title: Nitrate fertilization may delay autumn leaf senescence, while amino acid treatments do not

abstract:
Fertilization with nitrogen (N)-rich compounds leads to increased growth, but may compromise phenology and winter survival of trees in boreal regions. During autumn, N is remobilized from senescing leaves and stored in other parts of the tree to be used in the next growing season. However, the mechanism behind the N fertilization effect on winter survival is not well understood and it is unclear how N levels or forms modulate autumn senescence. We performed fertilization experiments and showed that treating Populus saplings with high or low levels of inorganic nitrogen resulted in a delay in senescence. In addition, by using precise delivery of solutes into the xylem stream of Populus trees in their natural environment, we found that delay of autumn senescence was dependent on the form of N administered: inorganic N (NO3-1) delayed senescence but amino acids (Arg, Glu, Gln, and Leu) did not. Metabolite profiling of leaves showed that the levels of tricarboxylic acids (TCA), arginine catabolites (ammonium, ornithine), glycine, glycine-serine ratio and overall carbon-to-nitrogen (C/N) ratio were affected differently by the way of applying NO3-1 and Arg treatments. In addition, the onset of senescence did not coincide with soluble sugar accumulation in any of the treatments. Taken together, metabolomic rearrangement under different N forms or experimental setups could modulate senescence process, but not initiation and progression in Populus. We propose that the different regulation of C and N status through direct molecular signaling of NO3-1 could account for the contrasting effects of NO3-1 and Arg on senescence.

One sentence summaryNitrate, administered by precision fertilization through injection into the trunk, may delay autumn senescence and change metabolism in Populus leaves, while the same amount of amino acids does not have the same effect.


title: Timing of gene expression and recruitment in independent origins of CAM in the Agavoideae (Asparagaceae).

abstract: CAM photosynthesis has evolved repeatedly across the plant tree of life, yet our understanding of the genetic convergence across independent origins remains hampered by the lack of comparative studies. CAM is furthermore thought to be closely linked to the circadian clock in order to achieve temporal separation of carboxylation and sugar production. Here, we explore gene expression profiles in eight species from the Agavoideae (Asparagaceae) encompassing three independent origins of CAM. Using comparative physiology and transcriptomics, we examined the variable modes of CAM in this subfamily and the changes in gene expression across time of day and between well-watered and drought-stressed treatments. We further assessed gene expression and molecular evolution of genes encoding phosphoenolpyruvate carboxylase (PPC), an enzyme required for primary carbon fixation in CAM. Most time-of-day expression profiles are largely conserved across all eight species and suggest that large perturbations to the central clock are not required for CAM evolution. In contrast, transcriptional response to drought is highly lineage specific. Yucca and Beschorneria have CAM-like expression of PPC2, a copy of PPC that has never been shown to be recruited for CAM in angiosperms, and evidence of positive selection in PPC genes implicates mutations that may have facilitated the recruitment for CAM function early in the evolutionary history of the Agavoideae. Together the physiological and transcriptomic comparison of closely related C3 and CAM species reveals similar gene expression profiles, with the notable exception of differential recruitment of carboxylase enzymes for CAM function.

category: plant biology


title: Human serum triggers antibiotic tolerance in Staphylococcus aureus

abstract: Staphylococcus aureus is a frequent cause of bloodstream infections. Treatment can be challenging, even when isolates appear to be drug susceptible, with high rates of persistent and relapsing infection. This is particularly the case with infections caused by methicillin resistant S. aureus (MRSA) strains, which are resistant to frontline antibiotics. To understand how the host environment influences treatment outcomes in MRSA infections, we studied the impact of human serum on staphylococcal susceptibility to daptomycin, an antibiotic of last resort. This revealed that serum triggered a very high degree of tolerance to daptomycin, as well as several other classes of antibiotics and antimicrobial peptides, including gentamicin, nitrofurantoin, vancomycin, nisin and gramicidin. Serum-induced daptomycin tolerance was due to two independent mechanisms. Firstly, the host defence peptide LL-37 present in serum induced tolerance by triggering the staphylococcal GraRS two component system. This led to increased cell wall accumulation that reduced access of daptomycin to its membrane target. Secondly, GraRS-independent changes to the membrane resulted in increased cardiolipin abundance that also contributed to daptomycin tolerance. When both mechanisms were blocked, serum exposed S. aureus cells were as susceptible to daptomycin as bacteria growing in laboratory media. These data demonstrate that host factors can significantly modulate antibiotic susceptibility via diverse mechanisms, which may in turn contribute to treatment failure. The inhibition of serum-induced cell wall accumulation by fosfomycin reduced tolerance, suggesting that this antibiotic may form a useful combination therapy with daptomycin.

category: microbiology


title: Learning induces coordinated neuronal plasticity of metabolic demands and functional brain networks

abstract: The neurobiological basis of learning is reflected in adaptations of brain structure, network organization and energy metabolism. However, it is still unknown how different neuroplastic mechanisms act together and if cognitive advancements relate to general or task-specific changes. To address these questions, we tested how hierarchical network interactions contribute to improvements in the performance of a visuo-spatial processing task by employing simultaneous PET/MR neuroimaging before and after a 4-week learning period. We combined functional PET with metabolic connectivity mapping (MCM) to infer directional interactions across brain regions and subsequently performed simulations to disentangle the role of functional network dynamics and glucose metabolism. As a result, learning altered the top-down regulation of the salience network onto the occipital cortex, with increases in MCM at resting-state and decreases during task execution. Accordingly, a higher divergence between resting-state and task-specific effects was associated with better cognitive performance, indicating that these adaptations are complementary and both required for successful skill learning. Simulations further showed that changes at resting-state were dependent on glucose metabolism, whereas those during task performance were driven by functional connectivity between salience and visual networks. Referring to previous work, we suggest that learning establishes a metabolically expensive skill engram at rest, whose retrieval serves for efficient task execution by minimizing prediction errors between neuronal representations of brain regions on different hierarchical levels.

category: neuroscience


title: Steroid-resistant nephrotic syndrome associated MYO1E mutations have differential effects on myosin 1e localization, dynamics, and activity

abstract: Myo1e is a non-muscle motor protein enriched in the podocyte foot processes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Here, we set out to differentiate between the pathogenic and neutral MYO1E variants identified in SRNS patients by exome sequencing. Based on protein sequence conservation and structural predictions, two mutations in the motor domain, T119I and D388H, were selected for this study. EGFP-tagged Myo1e constructs were delivered into the Myo1e-KO podocytes via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculoviral expression system and used to measure Myo1e ATPase and motor activity in vitro. Both mutants were expressed as full-length proteins in the Myo1e-KO podocytes. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs) in podocytes. In contrast, the D388H variant localization was similar to the WT. Surprisingly, the dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting that this mutation also affects Myo1e activity. The ATPase activity and the ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting the findings from cell-based experiments. The experimental pipeline developed in this study allowed us to determine that the T119I and D388H mutations appear to be pathogenic and gain additional knowledge in the Myo1e role in podocytes. This workflow can be applied to the future characterization of novel MYO1E variants associated with SRNS.

category: cell biology


title: Particle localization using local gradients and its application to nanometer stabilization of a microscope

abstract: Accurate localization of single particles plays an increasingly important role in a range of biological techniques, including single molecule tracking and localization-based superresolution microscopy. Such techniques require fast and accurate particle localization algorithms as well as nanometer-scale stability of the microscope. Here, we present a universal method for three-dimensional localization of single labeled and unlabeled particles based on local gradient calculation of microscopy images. The method outperforms current techniques in high noise conditions, and it is capable of nanometer accuracy localization of nano- and micro-particles with sub-ms calculation time. By localizing a fixed particle as fiducial mark and running a feedback loop, we demonstrate its applicability for active drift correction in sensitive nanomechanical measurements such as optical trapping and superresolution imaging. A multiplatform open software package comprising a set of tools for local gradient calculation in brightfield and fluorescence microscopy is shared to the scientific community.

category: biophysics


title: The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells.

abstract: The pathogenic fungus Candida albicans causes severe invasive candidiasis. C. albicans infection requires the action of the virulence factor Candidalysin (CL), which damages the plasma membrane of the target human cells. However, the molecular mechanism that CL uses to permeabilize membranes is poorly understood. We employed complementary biophysical, modeling, microscopy, and cell biology methods to reveal that CL forms membrane pores using a unique molecular mechanism. Unexpectedly, it was observed that CL readily assembles into linear polymers in solution. The basic structural unit in polymer formation is a CL 8-mer, which is sequentially added into a string configuration. Finally, the linear polymers can close into a loop. Our data indicate that CL loops spontaneously insert into the membrane to become membrane pores. We identified a CL mutation (G4W) that inhibited the formation of polymers in solution and prevented formation of pores in different synthetic lipid membranes systems. Studies in epithelial cells showed that G4W CL failed to activate the danger response signaling pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of thousands of CL pores by atomic force microscopy revealed the co-existence of simple depressions and complex pores decorated with protrusions. Imaging and modeling indicate that the two types of pores are formed by CL molecules assembled into alternate orientations. We propose that this structural rearrangement represents a maturation mechanism that might stabilize pore formation to achieve more robust cellular damage. Taken together, the data show that CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution directly leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.


