title: Characterisation of the phase-variable autotransporter Lav reveals a role in host cell adherence and biofilm formation in Non-Typeable Haemophilus influenzae

abstract: Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in non-typeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA(n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to host cells, and denser biofilm formation. A survey of Haemophilus spp. genome sequences showed lav is present in [~]60% of NTHi strains, but lav is not present in most typeable H. influenzae. Sequence analysis revealed a total of five distinct variants of the Lav passenger domain present in Haemophilus spp., with these five variants showing a distinct lineage distribution. Determining the role of Lav in NTHi will help understand the role of this protein during distinct pathologies.


title: The nitrogen phosphotransferase regulator PtsN (EIIANtr) regulates inorganic polyphosphate production in Escherichia coli

abstract:
Inorganic polyphosphate (polyP) is synthesized by bacteria under stressful environmental conditions and acts by a variety of mechanisms to promote cell survival. While the kinase that synthesizes polyP (PPK, enocoded by the ppk gene) is well known, little is understood about how environmental stress signals lead to activation of this enzyme. Previous work has shown that the transcriptional regulators DksA, RpoN ({sigma}54), and RpoE ({sigma}24) positively regulate polyP production, but not ppk transcription, in Escherichia coli. In this work, we set out to examine the role of the alternative sigma factor RpoN and nitrogen starvation stress response pathways in controlling polyP synthesis in more detail. In the course of these experiments, we identified GlnG, GlrR, PhoP, PhoQ, RapZ, and GlmS as proteins that affect polyP production, and uncovered a central role for the nitrogen phosphotransferase regulator PtsN (EIIANtr) in a polyP regulatory pathway, acting upstream of DksA, downstream of RpoN, and apparently independently of RpoE. However, none of these regulators appears to act directly on PPK, and the mechanism(s) by which they modulate polyP production remain unclear. Unexpectedly, we also found that the pathways that regulate polyP production vary depending not only on the stress condition applied, but also on the composition of the media in which the cells were grown before exposure to polyP-inducing stress. These results constitute substantial progress towards deciphering the regulatory networks driving polyP production under stress, but highlight the remarkable complexity of this regulation and its connections to a broad range of stress-sensing pathways.

IMPORTANCEBacteria respond to changes in their environments with a complex regulatory network that controls the expression and activity of a wide range of effectors important for their survival. This stress response network is critical for the virulence of pathogenic bacteria and for the ability of all bacteria to grow in natural environments. Inorganic polyphosphate (polyP) is an evolutionarily ancient and almost universally conserved stress response effector that plays multiple roles in virulence, stress response, and survival in diverse organisms. This work provides new insights into the connections between well characterized nitrogen starvation and cell envelope stress response signaling pathways and the production of polyP in Escherichia coli.

category: microbiology


title: Recurrent Independent Pseudogenization Events of the Sperm Fertilization Gene ZP3r in Apes and Monkeys

abstract: In mice, ZP3r/sp56 is a binding partner to the egg coat protein ZP3 and may mediate induction of the acrosome reaction. ZP3r, as a member of the RCA cluster, is surrounded by paralogs, some of which have been shown to be evolving under positive selection. Sequence divergence paired with paralogous relationships with neighboring genes, has complicated the accurate identification of the human ZP3r ortholog. Here, we phylogenetically and syntenically resolve that the human ortholog of ZP3r is the pseudogene C4BPAP1. We investigate the evolution of this gene within primates. We observe independent pseudogenization events of ZP3r in all Apes with the exception of Orangutans, and many monkey species. ZP3r in both primates that retain ZP3r and rodents contains positively selected sites. We hypothesize that redundant mechanisms mediate ZP3 recognition in mammals and ZP3rs relative importance to ZP recognition varies across species.

category: molecular biology


title: Intermittent fasting induces rapid hepatocyte proliferation

abstract: Nutrient availability fluctuates in most natural populations, forcing organisms to undergo periods of fasting and re-feeding. It is unknown how dietary change influences cell proliferation in the liver. Here, we show that intermittent fasting (IF) promotes rapid hepatocyte proliferation. Mechanistically, IF elicits intestinal production of endocrine FGF15, which activates the {beta}-KLOTHO receptor on hepatocytes to induce proliferation. Furthermore, IF-induced proliferation is locally controlled in hepatocytes by WNT signaling and the WNT target gene Tbx3. IF induces hepatocyte proliferation to re-establish a constant liver-to-body-mass ratio during periods of fasting and re-feeding and maintain the hepatostat. This study provides the first example of dietary influence on adult hepatocyte proliferation and identifies both systemic FGF and local WNT as regulators of this process.

