation of the binding pocket between Peramivir and neuraminidase. This study provides a basis for investigating the effects of other mutations on Peramivirs efficacy against the H5N1 virus.


title: Functional Determination of Calcium Binding Sites Required for the Activation of Inositol 1,4,5-trisphosphate receptors.

abstract: Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca2+) signals in response to various external cues. Notably, IP3R channel activity is determined by several obligatory factors including IP3, Ca2+ and ATP. The critical basic amino acid residues in the N-terminal IP3-binding core (IBC) region that facilitate IP3 binding are well characterized. In contrast, the residues conferring the biphasic regulation by Ca2+ are yet to be ascertained. Using comparative structural analysis of Ca2+ binding sites identified in two main families of intracellular Ca2+-release channels, ryanodine receptors (RyRs) and IP3Rs, we identified putative acidic residues coordinating Ca2+ in the cytosolic calcium sensor region in IP3Rs. We determined the consequences of substituting putative Ca2+ binding, acidic residues in IP3R family members. We show that the agonist-induced Ca2+ release, single channel open probability (P0) and Ca2+ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues are neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca2+ binding pocket shifted the Ca2+ required to activate IP3R to higher concentrations, indicating that these residues likely are a component of the Ca2+ activation site in IP3R. Taken together, our findings indicate that Ca2+ binding to a well conserved activation site is a common underlying mechanism resulted in increased channel activity shared by IP3Rs and RyRs.