title: Three-dimensional Cancer Cell Migration Directed by Dual Mechanochemical Guidance

abstract: Directed cell migration guided by external cues plays a central role in many physiological and pathophysiological processes. The microenvironment of cells often simultaneously contains various cues and the motility response of cells to multiplexed guidance is poorly understood. Here we combine experiments and mathematical models to study the three-dimensional migration of breast cancer cells in the presence of both contact guidance and a chemoattractant gradient. We find that the chemotaxis of cells is complicated by the presence of contact guidance as the microstructure of extracellular matrix (ECM) vary spatially. In the presence of dual guidance, the impact of ECM alignment is determined externally by the coherence of ECM fibers, and internally by cell mechanosensing Rho/Rock pathways. When contact guidance is parallel to the chemical gradient, coherent ECM fibers significantly increase the efficiency of chemotaxis. When contact guidance is perpendicular to the chemical gradient, cells exploit the ECM disorder to locate paths for chemotaxis. Our results underscores the importance of fully characterizing the cancer cell microenvironment in order to better understand invasion and metastasis.


title: POTATO: An automated pipeline for batch analysis of optical tweezers data

abstract:
Optical tweezers is a single-molecule technique that allows probing of intra- and intermolecular interactions that govern complex biological processes involving molecular motors, protein-nucleic acid interactions and protein/RNA folding. Recent developments in instrumentation eased and accelerated optical tweezers data acquisition, but analysis of the data remains challenging. Here, to enable high-throughput data analysis, we developed an automated python-based analysis pipeline called POTATO (Practical Optical Tweezers Analysis TOol). POTATO automatically processes the high-frequency raw data generated by force-ramp experiments and identifies (un)folding events using predefined parameters. After segmentation of the force-distance trajectories at the identified (un)folding events, sections of the curve can be fitted independently to worm-like chain and freely-jointed chain models, and the work applied on the molecule can be calculated by numerical integration. Furthermore, the tool allows plotting of constant force data and fitting of the Gaussian distance distribution over time. All these features are wrapped in a user-friendly graphical interface (https://github.com/REMI-HIRI/POTATO), which allows researchers without programming knowledge to perform sophisticated data analysis.

SIGNIFICANCEStudying (un)folding of biopolymer structures with optical tweezers under different conditions generates very large datasets for statistical data analysis. Recent technical improvements accelerated data acquisition by coupling modern instruments with microfluidic systems, at the same time creating the need for a high-throughput, and unbiased data analysis. We developed Practical Optical Tweezers Analysis TOol (POTATO); an open-source python-based tool that can process data gathered by any OT force-ramp experiment in an automated fashion. POTATO is principally designed for data preprocessing, identification of (un)folding events and the fitting of the force-distance curves. In addition, all parameters for preprocessing, statistical analysis and fitting of the curves can be adapted to suit the dataset under analysis in an easy-to-use graphical user interface.


title: RPRM negatively regulates ATM levels involving its phosphorylation mediated by CDK4/CDK6

abstract: Sensitizing cancer cells to radio- and chemotherapy remains a hot topic in cancer treatment. Here it is identified that Protein Reprimo (RPRM) negatively regulates the levels of ataxia-telangiectasia mutated (ATM) protein kinase, a master regulator of DNA damage response (DDR) in the presence of DNA double-strand breaks (DSBs), resulting in impaired DNA repair efficiency and enhanced cellular sensitivity to genotoxic agents. Mechanistically, although RPRM is primarily located in cytoplasm, it rapidly translocates to nucleus shortly after induced by X-irradiation, interacts with ATM and promotes the nuclear export and proteasomal degradation of ATM. The nuclear translocation of RPRM is associated with its phosphorylation at serine 98, which is mediated by cyclin-dependent kinases 4/6 (CDK4/6). Inhibition of CDK4/6 stabilizes RPRM and promotes its nuclear import, in turn enhances the nuclear export of ATM and the reduction of ATM levels. As a result, RPRM overexpression and its phosphorylation inhibition sensitize cells to genotoxic agents. Moreover, RPRM deficiency significantly increases resistance to radiation-induced damage both in vitro and in vivo. These findings establish a crucial regulatory mechanism in which ATM is negatively modulated by RPRM, suggesting that RPRM may serve as a novel target for both cancer therapy and radiation protection.

category: cancer biology


title: EBV LMP1-activated mTORC1 and mTORC2 Coordinately Promote Nasopharyngeal Cancer Stem Cell Properties

abstract:
Epstein-Barr Virus (EBV) is associated with several malignant diseases, including Burkitts lymphoma, nasopharyngeal carcinoma (NPC), certain types of lymphomas, and a portion of gastric cancers. Virus-encoded oncoprotein LMP1 induces the epithelial-to-mesenchymal transition (EMT), leading to cancer stem cell formation. In the current study, we investigated how LMP1 contributes to cancer stem cell development in NPC. We found that LMP1 plays an essential role in acquiring CSC characteristics, including tumor initiation, metastasis, and therapeutic resistance by activating the PI3K/mTOR/Akt signaling pathway. We dissected the functions of distinct signaling (mTORC1 and mTORC2) in the acquisition of different CSC characteristics. Side population (SP) formation, which represents the chemotherapy resistance feature of CSC, requires mTORC1 signaling. Tumor initiation capability is mainly attributed to mTORC2, which confers on NPC the capabilities of proliferation and survival by activating mTORC2 downstream genes c-Myc. Both mTORC1 and mTORC2 enhance cell migration and invasion of NPC cells, suggesting that mTORC1/2 co-regulate metastasis of NPC. The revelation of the roles of the mTOR signaling pathways in distinct tumorigenic features provides a guideline for designing efficient therapies by choosing specific mTOR inhibitors targeting mTORC1, mTORC2, or both to achieve durable remission of NPC in patients.

SignificanceLMP1 endows NPC to gain cancer stem cell characteristics through activating mTORC1 and mTORC2 pathways. The different mTOR pathways are responsible for distinct tumorigenic features. Rapamycin-insensitive mTORC1 is essential for CSC drug resistance. NPC tumor initiation capacity is mainly attributed to mTORC2 signaling. mTORC1 and mTORC2 co-regulate NPC cell migration and invasion. The revelation of the roles of mTOR signaling in NPC CSC establishment has implications for novel therapeutic strategies to treat relapsed and metastatic NPC and achieve durable remission.


title: The hexokinase ''HKDC1'' interaction with the mitochondria is essential for hepatocellular carcinoma progression

abstract: Hepatocellular carcinoma (HCC) is a leading cause of death from cancer malignancies. Recently, hexokinase domain containing 1 (HKDC1), was shown to have significant overexpression in HCC compared to healthy tissue. Using in vitro and in vivo tools, we examined the role of HKDC1 in HCC progression. Importantly, HKDC1 ablation stops HCC progression by promoting metabolic reprogramming by shifting glucose flux away from the TCA cycle. Next, HKDC1 ablation leads to mitochondrial dysfunction resulting in less cellular energy which cannot be compensated by enhanced glucose uptake. And finally, we show that the interaction of HKDC1 with the mitochondria is essential for its role in HCC progression, and without this mitochondrial interaction mitochondrial dysfunction occurs. In sum, HKDC1 is highly expressed in HCC cells compared to normal hepatocytes, therefore targeting HKDC1, specifically its interaction with the mitochondria, reveals a highly selective approach to target cancer cells in HCC.


title: Decoupling the molecular regulation of perenniality and flowering in bulbous barley (Hordeum bulbosum)

abstract:
Global crop production is being challenged by rapid population growth, declining natural resources, and dramatic climatic turnovers. These challenges have prompted plant breeders to explore new ventures to enhance adaptation and sustainability in crops. One intriguing approach to make agriculture more sustainable is by turning annual systems into perennial which offers many economic and biodiversity-friendly benefits. Previous attempts to develop a perennial cereal crop employed a classical breeding approach and extended over a long period with limited success. Thus, elucidating the genetic basis of perenniality at the molecular level can accelerate the breeding process.