category: cell biology


title: When are bacteria really gazelles? Comparing patchy ecologies with dimensionless numbers

abstract: From micro to planetary scales, spatial heterogeneity--patchiness--is ubiquitous in ecological systems, defining the environments in which organisms move and interact. While this fact has been recognized for decades, most large-scale ecosystem models still use spatially averaged "mean fields" to represent natural populations, while fine-scale, spatially explicit models are mostly restricted to particular organisms or systems. In a conceptual paper, Grunbaum (2012, Interface Focus 2: 150-155) introduced a heuristic framework, based on three dimensionless ratios quantifying movement, reproduction, and resource consumption, to characterize patchy ecological interactions and identify when mean-field assumptions are justifiable. In this paper, we calculated Grunbaums dimensionless numbers for 33 real interactions between consumers and their resource patches in terrestrial, aquatic, and aerial environments. Consumers ranged in size from bacteria to blue whales, and patches lasted from minutes to millennia, spanning spatial scales of mm to hundreds of km. We found that none of the interactions could be accurately represented by a purely mean-field model, though 26 of them (79%) could be partially simplified by averaging out movement, reproductive, or consumption dynamics. Clustering consumer-resource pairs by their non-dimensional ratios revealed several unexpected dynamic similarities between disparate interactions. For example, bacterial Pseudoalteromonas exploit nutrient plumes in a similar manner to Mongolian gazelles grazing on ephemeral patches of steppe vegetation. Our findings suggest that dimensional analysis is a valuable tool for characterizing ecological patchiness, and can link the dynamics of widely different systems into a single quantitative framework.

category: ecology


title: Escape from X-inactivation in twins exhibits intra- and inter-individual variability across tissues and is heritable

abstract: X-chromosome inactivation (XCI) silences one X-chromosome in female cells to balance sex-differences in X-dosage. A subset of X-linked genes escape XCI, but the extent to which this phenomenon occurs and how it varies across tissues and in a population is as yet unclear. In order to characterize the incidence and variability of escape across individuals and tissues, we conducted a large scale transcriptomic study of XCI escape in adipose, skin, lymphoblastoid cell lines (LCLs) and immune cells in 248 twins drawn from a healthy population cohort. We identify 159 X-linked genes with detectable escape, of which 54 genes, including 19 lncRNAs, were not previously known to escape XCI. Across tissues we find a range of tissue-specificity, with 11% of genes escaping XCI constitutively across tissues and 24% demonstrating tissue-restricted escape, including genes with cell-type specific escape between immune cell types (B, T-CD4+, T-CD8+ and NK cells) of the same individual. Escape genes interact with autosomal-encoded proteins and are involved in varied biological processes such as gene regulation. We find substantial variability in escape between individuals. 49% of genes show inter-individual variability in escape, indicating escape from XCI is an under-appreciated source of gene expression differences. We utilized twin models to investigate the role of genetics in variable escape. Overall, monozygotic (MZ) twin pairs share more similar escape than dizygotic twin pairs, indicating that genetic factors underlie differences in escape across individuals. However, we also identify instances of discordant XCI within MZ co-twin pairs, suggesting that environmental factors also influence escape. Thus, XCI escape may be shaped by an interplay of genetic factors with tissue- and cell type-specificity, and environment. These results illuminate an intricate phenotype whose characterization aids understanding the basis of variable trait expressivity in females.

category: genetics


title: Genomic insights into the population history and biological adaptation of Southwestern Chinese Hmong-Mien people

abstract: Hmong-Mien-speaking (HM) populations, widely distributed in South China, North of Thailand, Laos and Vietnam, have experienced different settlement environments, dietary habits and pathogen exposure. However, their specific biological adaptation also remained largely uncharacterized, which is important in the population evolutionary genetics and Trans-Omics for regional Precision Medicine. Besides, the origin and genetic diversity of HM people and their phylogenetic relationship with surrounding modern and ancient populations are unknown. Here, we reported genome-wide SNPs in 52 representative Miao people and combined them with 144 HM people from 13 geographically representative populations to characterize the full genetic admixture and adaptive landscape of HM speakers. We found that obvious genetic substructures existed in geographically different HM populations and also identified one new ancestral lineage specifically exited in HM people, which spatially distributed from Sichuan and Guizhou in the North to Thailand in the South and temporally dated to at least 500 years. The sharing patterns of the newly-identified homogeneous ancestry component combined the estimated admixture times via the decay of Linkage Disequilibrium and haplotype sharing in GLOBETROTTER suggested that the modern HM-speaking populations originated from Southwest China and migrated southward recently, which is consistent with the reconstructed phenomena of linguistic and archeological documents. Additionally, we identified specific adaptive signatures associated with several important human nervous system biological functions. Our pilot work emphasized the importance of anthropologically-informed sampling and deeply genetic structure reconstruction via whole-genome sequencing in the next step in the deep Chinese population genomic diversity project (CPGDP), especially in the ethnolinguistic regions.


title: Kernel-based genetic association analysis for microbiome phenotypes identifies host genetic drivers of beta-diversity

abstract: Understanding human genetic influences on the gut microbiota helps elucidate the mechanisms by which genetics affects health outcomes. We propose a novel approach, the covariate-adjusted kernel RV (KRV) framework, to map genetic variants associated with microbiome beta-diversity, which focuses on overall shifts in the microbiota. The proposed KRV framework improves statistical power by capturing intrinsic structure within the genetic and microbiome data while reducing the multiple-testing burden. We apply the covariate-adjusted KRV test to the Hispanic Community Health Study/Study of Latinos in a genome-wide association analysis (first gene-level, then variant-level) for microbiome beta-diversity. We have identified an immunity-related gene, IL23R, reported in previous association studies and discovered 3 other novel genes, 2 of which are involved in immune functions or autoimmune disorders. Our findings highlight the value of the KRV as a powerful microbiome GWAS approach and support an important role of immunity-related genes in shaping the gut microbiome composition.

category: genomics


title: SPOT-1D-LM: Reaching Alignment-profile-based Accuracy in Predicting Protein Secondary and Tertiary Structural Properties without Alignment.

abstract: Protein language models have emerged as an alternative to multiple sequence alignment for enriching sequence information and improving downstream prediction tasks such as biophysical, structural, and functional properties. Here we show that a combination of traditional one-hot encoding with the embeddings from two different language models (ProtTrans and ESM-1b) allows a leap in accuracy over single-sequence based techniques in predicting protein 1D secondary and tertiary structural properties, including backbone torsion angles, solvent accessibility and contact numbers. This large improvement leads to an accuracy comparable to or better than the current state-of-the-art techniques for predicting these 1D structural properties based on sequence profiles generated from multiple sequence alignments. The high-accuracy prediction in both secondary and tertiary structural properties indicates that it is possible to make highly accurate prediction of protein structures without homologous sequences, the remaining obstacle in the post AlphaFold2 era.

category: bioinformatics


title: Identification of binding interaction between Curcumin derivative and PERK13 Proline rich receptor like protein kinase protein using In silico docking techniques - to help plants tackle salt water

abstract:
IntroductionArabidopsis thaliana, mouse ear cress or thale cress are small flowering plants included in the cruciferae family. They comprise of various characteristics such as diploid genetics, small genome size, rapid growth cycle, and relatively low repetitive DNA content, making it a perfect model for plant genome projects.

ObjectiveThe aim of this present Insilico research study is to carry out molecular drug docking studies between PERK13-Proline rich receptor like protein kinase and De O Acetylated Curcumin Di Galactose or di-galactosylated curcumin, a derivative of curcumin. PERK13 protein is considered to play a significant role in helping plants tackle salinity levels in water.

MethodsIn this study, protein modelling tools and servers are used to model the 3D structure and the same is validated using Protein structure validation tools. Automated drug docking servers were used to dock the modelled protein with the chemical compound to analyse the electrostatic (H bond) interaction between PERK13 and De O Acetylated Curcumin Di Galactose, a better water-soluble compound than curcumin. The docked structure was visualized using an advanced molecular visualization tool.