Here, we investigated the genetic basis of bulb formation in the barley congener species Hordeum bulbosum by elucidating the transcripts presence/absence variation compared with other annual species in the Poaceae, and a differential expression analysis of meristem tissues. The PAV analysis recaptured the expected phylogeny and indicated that H. bulbosum is enriched with developmental and disease responsive genes that are absent among annual species. Next, the abundance of transcripts was quantified and allowed to identify differentially expressed genes that are associated with bulb formation pathways in addition to major circadian clock genes that regulate flowering. A first model for the bulb formation pathway is suggested and include developmental and starch biosynthesis genes. To the best of our knowledge this is the first transcriptome developed for H. bulbosum and the first attempt to describe the regulation of bulb initiation in cereals at the molecular level.


title: Frizzled-4 regulates β-catenin in endothelial cells exposed to disturbed flow via an atypical Wnt pathway leading to proinflammatory activation and increased permeability

abstract:
ObjectiveEndothelial cells are regulated by hemodynamic wall shear stress and multidirectional shear stress is known to promote endothelial dysfunction, although the molecular mechanisms are poorly defined. Wnt pathways play an important role in non-vascular mechanoresponsive cells. Here we investigated their role in endothelial mechanosignalling and endothelial dysfunction.

Approach & ResultsHuman aortic endothelial cells were exposed to shear stress using an orbital shaker. The expression of Frizzled-4 receptors was significantly increased in endothelial cells exposed to low magnitude multidirectional flow (LMMF) relative to high magnitude uniaxial flow (HMUF). Increased expression was also detected in regions of the murine aortic arch exposed to LMMF. The increased Frizzled-4 expression in cultured cells was abrogated following knockdown of R-spondin-3 (RSPO-3) using RNA interference. LMMF also increased the stabilisation and nuclear localisation of {beta}-catenin, an effect that was dependent on Frizzled-4 and RSPO-3. Inhibition of {beta}-catenin using a small molecule inhibitor (iCRT5), or knockdown of Frizzled-4 or R-spondin-3 resulted in a significant reduction of pro-inflammatory gene expression in endothelial cells exposed to LMMF. Stabilisation of the {beta}-catenin destruction complex using IWR-1 under LMMF also reduced pro-inflammatory gene expression, as did inhibition of Wnt5a signalling. Interestingly, inhibition of the canonical Wnt pathway had no effect. Inhibition of {beta}-catenin signalling also reduced endothelial permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling.

ConclusionsThese data suggest the presence of an atypical Wnt-{beta}-catenin pathway in endothelial cells that promotes inflammatory activation and barrier disruption in response to LMMF.


title: Disruption of Mitochondrial Quality Control Genes Promotes Caspase-Resistant Cell Survival Following Apoptotic Stimuli

abstract: In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases a subpopulation of the treated cells can exhibit a sublethal response, termed "minority MOMP". In this phenomenon, the affected cells survive, despite a low level of caspase activation and a subsequent limited activation of the endonuclease CAD (DFFB). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. To discover genes affecting MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells. Among these were genes related to mitochondrial dynamics and mitophagy, which participate in the mitochondrial quality control (MQC) system. To test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.


title: Optogenetic manipulation of cGMP highlights PDE5 as the predominant cGMP-hydrolyzing PDE in megakaryocytes

abstract: Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatiotemporal regulation in megakaryocytes (MKs) is lacking. We expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide (NO) triggered cGMP generation in BM MKs. In summary, we established for the first time optogenetics in primary MKs and identified PDE5 as the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.


title: Identification and Characterization of Stem Cells in Mammalian Esophageal Stratified Squamous Epithelia

abstract: Somatic stem cells are essential for maintenance of cell proliferation-differentiation homeostasis in organs. Despite the importance, how the esophageal epithelium that executes its self-renewal and maintenance remains elusive. In this study, using 5-bromo-2-deoxyuridine (BrdU) label-chase in rat and rat esophageal keratinocyte cell line-derived organoids together with genome-wide DNA methylation profiling and single-cell RNA sequencing (scRNA-seq), we identify slow cycling/quiescent stem cell population that contain high levels of hemidesmosome (HD)s and low levels of Wnt signaling localized spatially and randomly at the basal layer of the esophageal epithelium. Pseudo-time cell trajectory from scRNA-seq indicates that cell fates begin from quiescent basal cells (the stem cells) of the basal layer that produce proliferating and/or differentiating cells in the basal layer, which, in turn, progress into differentiating cells in the suprabasal layer, ultimately transforming into differentiated keratinocytes in the differentiated layer. Perturbations of HD component expressions and/or Wnt signaling reduce stem cell in the basal layer of esophageal keratinocyte organoids, resulting in alterations of organoid formation rate, size, morphogenesis and proliferation-differentiation homeostasis. Furthermore, we show that not only high levels of HDs and low levels of Wnt signaling but also an interplay between HD and Wnt signaling defined stem cells of the basal layer in the esophageal squamous epithelium. Hence, HDs and Wnt signaling are the critical determinants for defining stem cells of the basal layer required for proliferation-differentiation homeostasis and maintenance in the mammalian esophageal squamous epithelium.


title: G6b-B regulates an essential step in megakaryocyte maturation

abstract: G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null-mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, while proplatelet release is hampered, but the underlying molecular mechanism remains unclear. Here, we report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis and osteosclerosis. As the mutation is based on a single nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller in size, displayed a less developed demarcation membrane system and reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1, and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.


title: Abolishing the prelamin A ZMPSTE24 cleavage site leads to progeroid phenotypes with near-normal longevity in mice

abstract: Prelamin A is a farnesylated precursor of lamin A, a nuclear lamina protein. Accumulation of the farnesylated prelamin A variant progerin, with an internal deletion including its processing site, causes Hutchinson-Gilford progeria syndrome. Loss of function mutations in ZMPSTE24, which encodes the prelamin A processing enzyme, lead to accumulation of full-length farnesylated prelamin A and cause related progeroid disorders. Some data suggest that prelamin A also accumulates with physiological aging. Zmpste24-/- mice die young, at ~20 weeks. Because ZMPSTE24 has functions in addition to prelamin A processing, we generated a mouse model to examine effects solely due to the presence of permanently farnesylated prelamin A. These mice have an L648R amino acid substitution in prelamin A that blocks ZMPSTE24-catalyzed processing to lamin A. The LmnaL648R/L648R mice express only prelamin and no mature protein. Notably, nearly all survive to 65-70 weeks, with approximately 40% of male and 75% of female LmnaL648R/L648R mice having near-normal lifespans of 90 weeks (almost 2 years). Starting at ~10 weeks of age, LmnaL648R/L648R mice of both sexes have lower body masses and body fat than controls. By ~20-30 weeks of age, they exhibit detectable cranial, mandibular and dental defects similar to those observed in Zmpste24-/- mice, and have decreased vertebral bone density compared to age- and sex-matched controls. Cultured embryonic fibroblasts from LmnaL648R/L648R mice have aberrant nuclear morphology that is reversible by treatment with a protein farnesyltransferase inhibitor. These novel mice provide a robust model to study the effects of farnesylated prelamin A during physiological aging.


title: Multi-omic rejuvenation and lifespan extension upon exposure to youthful circulation

abstract: Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on the biological age of organisms and their long-term health remains unknown. Here, we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the biological age of blood and liver based on epigenetic analyses across several clock models on two independent platforms; remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a comprehensive multi-omic rejuvenation effect. In addition, old HPB mice showed transcriptome changes opposite to aging, but akin to several lifespan-extending interventions. Altogether, we reveal that long-term HPB can decrease the biological age of mice, in part through long-lasting epigenetic and transcriptome remodeling, culminating in the extension of lifespan and healthspan.


title: BeeDNA: microfluidic environmental DNA metabarcoding as a tool for connecting plant and pollinator communities

abstract: Pollinators are imperiled by global declines that can impair plant reproduction, erode essential ecosystem services and resilience, and drive economic losses. Monitoring pollinator biodiversity trends is key for adaptive conservation and management, but conventional surveys are often costly, time consuming, and require taxonomic expertise. Environmental DNA (eDNA) metabarcoding surveys are booming due to their rapidity, non-invasiveness, and cost efficiency. Microfluidic technology allows multiple primer sets from different markers to be used in eDNA metabarcoding for more comprehensive species inventories whilst minimizing biases associated with individual primer sets. We evaluated microfluidic eDNA metabarcoding for pollinator community monitoring by introducing a bumblebee colony to a greenhouse flower assemblage and sampling natural flower plots. We collected nectar draws, flower swabs, or whole flower heads from four flowering species, including two occurring in both the greenhouse and field. Samples were processed using two eDNA isolation protocols before amplification with 15 primer sets for two markers (COI and 16S). Microfluidic eDNA metabarcoding detected the target bumblebee and greenhouse insects as well as common regional arthropods. Pollinator detection was maximized using whole flower heads preserved in ATL buffer and extracted with a modified Qiagen(R) DNeasy protocol for amplification with COI primers. eDNA surveillance could enhance pollinator assessment by detecting protected and endangered species and being more applicable to remote, inaccessible locations, whilst reducing survey time, effort, and expense. Microfluidic eDNA metabarcoding requires optimization but shows promise in revealing complex networks underpinning critical ecosystem functions and services, enabling more accurate assessments of ecosystem resilience.

category: ecology


title: The dimensionality of plant-plant competition

abstract: To avoid extinction, every species must be able to exploit available resources at least as well as the other species in its community. All else being equal, theory predicts that the more distinct the niches of such co-occurring and competing species, the more species that can persist in the long run. However, both theoretical and experimental studies define a priori the nature and number of resources over which species compete. It therefore remains unclear whether or not species in empirically realistic contexts are actually exploiting all or some of the niches available to them. Here we provide a mathematical solution to this long-standing problem. Specifically, we show how to use the interactions between sets of co-occurring plant species to quantify their implied "niche dimensionality": the effective number of resources over which those species appear to be competing. We then apply this approach to quantify the niche dimensionality of 12 plant assemblages distributed across the globe. Contrary to conventional wisdom, we found that the niche dimensionality in these systems was much lower than the number of competing species. However, two high-resolution experiments also show that changes in the local environment induce a reshuffling of plants competitive roles and hence act to increase the assemblages effective niche dimensionality. Our results therefore indicate that homogeneous environments are unlikely to be able to maintain high diversity and also shows how environmental variation impacts species niches and hence their opportunities for long-term survival.


title: Legume plant defenses and nutrients mediate indirect interactions between soil rhizobia and chewing herbivores

abstract: Soil bacteria that form mutualisms with plants, such as rhizobia, affects susceptibility of plants to herbivores and pathogens. Soil rhizobia also promote nitrogen fixation, which mediates host nutrient levels and defenses. However, whether aboveground herbivores affect the function of soil rhizobia remains poorly understood. We assessed reciprocal interactions between Sitona lineatus, a chewing herbivore, and pea (Pisum sativum) plants grown with or without rhizobia (Rhizobium leguminosarum biovar viciae). We also examined the underlying plant-defense and nutritional mechanisms of these interactions. In our experiments, soil rhizobia influenced feeding and herbivory by chewing herbivores. Leaf defoliation by S. lineatus was lower on plants treated with rhizobia, but these insects had similar amino acid levels compared to those on un-inoculated plants. Plants grown with soil rhizobia had increased expression of gene transcripts associated with phytohormone-mediated defense, which may explain decreased susceptibility to S. lineatus. Rhizobia also induced expression of gene transcripts associated with physical and antioxidant-related defense pathways in P. sativum. Conversely, S. lineatus feeding reduced the number of root nodules and nodule biomass, suggesting a disruption of the symbiosis between plants and rhizobia. Our study shows that aboveground herbivores can engage in mutually antagonistic interactions with soil microbes mediated through a multitude of plant-mediated pathways.


title: The abiotic and biotic environment together predict plant, mammal, and bird diversity and turnover across the United States

abstract: The distribution of taxonomic, phylogenetic, and functional biodiversity results from a combination of abiotic and biotic drivers which are scale dependent. Parsing the relative influence of these drivers is critical to understanding the processes underlying species assembly and generating predictions of biodiversity across taxonomic groups and for novel sites. However, doing so requires data that capture a spatial extent large enough to reflect broad-scale dynamics such as speciation and biogeography, and a spatial grain fine enough to detect local-scale dynamics like environmental filtering and biotic interactions. We used species inventories of vascular plants, birds, and mammals collected by the U.S. National Ecological Observatory Network (NEON) at 38 terrestrial field sites, to explore the processes underlying taxonomic, phylogenetic, and functional diversity and turnover. We found that, for both species richness (alpha-diversity) and turnover (beta-diversity), taxonomic, phylogenetic, and functional diversity are weak proxies for one-another, and thus may capture different species assembly processes. All diversity metrics were best predicted by a combination of abiotic and biotic variables. Taxonomic and phylogenetic richness tended to be higher at warmer, wetter sites, reflecting the role energy inputs play in driving broad-scale diversity. However, plant diversity was negatively correlated with bird phylogenetic and mammal functional diversity, implying trait conservation in plant communities may limit niche availability for consumer species. Equally, turnover in bird and mammal species across sites were associated with plant turnover. That the biodiversity of one taxon is predictive of another across these North American sites, even when controlling for environment, supports a role for the cross-clade biotic environment in driving species assembly.


title: Are island plants really poorly defended? No support for the loss of defense hypothesis in domatia-bearing plants

abstract: 1.The loss of defense hypothesis posits that island colonizers experience a release from predation on the mainland and subsequently lose their defensive adaptations. While support for the hypothesis is abundant, it has never been tested in domatia-bearing plants. Leaf domatia are cave-like structures produced on the underside of leaves that facilitate a defensive mutualism with predatory and fungivorous mites. I tested the loss of defense hypothesis in six domatia-bearing taxa inhabiting New Zealand and its offshore islands. No support for the loss of defense hypothesis was found. Changes in domatia investment were instead associated with changes in leaf size - a trait that has been repeatedly observed to undergo rapid evolution on islands. Overall results demonstrate that not all types of defense are lost on islands, suggesting a higher-resolution approach is needed when studying the evolution of defense on islands.

category: evolutionary biology


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

abstract:
Genetic deficiency of acid -glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe disease (PD) encompassing at least four clinical subtypes of varying severity (infantile; childhood, juvenile and late onset). Our objective is to develop an innovative and affordable approach for enzyme replacement therapy (ERT) via oral administration (Oral-ERT) to maintain a sustained, therapeutic level of enzyme on a daily basis to improve efficacy of treatment and quality of life for people living with Pompe disease. A consensus at a 2019 US Acid Maltase Deficiency (AMDA) conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment of Pompe disease. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. Previously, we have shown that a lysate from transgenic tobacco seeds expressing human GAA (tobrhGAA) was enzymatically active and can correct enzyme deficiency in cultured PD cells and in adult lymphocytes of Pompe patients and in vivo in disease-relevant tissues in GAA knockout (KO) mice when administered IP.

We have extended these pre-clinical studies in PD knockout (KO) mice with ground tobrhGAA seeds that supports proof-of-concept for Oral-ERT for future clinical trials. Briefly in GAA KO mice, Oral-ERT with ground tobrhGAA seeds showed significant reversal of fore-limb and hind-limb muscle weakness, increased motor coordination/balance/strength and mobility, improved spontaneous learning, increased GAA baseline activity in tissues, reduced glycogen in tissues and negible serum titers to GAA. Pharmacokinetics showed maximum serum GAA concentration (Cs) at 8-10 hr and peak urine excretion at 10-12 hr. The tobrhGAA was taken up in PD fibroblast, lymphoid and myoblast cells. Enzyme kinetics compared favorably or superior to placental hGAA, plus alglucosidase alfa or other rhGAAs for Km, Vmax, pH optima, thermal heat stability and IC50 for inhibitors. The tobrhGAA in seeds was extremely stable stored for 15 years at room temperature. NGS-genome sequencing of the tobrhGAA and wild-type plants and RNA expression profiles was performed and will be posted on our website. Thus, Oral-ERT with ground tobrhGAA seeds is an innovative approach that overcomes some of the challenges of alglucosidase alfa-ERT and provides a more effective, safe and significantly less expensive treatment.

category: genetics


title: A DNA barcode database for the woody plants of Japan

abstract: DNA barcode databases are increasingly available for a range of organisms facilitating the wide application of DNA barcode-based pursuits. Here we announce the development of a comprehensive DNA barcode database of the Japanese woody flora representing 43 orders, 99 families, 303 genera and 834 species and comprising 77.3% of genera and 72.2% of species of woody plants in Japan. A total of 6,216 plant specimens were collected from 223 sites (municipalities, i.e. city, town, village) across the subtropical, temperate, boreal and alpine biomes in Japan with most species represented by multiple accessions. This database utilised three chloroplast DNA regions (rbcL, trnH-psbA and matK) and consists of 14,404 barcode sequences. Individual regions varied in their identification rates with species-level and genus-level rates for rbcL, trnH-psbA and matK being 57.4%/ 96.2%, 78.5%/ 99.1 % and 67.8%/ 98%, respectively. Identification rates were higher using region combinations with total species level rates for two region combinations (rbcL & trnH, rbcL & matK, and trnH-psbA & matK) ranging between 90.6-95.8%, and for all three regions equal to 98.6%. Genus level identification rates were even higher ranging between 99.7-100% for two region combinations and being 100% for the three regions. These results indicate that this DNA barcode database is an effective resource for investigations of woody plants in Japan using DNA barcodes and provides a useful template for development of libraries for other components of the Japanese flora.