Results and DiscussionThe overall results obtained from this study on De O Acetylated Curcumin Di Galactose and PERK13 protein shows that De O Acetylated Curcumin Di Galactose, directly binds with the active site and other potentially binding regions of PERK13. Hence, it is concluded that De O Acetylated Curcumin Di Galactose could potentially play a vital role in future research related to the problem of helping the plants tackle increased saline levels in water.


title: iHypoxia: an integrative database of the expression dynamics of proteins in response to hypoxia in animals

abstract: Decreased oxygen concentrations (hypoxia) outside of the physiological range may severely subvert cell, tissue, and organism survival. Mammals have evolved mechanisms to sense hypoxia and induce a series of hypoxic responses. In recent years, high-throughput techniques have greatly promoted global perturbation studies of protein expression during hypoxia, and these studies have contributed to the understanding of the complex regulatory networks of hypoxia. In this study, we developed an integrated resource for the expression dynamics of proteins in response to hypoxia (iHypoxia), which contains 1,629 expression events of 1,215 proteins identified by low-throughput experiments (LTEs) and 154,953 quantitative expression events of 36,194 proteins identified by high-throughput experiments (HTEs) from five mammals that exhibit a response to hypoxia. Various experimental details such as the hypoxic experimental conditions, expression patterns, and samples were carefully collected and integrated. In addition, we conducted an orthologous search and identified 581,763 proteins that may respond to hypoxia among 50 animals. An enrichment analysis of human proteins identified from LTEs showed that these proteins were enriched in certain drug targets and cancer genes. The annotation of known posttranslational modification (PTM) sites to proteins identified by LTEs revealed that these proteins underwent extensive PTMs, particularly phosphorylation, ubiquitination and acetylation. Based on the results, iHypoxia provides a convenient and user-friendly method for users to obtain hypoxia-related information of interest. We anticipate that iHypoxia, which is freely accessible at http://ihypoxia.omicsbio.info, will advance the understanding of hypoxia and serve as a valuable data resource.


title: Pharmacological mechanisms on the anti-breast cancer property of Coix lacryma-jobi: A network-based analysis

abstract: Breast cancer is considered as one of the three most common cancers around the world and the second leading cause of cancer deaths among women. Coix lachrymal jobi, commonly known as Jobs tears or adlay has been reported to possess anti-cancer properties. Despite evidences provided by clinical data, the usage of Coix lacryma-jobi in treating cancer, particularly breast cancer, has been scarce. Thus, this study was conducted to determine the pharmacological mechanisms underlying its anti-breast cancer property using various network pathway analyses. Bioactive compounds from Coix lacryma-jobi and its potential target genes were obtained from SymMap. Breast cancer-related target genes were collected from CTD. Protein-protein interaction network was analyzed using the STRING database. GO and KEGG pathway enrichment analyses were performed using DAVID to further explore the mechanisms of Coix lacryma-jobi in treating breast cancer. PPI and compound-target-pathway were visualized using Cytoscape. A total of 26 bioactive compounds, 201 corresponding targets, 36625 breast cancer-associated targets were obtained, and 200 common targets were considered potential therapeutic targets. The 9 bioactive compounds identified were berberine, oleic acid, beta-sitosterol, sitosterol, linoleic acid, berberrubine, jatrorrhizine, thalifendine, and stigmasterol. The identified 5 core targets were ESR1, JUN, MAPK14, and RXRA. Coix lacryma-jobi targets enriched in GO terms were mostly involved in regulation of transcription from RNA polymerase II promoter, drug response, steroid hormone receptor activity, and protein binding. This study elucidates on the pharmacological underpinnings on the potency of adlay against breast cancer. Its subsequent drug development will be worth a step forward for a breast cancer-free society.

category: pharmacology and toxicology


title: Blood flow synchronization in renal microcirculation - a high-resolution imaging study.

abstract:
AimsInternephron signalling and interaction are fundamental for kidney function. Earlier studies have shown that nephrons signal to each other over short distances and adjust their activity accordingly. Micropuncture experiments revealed synchronous clusters of 2-3 nephrons formed from such interactions, while imaging and modelling results suggested the possibility of larger clusters. Such clusters are expected to play an important role in renal autoregulation, but their presence has not been confirmed and their size has not been estimated. In this study, we present methodology for high resolution renal blood flow imaging and apply it to estimate frequency and phase angle differences in kidney blood vessels under normal conditions and after administration of the vasoactive agents angiotensin II and acetylcholine.