title: Multiscale profiling of enzyme activity in cancer

abstract: Diverse processes in cancer are mediated by enzymes, which most proximally exert their function through their activity. Methods to quantify enzyme activity, rather than just expression, are therefore critical to our ability to understand the pathological roles of enzymes in cancer and to harness this class of biomolecules as diagnostic and therapeutic targets. Here we present an integrated set of methods for measuring specific enzyme activities across the organism, tissue, and cellular levels, which we unify into a methodological hierarchy to facilitate biological discovery. We focus on proteases for method development and validate our approach through the study of tumor progression and treatment response in an autochthonous model of Alk-mutant lung cancer. To quantitatively measure activity dynamics over time, we engineered multiplexed, peptide-based nanosensors to query protease activity in vivo. Machine learning analysis of sensor measurements revealed dramatic protease dysregulation in lung cancer, including significantly enhanced proteolytic cleavage of one peptide, S1 (Padj < 0.0001), which returned to healthy levels within three days after initiation of targeted therapy. Next, to link these organism-level observations to the in situ context, we established a multiplexed assay for on-tissue localization of enzyme activity and pinpointed S1 cleavage to endothelial cells and pericytes of the tumor vasculature. Lastly, to directly link enzyme activity measurements to cellular phenotype, we designed a high-throughput method to isolate and characterize proteolytically active cells, uncovering profound upregulation of pro-angiogenic transcriptional programs in S1-positive cells. Together, these methods allowed us to discover that protease production by angiogenic vasculature responds rapidly to targeted therapy against oncogene-addicted tumor cells, identifying a highly dynamic interplay between tumor cells and their microenvironment. This work provides a generalizable framework to functionally characterize enzyme activity in cancer.

category: bioengineering


title: Functionalizing silica sol-gel with entrapped plant virus-based immunosorbent nanoparticles

abstract: Advancements in understanding and engineering of virus-based nanomaterials (VBNs) for biomedical applications motivate a need to explore the interfaces between VBNs and other biomedically-relevant chemistries and materials. While several strategies have been used to investigate some of these interfaces with promising initial results, including VBN-containing slow-release implants and VBN-activated bioceramic bone scaffolds, there remains a need to establish VBN-immobilized three dimensional materials that exhibit improved stability and diffusion characteristics for biosensing and other analyte-capture applications. Silica sol-gel chemistries have been researched for biomedical applications over several decades and are well understood; various cellular organisms and biomolecules (e.g., bacteria, algae, enzymes) have been immobilized in silica sol-gels to improve viability, activity, and form factor (i.e., ease of use). Here we present the immobilization of an antibody-binding VBN in silica sol-gel by pore confinement. We have shown that the resulting system is sufficiently diffuse to allow antibodies to migrate in and out of the matrix. We also show that the immobilized VBN is capable of antibody binding and elution functionality under different buffer conditions for multiple use cycles. The promising results of the VBN and silica sol-gel interface indicate a general applicability for VBN-based bioseparations and biosensing applications.


title: Self-organization of kinetochore-fibers in human mitotic spindles

abstract: During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.


title: Cells use molecular working memory to navigate in changing chemoattractant fields

abstract: In order to migrate over large distances, cells within tissues and organisms rely on sensing local gradient cues. These cues however are multifarious, irregular or conflicting, changing both in time and space. Here we find that single cells utilize a molecular mechanism akin to a working memory, to generate persistent directional migration when signals are disrupted by temporally memorizing their position, while still remaining adaptive to spatial and temporal changes of the signal source. Using dynamical systems theory, we derive that these information processing capabilities are inherent for protein networks whose dynamics is maintained away from steady state through organization at criticality. We demonstrate experimentally using the Epidermal growth factor receptor (EGFR) signaling network, that the memory is maintained in the prolonged receptors activity via a slow-escaping remnant, a dynamical "ghost" of the attractor of the polarized signaling state, that further results in memory in migration. As this state is metastable, it also enables continuous adaptation of the migration direction when the signals vary in space and time. We therefore show that cells implement real-time computations without stable-states to navigate in changing chemoattractant fields by memorizing position of disrupted signals while maintaining sensitivity to novel chemical cues.


title: Merlin Tumor Suppressor Function is Regulated by PIP2-Mediated Dimerization

abstract: Neurofibromatosis Type 2 is an inherited disease characterized by Schwann cell tumors of cranial and peripheral nerves. The NF2 gene encodes Merlin, a member of the ERM family consisting of an N-terminal FERM domain, a central -helical region and a C-terminal domain that binds to the FERM domain. Changes in the intermolecular FERM-CTD interaction allow Merlin to transition between an open, FERM accessible conformation and a closed, FERM-inaccessible conformation, modulating Merlin activity. These conformational transitions are regulated by both phosphorylation and phosphoinositide binding. Merlin has been shown to dimerize, but the regulation and function Merlin dimerization is not clear. We used a nanobody based binding assay and found that Merlin dimerizes via a FERM-FERM interaction in a parallel orientation that requires an uncovered N-terminus and the first 18 amino acids of the FERM domain. Patient derived and structural mutants show that dimerization controls interactions with specific binding partners, including HIPPO pathway components, and correlates with tumor suppressor activity. Dimerization requires an open conformation, is inhibited by phosphorylation at serine 518 and is enhanced by PIP2 binding. The discovery that active, open conformation Merlin is a dimer represents a new paradigm for Merlin function with significant implications for the development of therapies designed to compensate for Merlin loss.


title: Marchantia polymorpha gene expression atlas reveals the hierarchy of abiotic stress responses and conservation of diurnal gene expression

abstract: Abiotic stress has a dramatic impact on ecosystems and the yield of crops, and global warming will likely result in more frequent and intense stresses. However, due to the complexity of the underlying signalling pathways and the fact that stresses often occur in combinations in nature, our knowledge of how plants acclimatize to abiotic stress is still lacking. Here, we used a plant with minimal regulatory network redundancy, Marchantia polymorpha, to study how seven abiotic stresses alone and in combination affect the phenotype, gene expression, and activity of biological pathways. Worryingly, we see no evidence of conservation of gene expression to abiotic stress between model organisms, suggesting that each species develops unique strategies to cope with abiotic stress. Interestingly, we observed that certain stresses are able to suppress others, while specific combinations can elucidate novel transcriptomic responses. Finally, we provide two online databases to study abiotic stress responses and diurnal gene expression data in Marchantia.


title: Physiological and molecular mechanism of tolerance of two maize genotypes under multiple abiotic stresses.

abstract: Abiotic stresses are the major threat to crops regardless of their nature, duration, and frequency, their occurrence either singly, and or combination is deleterious for the plant growth and development. Maize is most important crop largely grown in tropical region in summer rainy season, often face a stress combination of drought and waterlogging. We previously showed under multiple stresses up-regulated leaf proteins of maize plants were involved to enhance the tolerance mechanism of tolerant genotype. Whereas, in susceptible genotypes up-regulated proteins ameliorate to survive the stressful condition. Further to understand the response of roots proteome under multiple stresses was determined using the 2DE technique. The results of the root proteome show the up-regulated proteins of CML49 genotype (tolerant) are involved in enhancing the N content, cell wall remodeling, and acclimatization during the stresses. Up-regulated proteins of CML100 genotype (sensitive) are stressed marker of roots primary and secondary metabolism. However, the root proteome of both genotypes correlates with the leaf proteome (previous). Therefore, the present study and our previous results provide comprehensive insight into the molecular mechanisms of tolerance in multiple abiotic stresses of maize plants.


title: The rice OsERF101 transcription factor regulates the NLR Xa1-mediated perception of TAL effectors and Xa1-mediated immunity

abstract:
O_LIPlant nucleotide-binding leucine-rich repeat receptors (NLRs) initiate immune responses and the hypersensitive response by recognizing pathogen effectors. Xa1 encodes an NLR with an N-terminal BED domain, and recognizes transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo). The molecular mechanisms controlling the recognition of TAL effectors by Xa1 and the subsequent induction of immunity remain poorly understood.
C_LIO_LIXa1 interacts in the nucleus with two TAL effectors via the BED domain. We identified the AP2/ERF-type transcription factor OsERF101/OsRAP2.6 as an interactor with Xa1, and found that it also interacts with the TAL effectors.
Overexpression of OsERF101 exhibited an enhanced resistance to an incompatible Xoo strain only in the presence of Xa1, indicating that OsERF101 functions as a positive regulator of Xa1-mediated immunity. Unexpectedly, oserf101 mutants also showed enhanced Xa1-dependent resistance, but in a different manner from the overexpressing plants. This result revealed an additional Xa1-mediated immune pathway that is negatively regulated by OsERF101. Furthermore, OsERF101 directly interacted with the TAL effectors.
C_LIO_LIOur results show that OsERF101 regulates the recognition of TAL effectors and the Xa1-mediated activation of the immune response. These data provide new insights into the molecular mechanism of NLR-mediated immunity in plants.
C_LI


title: Computational design of nanoscale rotational mechanics in de novo protein assemblies

abstract:
Natural nanomachines like the F1/F0-ATPase contain protein components that undergo rotation relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein rotary machinery from designed axle and ring components. Using cryoelectron microscopy, we find that axle-ring systems assemble as designed and populate diverse rotational states depending on symmetry match or mismatch and the designed interface energy landscape. These mechanical systems with internal rotational degrees of freedom are a step towards the systematic design of genetically encodable nanomachines.

One-Sentence SummaryComputationally designed self-assembling protein rotary machines sample internal degrees of freedom sculpted within the energy landscape.

category: synthetic biology


title: Multi-regional characterisation of renal cell carcinoma and microenvironment at single cell resolution

abstract:
Tumour behaviour is dependent on the oncogenic properties of cancer cells and their multicellular interactions. These dependencies were examined through 270,000 single cell transcriptomes and 100 micro-dissected whole exomes obtained from 12 patients with kidney tumours. Tissue was sampled from multiple regions of tumour core, tumour-normal interface, normal surrounding tissues, and peripheral blood. We found the principal spatial location of CD8+ T cell clonotypes largely defined exhaustion state, with clonotypic heterogeneity not explained by somatic intra-tumoural heterogeneity. De novo mutation calling from single cell RNA sequencing data allows us to lineagetrace and infer clonality of cells. We discovered six meta-programmes that distinguish tumour cell function. An epithelial-mesenchymal transition meta-programme, enriched at the tumour-normal interface appears modulated through macrophage expressed IL1B, potentially forming a therapeutic target.

Single sentence summaryKidney cancer evolution, prognosis and therapy are revealed by a single cell multi-regional study of the microenvironment.

category: genomics


title: Abnormal (hydroxy)prolines deuterium content redefines hydrogen chemical mass

abstract:
Analysing the {delta}2H in individual amino acids of proteins extracted from vertebrates, we unexpectedly found in some samples, notably bone collagen from seals, more than twice as much deuterium in proline and hydroxyproline residues than in seawater. This corresponds to at least four times higher {delta}2H than in any previously reported biogenic sample. We ruled out diet as a plausible mechanism for such anomalous enrichment. This finding puts into question the old adage that you are what you eat.

SUMMARYThe chemical mass of hydrogen is defined as an interval from the lowest to the highest content of deuterium 2H, hydrogens heavy stable isotope. Measurements of the deviations {delta}2H in the deuterium content from the standard (ocean water, {delta}2H = 0{per thousand}) are used to characterise biological samples, such as animal bone collagen. The results are often interpreted in terms of the trophic level and diet of the animal as well as prevailing climate during its lifetime. The majority of the published bone collagen {delta}2H data fall into a narrow {delta}2H range limited to {+/-}100{per thousand}. Using novel analysis method, we unexpectedly found greatly higher {delta}2H values, up to 1500{per thousand}, in seal bone collagen. Such anomalous deuterium enrichment is detected only in two amino acid residues, proline and its derivative hydroxyproline, while other residues show much smaller {delta}2H values. Anomalously high {delta}2H values, albeit of lower magnitudes, are also found for these residues in other biological sources. This finding substantially expands the upper bound of the hydrogen chemical mass for biogenic sources. Since neither diet nor environment explain these mysteriously high enrichment levels amounting to more than twice deuterium content in sea water, our understanding of stable isotopes in nature, as well as the old adage "you are what you eat", are put in question.

category: biochemistry


title: G6PD deficiency sensitizes metastasizing melanoma cells to oxidative stress and glutaminolysis

abstract:
The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To test this, we mutated the substrate binding site of Glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative branch of the pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors but when these tumors spontaneously metastasized the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species (ROS), decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing the production of NADPH through glutaminolysis. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutamine as compared to control melanomas. The oxidative pentose phosphate pathway and compensatory glutaminolysis thus confer layered protection against oxidative stress during metastasis.

SignificanceMelanoma metastasis is limited by oxidative stress. Cells that enter the blood experience high levels of ROS and usually die of ferroptosis. We found that melanoma cells become more dependent upon the oxidative branch of the pentose phosphate pathway to manage oxidative stress during metastasis. When pentose phosphate pathway function was disabled by G6PD mutation, the melanoma cells increased their utilization of malic enzyme, fueled by increased consumption of glutamine in the tricarboxylic acid cycle. Melanoma cells thus have redundant and layered protection against oxidative stress.


title: An instance-specific causal framework for learning intercellular communication networks that define microenvironments of individual tumors

abstract: Cells within a tumor microenvironment (TME) dynamically communicate and influence each others cellular states through an intercellular communication network (ICN). In cancers, intercellular communications underlie immune evasion mechanisms of individual tumors. We developed an individualized causal analysis framework for discovering tumor-specific ICNs. Using head and neck squamous cell carcinoma (HNSCC) tumors as a testbed, we first mined single-cell RNA-sequencing data to discover gene expression modules (GEMs) that reflect the states of transcriptomic processes within tumor and stromal single cells. By deconvoluting bulk transcriptomes of HNSCC tumors profiled by The Cancer Genome Atlas (TCGA), we estimated the activation states of these transcriptomic processes in individual tumors. Finally, we applied individualized causal network learning to discover an ICN within each tumor. Our results show that cellular states of cells in TMEs are coordinated through ICNs that enable multi-way communications among epithelial, fibroblast, endothelial, and immune cells. Further analyses of individual ICNs revealed structural patterns that were shared across subsets of tumors, leading to the discovery of 4 different subtypes of networks that underlie disparate TMEs of HNSCC. Patients with distinct TMEs exhibited significantly different clinical outcomes. Our results show that the capability of estimating individual ICNs reveals heterogeneity of ICNs and sheds light on the importance of intercellular communication in impacting disease development and progression.


title: An individualized causal framework for learning intercellular communicationnetworks that define microenvironments of individual tumors


title: Fingerprinting of skin cells by live cell Raman spectroscopy reveals melanoma cell heterogeneity and cell-type specific responses to UVR

abstract: Raman spectroscopy is an emerging dermatological technique with the potential to discriminate biochemically between cell types in a label free and non-invasive manner. Here we use live single cell Raman spectroscopy and principal component analysis (PCA) to fingerprint mouse melanoblasts, melanocytes, keratinocytes and melanoma cells. We show the differences in their spectra are attributable to biomarkers in the melanin biosynthesis pathway and that melanoma cells are a heterogeneous population that sit on a trajectory between undifferentiated melanoblasts and differentiated melanocytes. We demonstrate the utility of Raman spectroscopy as a highly sensitive tool to probe the melanin biosynthesis pathway and its immediate response to UV irradiation revealing previously undescribed opposing responses to UVA and UVB irradiation in melanocytes. Finally, we identify melanocyte specific accumulation of {beta}-carotene correlated with a stabilisation of the UVR response in lipids and proteins consistent with a {beta}-carotene mediated photoprotective mechanism. In summary our data show that Raman spectroscopy can be used to determine the differentiation status of cells of the melanocyte lineage and describe the immediate and temporal biochemical changes associated with UV exposure which differ depending on cell type, differentiation status and competence to synthesise melanin.


title: Centrosomal Enrichment and Proteasomal Degradation of SYS-1/-β-catenin Requires the Microtubule Motor Dynein

abstract: The C. elegans Wnt/{beta}-catenin Asymmetry (W{beta}A) pathway utilizes asymmetric regulation of SYS- 1/{beta}-catenin and POP-1/TCF coactivators. W{beta}A differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased towards their appropriate cell fate. Despite the induction of asymmetry, {beta}-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Due to the mitosis-specific localization of SYS-1 to centrosomes and enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNAi-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe depletion of microtubules by nocodazole treatment or RNAi of dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose a model whereby retrograde microtubule-mediated trafficking enables SYS-1 enrichment at centrosomes, enhancing its eventual proteasomal degradation. These studies support the link between centrosomal localization and enhancement of proteasomal degradation, particularly for proteins not generally considered  centrosomal.


title: Molecular basis of unidirectional information transmission in two-component systems: lessons from the DesK-DesR thermosensor

abstract: Cellular signaling systems transmit information over long distances using allosteric transitions and/or post-translational modifications. In two-component systems the sensor histidine kinase and response regulator are wired through phosphoryl-transfer reactions, using either a uni- or bi-directional transmission mode, allowing to build rich regulatory networks. Using the thermosensor DesK-DesR two-component system from Bacillus subtilis and combining crystal structures, QM/MM calculations and integrative kinetic modeling, we uncover that: i) longer or shorter distances between the phosphoryl-acceptor and -donor residues can shift the phosphoryl-transfer equilibrium; ii) the phosphorylation-dependent dimerization of the regulator acts as a sequestering mechanism by preventing the interaction with the histidine kinase; and iii) the kinases intrinsic conformational equilibrium makes the phosphotransferase state unlikely in the absence of histidine phosphorylation, minimizing backwards transmission. These mechanisms allow the system to control the direction of signal transmission in a very efficient way, showcasing the key role that structure-encoded allostery plays in signaling proteins to store and transmit information.