Methods and resultsTo resolve signals from separate arterioles in a sufficiently large field of view, we developed a method for renal laser speckle contrast imaging. Our setup provides imaging of blood flow in the kidney cortex with a limit of image resolution at 0.8m per pixel and imaging frequency of 160Hz. We used the method to record from 1.5x1.5 mm2 sections of the renal surface in anaesthetised Sprague-Dawley rats in unstimulated conditions and during IV infusion of the vasoconstrictor angiotensin II or the vasodilator acetylcholine. In each section, we resolved and segmented 94.8{+/-}15.66 individual arterioles and venules, and analyzed blood flow using wavelet spectral analysis to identify clusters of synchronized blood vessels.

ConclusionsWe observed spatial and temporal evolution of blood vessel clusters of various sizes, including the formation of large (>90 vessels) long-lived clusters (>10 periods) locked at the frequency of the tubular glomerular feedback (TGF) mechanism. The analysis showed that synchronization patterns and thus the co-operative dynamics of nephrons change significantly when either of the vasoactive agents is administered. On average, synchronization was stronger (larger clusters, longer duration) with angiotensin II administration than in the unstimulated state or with acetyl choline. While it weakens with distance, increased synchronization duration spanned the whole field of view, and likely, beyond it. Neighbouring vessels tend to demonstrate in-phase synchronization, especially in the vasoconstricted condition, which is expected to cause locally increased pressure variation. Our results confirm both the presence of the local synchronization in the renal microcirculatory blood flow and the fact that it changes depending on the condition of the vascular network and the blood pressure, which might have further implications for the role of such synchronization in pathologies development.

category: physiology


title: Single-cell transcriptomics unveils xylem cell development and evolution

abstract: As the most abundant tissue on Earth1, xylem is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells2. In most angiosperms, fusiform cells are a combination of vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed together by tracheids in other vascular plants2. However, little is known about the developmental programs and evolutionary relationships of these xylem cell types. Through both single-cell and laser-capture microdissection transcriptomic profiling, here we demonstrate the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Cross-species analyses of single-cell trajectories reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed character3, 4, exhibit strong transcriptomic similarity to vessel elements but not libriform fibers, suggesting that water transportation, instead of mechanical support, is the major feature. We also found that the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. This evo-developmental framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history5.

category: plant biology


title: Long-term imaging of the ventral nerve cord in behaving adult Drosophila

abstract: The dynamics and connectivity of neural circuits continuously change during an animals lifetime on timescales ranging from milliseconds to days. Therefore, to investigate how biological networks accomplish remarkable cognitive and behavioral tasks, minimally invasive methods are needed to perform repeated measurements, or perturbations of neural circuits in behaving animals across time. Such tools have been developed to investigate the brain but similar approaches are lacking for comprehensively and repeatedly recording motor circuits in behaving animals. Here we describe a suite of microfabricated technologies that enable long-term, minimally invasive optical recordings of the adult Drosophila melanogaster ventral nerve cord (VNC)--neural tissues that are functionally equivalent to the vertebrate spinal cord. These tools consist of (i) a manipulator arm that permits the insertion of (ii) a compliant implant into the thorax to expose the imaging region of interest; (iii) a numbered, transparent polymer window that encloses and provides optical access to the inside of the thorax, and (iv) a hinged remounting stage that allows gentle and repeated tethering of an implanted animal for two-photon imaging. We validate and illustrate the utility of our toolkit in several ways. First, we show that the thoracic implant and window have minimal impact on animal behavior and survival while also enabling neural recordings from individual animals across at least one month. Second, we follow the degradation of chordotonal organ mechanosensory nerve terminals in the VNC over weeks after leg amputation. Third, because our tools allow recordings of the VNC with the gut intact, we discover waves of neural population activity following ingestion of a high-concentration caffeine solution. In summary, our microfabricated toolkit makes it possible to longitudinally monitor anatomical and functional changes in premotor and motor neural circuits, and more generally opens up the long-term investigation of thoracic tissues.