title: Identifying common transcriptome signatures of cancer by interpreting deep learning models

abstract: Cancer is a set of diseases characterized by unchecked cell proliferation and invasion of surrounding tissues. The many genes that have been genetically associated with cancer or shown to directly contribute to oncogenesis vary widely between tumor types, but common gene signatures that relate to core cancer pathways have also been identified, signifying that cancer cases display common hallmark molecular features. It is not clear however whether there exist additional sets of genes or transcriptomic features that are less well known in cancer biology but that are also commonly deregulated across several cancer types. Here, in order to agnostically identify transcriptomic features that are commonly shared between cancer types, we used RNA-Seq datasets encompassing thousands of samples from 19 healthy tissue types and 18 solid tumor types to train three feed-forward neural networks, based either on protein-coding gene expression, lncRNA expression or splice junction use, to distinguish between healthy and tumor samples. All three models achieve high precision, recall and accuracy on test sets derived from 13 datasets used during training and on an independent test dataset, indicating that our models recognize transcriptome signatures that are consistent across tumors. Analysis of attribution values extracted from our models reveals that genes that are commonly altered in cancer by expression or splicing variations are under strong evolutionary and selective constraints, suggesting that they have important cellular functions. Importantly, we found that genes composing our cancer transcriptome signatures are not frequently affected by mutations or genomic alterations and that their functions differ widely from the genes genetically associated with cancer. Finally, our results also highlighted that deregulation of RNA-processing genes and aberrant splicing are pervasive features across a large array of solid tumor types. The transcriptomic features that we highlight here define cancer signatures that may reflect causal variations or consequences of disease state, or a combination of both.


title: Hydrodynamic model of fish orientation in a channel flow

abstract: For over a century, scientists have sought to understand how fish orient against an incoming flow, even without visual and flow cues. Here, we elucidate a potential hydrodynamic mechanism of rheotaxis through the study of the bidirectional coupling between fish and the surrounding fluid. By modeling a fish as a vortex dipole in an infinite channel with an imposed background flow, we establish a planar dynamical system for the cross-stream coordinate and orientation. The system dynamics captures the existence of a critical flow speed for fish to successfully orient while performing cross-stream, periodic sweeping movements. Model predictions are examined in the context of experimental observations in the literature on the rheotactic behavior of fish deprived of visual and lateral line cues. The crucial role of bidirectional hydrodynamic interactions unveiled by this model points at an overlooked limitation of existing experimental paradigms to study rheotaxis in the laboratory.


title: Transcriptomic plasticity and symbiont shuffling underpin Pocillopora acclimatization across heat-stress regimes in the Pacific Ocean

abstract: The characterization of adaptation and acclimation capacities of coral holobionts is crucial for anticipating the impact of global climate change on coral reefs. Understanding the extent to which the coral host and its photosymbionts contribute to adaptive and/or plastic responses in the coral metaorganism is important. In this study, we highlight new and complex links between coral genomes, transcriptomes, and environmental features in Pocilloporid corals at basin-wide scale. We analyzed metagenomic and metatranscriptomic sequence data from Pocillopora colonies sampled from 11 islands across the Pacific Ocean in order to investigate patterns of gene expression in both the host and photosymbiont across an environmental gradient. Single nucleotide polymorphisms (SNPs) analysis partitioned coral hosts and algal photosymbionts into five genetic lineages each. We observed strong host-symbiont fidelity across environments except at islands where recent and/or historical heat stress may have induced a symbiont shift towards more heat-tolerant lineages in some colonies. Host gene expression profiles were strongly segregated by genetic lineage and environment, and were significantly correlated with several historical sea surface temperature (SST) traits. Symbiont expression profiles were less dependent on environmental context than the host and were primarily driven by algal genotype. Overall, our results suggest a three-tiered strategy underpinning thermal acclimatization in Pocillopora holobionts with 1) host-photosymbiont fidelity, 2) host transcriptomic plasticity, and 3) photosymbiont shuffling playing progressive roles in response to elevated SSTs. Our data provide a reference for the biological state of coral holobionts across the Indo-Pacific and demonstrate the power of disentangling environmental and genetic effects to provide new insights into corals capacities for acclimatization and adaptation under environmental change.

category: physiology


title: Experimental and Biophysical Modeling of Transcription and Translation Dynamics in Bacterial- and Mammalian-based Cell-Free Expression Systems

abstract: Cell-free expression (CFE) systems have been used extensively in system and synthetic biology as a promising platform for manufacturing proteins and chemicals. Currently, the most widely used CFE system is in vitro protein transcription and translation platform. As the rapidly increased applications and uses, it is crucial to have a standard biophysical model for quantitative studies of gene circuits, which will provide a fundamental understanding of basic working mechanisms of CFE systems. Current modeling approaches mainly focus on the characterization of E. coli-based CFE systems, a computational model that can be utilized to both bacterial- and mammalianbased CFE has not been investigated. Here, we developed a simple ODE (ordinary differential equation)-based biophysical model to simulate transcription and translation dynamics for both bacterial- and mammalian-based CFE systems. The key parameters were estimated and adjusted based on experimental results. We next tested four gene circuits to characterize kinetic dynamics of transcription and translation in E. coli- and HeLa-based CFE systems. The real-time transcription and translation were monitored using Broccoli aptamer, double stranded locked nucleic acid (dsLNA) probe and fluorescent protein. We demonstrated the difference of kinetic dynamics for transcription and translation in both systems, which will provide valuable information for quantitative genomic and proteomic studies. This simple biophysical model and the experimental data for both E. coli- and HeLa-based CFE will be useful for researchers that are interested in genetic engineering and CFE bio-manufacturing.


title: A de novo matrix for macroscopic living materials from bacteria

abstract: Summary ParagraphEngineered living materials (ELMs) are composites of living cells embedded in a biopolymer matrix that combine the desirable properties of natural biomaterials with non-natural, tailored properties. ELMs with a wide range of sophisticated biological functions have been created by engineering the embedded cells using synthetic biology. Engineering a de novo biomolecular matrix would offer control over material assembly, structure, and composition, thus enabling us to grow macroscopic ELMs with customizable mechanical properties. However, we have lacked the genetic tools and design rules to genetically encode a synthetic matrix that programs collective cell self-organization into macroscopic structures. Here we report growth of macroscopic ELMs from Caulobacter crescentus cells that display and secrete an engineered self-interacting protein. This protein formed an extracellular de novo matrix and assembled cells into hierarchically-ordered, centimeter-scale ELMs. We showed that the mechanical, catalytic, and morphological properties of these ELMs can be tuned through genetic modification of the matrix. Our work identifies novel genetic tools, design and assembly rules for growing macroscopic ELMs with both wide-ranging mechanical properties and customizable functions. We anticipate the modularity of this approach will permit the incorporation of different protein polymers in the de novo matrix, thus allowing to generate ELMs with a variety of desired structures and compositions of the bulk material. We envision specific matrix properties that can be combined synergistically with existing cellular functions to greatly expand the opportunities for ELMs in human health, energy, and the environment.


title: Regulatory T cells suppress the formation of super-effector CD8 T cells by limiting IL-2

abstract: Regulatory T cells (Tregs) are indispensable for maintaining self-tolerance by suppressing conventional T cells. On the other hand, Tregs may promote tumor growth by inhibiting anti-cancer immunity. In this study, we identified that Tregs increase the quorum of self-reactive CD8+ T cells required for the induction of experimental autoimmune diabetes. Their major suppression mechanism is limiting available IL-2, an essential T-cell cytokine. Specifically, Tregs inhibit the formation of a previously uncharacterized subset of antigen-stimulated KLRK1+ IL7R+ (KILR) CD8+ effector T cells, which are distinct from conventional effector CD8+ T cells. KILR CD8+ T cells show a superior cell killing abilities in vivo. The administration of agonistic IL-2 immunocomplexes phenocopies the absence of Tregs, i.e., it induces KILR CD8+ T cells, promotes autoimmunity, and enhances anti-tumor responses. Counterparts of KILR CD8+ T cells were found in the human blood, revealing them as a potential target for immunotherapy.

category: immunology


title: Regulatory T cells suppress the formation of potent KLRK1 and IL-7R expressing effector CD8 T cells by limiting IL-2


title: An interplay between viruses and bacteria associated with marine sponges from the White Sea revealed by metagenomics

abstract: Sponges are remarkable holobionts harboring extremely diverse microbial and viral communities. However, the interactions between the components within holobionts and between a holobiont and environment are largely unknown, especially for polar organisms. To investigate possible interactions within the sponge-associated communities and between them, we probed the microbiomes and viromes of cold-water sympatric sponges Isodictya palmata, Halichondria panicea, and Halichondria sitiens by 16S and shotgun metagenomics. We showed that the bacterial and viral communities associated with these White Sea sponges are species-specific and different from the surrounding water. Extensive mining of bacterial antiphage defense systems in the metagenomes revealed a variety of defense mechanisms. The abundance of defense systems was comparable in the metagenomes of the sponges and the surrounding water, thus distinguishing the White Sea sponges from those inhabiting the tropical seas. We developed a network-based approach for the combined analysis of CRISPR-spacers and protospacers. Using this approach, we showed that the virus-host interactions within the sponge-associated community are typically more abundant than the inter-community interactions. Additionally, we detected the occurrence of viral exchanges between the communities. Our work provides the first insight into the metagenomics of the three cold-water sponge species from the White Sea and paves the way for a comprehensive analysis of the interactions between microbial communities and associated viruses.