category: neuroscience


title: Dynamic Functional Brain Reconfiguration During Sustained Pain

abstract: AO_SCPLOWBSTRACTC_SCPLOWPain is constructed through complex interactions among multiple brain systems, but it remains unclear how functional brain network representations are dynamically reconfigured over time while experiencing pain. Here, we investigated the dynamic changes in the functional brain networks during 20-min capsaicin-induced sustained orofacial pain. In the early stage, the orofacial areas of the primary somatomotor cortex were separated from the other primary somatomotor cortices and integrated with subcortical and frontoparietal regions, constituting a brain-wide pain supersystem. As pain decreased over time, the subcortical and frontoparietal regions were separated from this pain supersystem and connected to multiple cerebellar regions. Machine-learning models based on these dynamic network features showed significant predictions of changes in pain experience across two independent datasets (n = 48 and 74). This study provides new insights into how multiple brain systems dynamically interact to construct and modulate pain experience, potentially advancing our mechanistic understanding of chronic pain.


title: Ionizing radiation improves RIG-I mediated immunotherapy through enhanced p53 activation in malignant melanoma

abstract: Radiation therapy induces cytotoxic DNA damage, which results in cell-cycle arrest and activation of cell-intrinsic death pathways, but its application has been limited by the radioresistance of tumors, such as in malignant melanoma. RIG-I is a cytosolic immune receptor expressed in all somatic cells, including tumor cells, with a key role in sensing viral RNA. RIG-I specific oligonucleotide ligands elicit a robust cell-intrinsic antiviral response and immunogenic cell death in tumor cells and are being tested in clinical trials. Nonetheless, their potential to overcome radioresistance has not yet been explored. Here, we demonstrate that activation of RIG-I enhances the extent and immunogenicity of irradiation-induced tumor cell death in human and murine melanoma cell lines in vitro and improved survival in the murine B16 melanoma model. Pathway analysis of transcriptomic data revealed a central role for p53 downstream of the combination treatment, which was corroborated using p53-/- B16 cells. In vivo, the effect of irradiation on immune-cell activation and inhibition of tumor growth was absent in mice carrying p53-/- B16 tumors, while the response to RIG-I stimulation in those mice was maintained. Our results identify p53 as pivotal for the synergistic antitumoral effect of RIG-I and irradiation, resulting in potent induction of immunogenic tumor-cell death. Thus, the administration of RIG-I ligands in combination with radiotherapy is a promising therapeutic approach to treating radioresistant tumors with a functional p53 pathway, such as malignant melanoma.

category: immunology


title: RIG-I immunotherapy overcomes radioresistance in p53-positive malignant melanoma


title: Antibiotic resistance via bacterial cell shape-shifting

abstract: Bacteria have evolved to develop multiple strategies for antibiotic resistance by effectively reducing intra-cellular antibiotic concentrations or antibiotic binding affinities, but the role of cell morphology on antibiotic resistance remains poorly characterized. By analyzing cell morphological data of different bacterial species under antibiotic stress, we find that bacterial cells robustly reduce surface-to-volume ratio in response to most types of antibiotics. Using quantitative modelling we show that by reducing surface-to-volume ratio, bacteria can effectively reduce intracellular antibiotic concentration by decreasing antibiotic influx. The model predicts that bacteria can increase surface-to-volume ratio to promote antibiotic dilution if efflux pump activity is reduced, in agreement with data on membrane-transport inhibitors. Using the particular example of ribosome-targeting antibiotics, we present a systems-level model for the regulation of cell shape under antibiotic stress, and discuss feedback mechanisms that bacteria can harness to increase their fitness in the presence of antibiotics.


title: Effects of dual deletion of glnR and mtrA on expression of nitrogen metabolism genes in Streptomyces venezuelae

abstract: GlnR activates nitrogen metabolism genes under nitrogen-limited conditions whereas MtrA represses these genes under nutrient-rich conditions in Streptomyces. In this study, we compared the transcription patterns of nitrogen metabolism genes in a double deletion mutant ({Delta}mtrA-glnR) lacking both mtrA and glnR and in mutants lacking either mtrA ({Delta}mtrA) or glnR ({Delta}glnR). The nitrogen metabolism genes were expressed similarly in {Delta}mtrA-glnR and {Delta}glnR under both nitrogen-limited and nutrient-rich conditions, with patterns distinctly different from that of {Delta}mtrA, suggesting a decisive role for GlnR in the control of nitrogen metabolism genes and further suggesting that regulation of these genes by MtrA is GlnR-dependent. MtrA and GlnR utilize the same binding sites upstream of nitrogen metabolism genes, and we showed stronger in vivo binding of MtrA to these sites under nutrient-rich conditions and of GlnR under nitrogen-limited conditions, consistent with the higher levels of MtrA or GlnR under those respective conditions. In addition, we showed that both mtrA and glnR are auto-regulatory. Our study provides new insights into the regulation of nitrogen metabolism genes in Streptomyces.


title: Oxidative stress induces inflammation of lens cells and triggers immune surveillance of ocular tissues

abstract:
Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1- aged Gclcf/f;Le-CreTg/- (KO) and Gclcf/f;Le-Cre-/- control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclcw/w;Le-CreTg/- (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-L-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), -SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures.

HighlightsO_LIImmune surveillance of ocular structures occurs in mouse eyes deficient in glutathione.
C_LIO_LIOxidative stress upregulates the expression of pro-inflammatory cytokines (e.g., GDF15, CSF1) in lens cells in vitro and in vivo.
C_LIO_LIThe upregulation of cytokines in lens cells is associated with markers of an epithelial-to-mesenchymal transition phenotype.
C_LIO_LIOxidative stress-induced inflammation and associated epithelial-to-mesenchymal transition may play a role in the development of posterior capsule opacification.
C_LI

category: developmental biology


title: PySOFI: an open source Python package for SOFI

abstract: Super-resolution optical fluctuation imaging (SOFI) is a highly democratizable technique that provides optical super-resolution (SR) without requirement of sophisticated imaging instruments. An open source package for SOFI algorithm is needed to support not only the utilization of SOFI, but also the community adoption and participation for further development of SOFI. In this work, we developed PySOFI, an open source python package for SOFI analysis that offers the flexibility to inspect, test, modify, improve and extend the algorithm. We provide a complete documentation for the package and a collection of Jupyter Notebooks to demonstrate the usage of the package. We discuss the architecture of PySOFI, illustrate how to use each functional module, and demonstrate how to extend the PySOFI package with additional modules. We expect PySOFI to facilitate efficient adoption, testing, modification, dissemination and prototyping of new SOFI-relevant algorithms.

category: biophysics


title: Cellular perception of growth rate and the mechanistic origin of bacterial growth laws

abstract: Bacteria organize many activities according to their grow rate via the ppGpp signaling system. Yet it is not clear how this signaling system "knows" how fast cells grow. Through quantitative experiments, we show that ppGpp responds inversely to the rate of translational elongation in E. coli. Together with its roles in inhibiting ribosome biogenesis and activity, ppGpp closes a key regulatory circuit that enables the cell to perceive and control the rate of its growth across conditions. The celebrated linear growth law relating the ribosome content and growth rate emerges as a consequence of keeping a supply of ribosome reserves while maintaining elongation rate in slow growth conditions. Further analysis suggests the elongation rate itself is detected by sensing the ratio of dwelling and translocating ribosomes, a strategy employed to collapse the complex, high-dimensional dynamics of the molecular processes underlying cell growth to perceive the physiological state of the whole.

category: systems biology


title: Formation of multinucleated globoid cells in Krabbe iPSC-derived microglia cultures: a preliminary investigation

abstract: Globoid cell leukodystrophy (Krabbe disease) is a severe demyelinating, neurodegenerative lysosomal storage disorder caused by deficiency in glycosphingolipid catabolic enzyme galactosylceramidase (GALC). Histologically, Krabbe disease is characterized by the appearance of large multinucleated globoid cells that express classical macrophage markers (both of brain-resident microglia and peripheral monocyte-derived). Globoid cells reside near areas of degeneration; however, their functional significance in disease progression remains unclear. In the current study, we differentiated microglia-like cells from iPSCs from a donor with infantile Krabbe disease and compared them to microglia generated from two healthy controls and two donors with the lysosomal storage disorder metachromatic leukodystrophy (MLD), which is genetically distinct from Krabbe disease but presents similarly in terms of severity of demyelination and neurodegeneration. We report the novel finding of prominent formation of giant multinucleated globoid cells from the microglia derived from the Krabbe donor, but not from healthy control or MLD donors. The Krabbe microglia displayed reduced IL-6 protein expression upon stimulation with lipopolysaccharide, and the multinucleated globoid cells themselves appeared deficient in phagocytosis of both disease-relevant myelin debris and E. coli, together hinting at an impairment of normal function. The formation of the globoid cells could be attenuated by fully replacing the medium following passaging, suggesting that yet-to-be determined secreted factors are influencing cell fusion in our culture system. While preliminary, our results imply that globoid cells may be detrimental in Krabbe disease by hindering the normal function of brain-residing macrophages.