title: T1SEstacker: A tri-layer stacking model effectively predicts bacterial type 1 secreted proteins based on C-terminal non-RTX-motif sequence features

abstract: The proteins secreted through type 1 secretion systems often play important roles in pathogenicity of various gram-negative bacteria. However, the type 1 secretion mechanism remains unknown. In this research, we observed the sequence features of RTX proteins, a major class of type 1 secreted substrates. We found striking non-RTX-motif amino acid composition patterns at the C-termini, most typically exemplified by the enriched  [FLI][VAI] at the most C-terminal two positions. Machine-learning models, including deep-learning models, were trained using these sequence-based non-RTX-motif features, and further combined into a tri-layer stacking model, T1SEstacker, which predicted the RTX proteins accurately, with a 5-fold cross-validated sensitivity of ~0.89 at the specificity of ~0.94. Besides substrates with RTX motifs, T1SEstacker can also well distinguish non-RTX-motif type 1 secreted proteins, further suggesting their potential existence of common secretion signals. In summary, we made comprehensive sequence analysis on the type 1 secreted RTX proteins, identified common sequence-based features at the C-termini, and developed a stacking model that can predict type 1 secreted proteins accurately.


title: Phage-encoded sigma factors alter bacterial dormancy

abstract: By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to tolerate suboptimal conditions that would otherwise reduce their fitness. Dormancy may also benefit bacteria by serving as a refuge from parasitic infections. Here we focus on dormancy in the Firmicutes, where endospore development is transcriptionally regulated by the expression of sigma factors. A disruption of this process could influence the survivorship and reproduction of phages that infect spore-forming hosts with implications for coevolutionary dynamics. Here, we characterized the distribution and diversity of sigma factors in nearly 3,500 phage genomes. Homologs of sporulation-specific sigma factors were identified in phages that infect spore-forming hosts. Unlike sigma factors required for phage reproduction, the sporulation-like sigma factors were non-essential for lytic infection. However, when expressed in the spore-forming Bacillus subtilis, sigma factors from phages activated the bacterial sporulation gene network and reduced spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate a complex and ancient trait in one of the most abundant cell types on Earth.


title: Differential regulation of degradation and immune pathways underlies adaptation of the symbiotic nematode Laxus oneistus to oxic-anoxic interfaces

abstract:
Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also upregulated when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worms Toll- like innate immunity pathway and several immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides).

In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires oxidative phosphorylation and by reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.


title: Differential regulation of degradation and immune pathways underlies adaptation of the ectosymbiotic nematode Laxus oneistus to oxic-anoxic interfaces


title: Genomic landscape of drug response reveals novel mediators of anthelmintic resistance

abstract:
Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to improving the efficacy and sustainability of parasite control. Here, we use a genetic cross in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies novel alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor, cky-1, in ivermectin resistance. This gene is within a locus under selection in ivermectin resistant populations worldwide; functional validation using knockout experiments supports a role for cky-1 overexpression in ivermectin resistance. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode, and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

One-Sentence SummaryGenetic mapping of known and novel anthelmintic resistance-associated alleles in a multi-drug resistant parasitic nematode


title: Systematic assessment of lipid profiles for the discovery of tissue contributors to the circulating lipid pool in cold exposure

abstract:
Plasma lipid levels are altered in chronic conditions such as type 2 diabetes and cardiovascular disease as well as acute stresses such as fasting and cold exposure. Advances in mass spectrometry based lipidomics have uncovered the complexity of the plasma lipidome which includes over 500 lipids that serve functional roles including energy substrate and signaling molecule. The plasma lipid pool is maintained through regulation of tissue production, secretion, and uptake. A major challenge is establishing the tissues of origin and uptake for various plasma lipids, which is necessary to determine the lipid function. Using cold exposure as an acute stress, we performed global lipidomics on the plasma and nine tissues that may contribute to the circulating pool. We found that numerous species of plasma acylcarnitines (ACars) and ceramides were significantly changed with cold exposure. Through computational assessment, we identified the liver and brown adipose tissue (BAT) as major contributors and consumers of circulating ACars, in agreement with our previous work. We further identified the kidney and intestine as novel contributors to the circulating ACar pool and validated these findings with gene expression analysis. Regression analysis also identified that the BAT and kidney as regulators of the plasma ceramide pool. These studies provide an adaptable computational tool to assess tissue contribution to the plasma lipid pool. Our findings have implications in understanding the function of plasma ACars and ceramides, which are elevated in metabolic diseases.

SummaryThere are over 500 identified lipids in circulating plasma, many without known origin or function. Using untargeted lipidomics on plasma and nine other tissues of cold exposed mice, we identified novel regulation of circulating acylcarnitines through the kidney and intestine, and a multiorgan system that regulates plasma ceramides. Our findings offer new targets for the study and functional characterization of circulating lipids in acute cold exposure and a computational resource for other investigators to explore multi-tissue lipidome remodeling during cold exposure.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

HighlightsO_LIGlobal lipidomics atlas of 9 tissues and plasma demonstrate dynamic shift with cold exposure.
C_LIO_LIAdaptive resource for the selection of extraction method, data processing, and data analysis of multi-tissue global lipidomics data.
C_LIO_LIRegression analysis identified the liver, BAT, intestine, and kidney as regulators of the plasma acylcarnitine pool that are not apparent by lipid levels alone.
C_LIO_LIAcute cold exposure increases plasma ceramide levels, with the BAT and kidney as major contributors
C_LI


title: A novel non-genetic murine model of hyperglycemia and hyperlipidemia-associated accelerated atherosclerosis

abstract:
ObjectiveAtherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s).

Methods and ResultsWild-type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less -SMA-positive area) and had more inflammation (increased plasma IL-1{beta} level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemic mice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks.

ConclusionWe established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice.

category: pharmacology and toxicology


title: A novel nongenetic murine model of hyperglycemia and hyperlipidemia-associated aggravated atherosclerosis


title: Behavioral and postural analyses establish sleep-like states for mosquitoes that can impact interactions with hosts

abstract: Sleep is an evolutionarily conserved process that has been described in different animal systems. For insects, sleep characterization has been primarily achieved using behavioral and electrophysiological correlates in a few systems. Sleep in mosquitoes, which are important vectors of disease-causing pathogens, has not been directly examined. This is surprising as circadian rhythms, which have been well studied in mosquitoes, influence sleep in other systems. In this study, we characterized sleep in mosquitoes using body posture analysis and behavioral correlates, and quantified the effect of sleep deprivation on sleep rebound, host landing and blood-feeding propensity. Body and appendage position metrics revealed a clear distinction between the posture of mosquitoes in their putative sleep and awake states for multiple species, which correlate with a reduction in responsiveness to host cues. Sleep assessment informed by these posture analyses indicated significantly more sleep during periods of low activity. Nighttime and daytime sleep deprivation resulting from the delivery of vibration stimuli induced sleep rebound in the subsequent phase in day and night active mosquitoes, respectively. Lastly, sleep deprivation suppressed host landing in both laboratory and field settings and also impaired blood feeding of a human host when mosquitoes would normally be active. These results suggest that quantifiable sleep states occur in mosquitoes, and highlight the potential epidemiological importance of mosquito sleep.


title: Behavioral and postural analyses establish sleep-like states for mosquitoes that can impact host landing and blood feeding


title: STAT1 Gain-of-Function Variants Drive Altered T Cell Prevalence, Metabolism, and Heightened IL-6 Sensitivity

abstract: Patients with Signal Transducer and Activator of Transcription 1 (STAT1) gain-of-function (GOF) pathogenic variants exhibit susceptibility to infections, autoimmunity, and cancer due to enhanced or prolonged STAT1 phosphorylation following cytokine stimulation. While interferons (IFNs) are canonical STAT1 activators, other cytokines that may also contribute to pathology in STAT1 GOF patients have been less well defined. Here we analyzed the immune profiles and cytokine responses of two patients with heterozygous GOF mutations in the STAT1 coiled-coil domain. A systems immunology approach revealed major changes in the T cell compartment and minor changes in the B cells, NK cells, and myeloid cells. Both patients with STAT1 GOF differed from healthy individuals in the abundance and phenotype of effector memory, Th17, and Treg populations. STAT1 GOF T cells displayed a pattern of increased activation and had elevated markers of glycolysis and lipid oxidation. Hypersensitivity of T cells to IL-6 was observed with intense, sustained STAT1 phosphorylation in memory T cell populations that exceeded that induced by IFNs. Together, these results show a role for STAT1 in T cell metabolism and suggest that IL-6 may play a critical role to promote T cell memory formation and activation in patients with STAT1 GOF.