title: Adaptive coding of pain prediction error in the anterior insula

abstract:
BackgroundTo reduce the computational demands of the task of determining values, the brain is thought to engage in  adaptive coding, where the sensitivity of some neurons to value is modulated by contextual information. There is good behavioural evidence that pain is coded adaptively, but controversy regarding the underlying neural mechanism. Additionally, there is evidence that reward prediction errors are coded adaptively, but no parallel evidence regarding pain prediction errors.

MethodsWe tested the hypothesis that pain prediction errors are coded adaptively by scanning 19 healthy adults with fMRI while they performed a cued pain task. Our analysis followed an axiomatic approach.

ResultsWe found that the left anterior insula was the only region which was sensitive both to predicted pain magnitudes and the unexpectedness of pain delivery, but not to the magnitude of delivered pain.

ConclusionsThis pattern suggests that the left anterior insula is part of a neural mechanism that serves the adaptive prediction error of pain.

Significance statementThere is behavioural evidence that pain is coded adaptively but controversy as to the neural mechanism that serves this process, and limited understanding of how adaptive coding of pain relates to adaptively-coded pain prediction error. We provide evidence that the left anterior insula complies with axioms that describe adaptive coding of pain prediction error, adding to the understanding of the role it plays in subjective pain representation.


title: Spatial proteogenomics reveals distinct and evolutionarily-conserved hepatic macrophage niches

abstract: The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here, we present a spatial proteogenomic atlas of the healthy human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and bile-duct macrophages. We also uncover the respective spatially-resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that bile-duct macrophages are induced by local lipid exposure, while Kupffer cells crucially depend on their crosstalk with hepatic stellate cells via the evolutionarily-conserved ALK1-BMP9/10 axis.


title: Automatic identification and annotation of MYB gene family members in plants

abstract:
BackgroundMYBs are among the largest transcription factor families in plants. Consequently, members of this family are involved in a plethora of processes including development and specialized metabolism. The MYB families of many plant species were investigated in the last two decades since the first investigation looked at Arabidopsis thaliana. This body of knowledge and characterized sequences provide the basis for the identification, classification, and functional annotation of candidate sequences in new genome and transcriptome assemblies.

ResultsA pipeline for the automatic identification and functional annotation of MYBs in a given sequence data set was implemented in Python. MYB candidates are identified, screened for the presence of a MYB domain and other motifs, and finally placed in a phylogenetic context with well characterized sequences. In addition to technical benchmarking based on existing annotation, the transcriptome assembly of Croton tiglium and the annotated genome sequence of Castanea crenata were screened for MYBs. Results of both analyses are presented in this study to illustrate the potential of this application. The analysis of one species takes only a few minutes depending on the number of predicted sequences and the size of the MYB gene family. This pipeline, the required bait sequences, and reference sequences for a classification are freely available on github: https://github.com/bpucker/MYB_annotator.

ConclusionsThis automatic annotation of the MYB gene family in novel assemblies makes genome-wide investigations consistent and paves the way for comparative studies in the future. Candidate genes for in-depth analyses are presented based on their orthology to previously characterized sequences which allows the functional annotation of the newly identified MYBs with high confidence. The identification of orthologs can also be harnessed to detect duplication and deletion events.


title: Amoeboid-like neuronal migration ensures correct horizontal cell layer formation in the developing vertebrate retina

abstract: As neurons are often born at positions different than where they ultimately function, neuronal migration is key to ensure successful nervous system development. Radial migration during which neurons featuring unipolar and bipolar morphology, employ pre-existing processes or underlying cells for directional guidance, is the most well explored neuronal migration mode. However, how neurons that display multipolar morphology, without such processes, move through highly crowded tissue environments towards their final positions remains elusive. To understand this, we here investigated multipolar migration of horizontal cells in the zebrafish retina. We found that horizontal cells tailor their movements to the environmental spatial constraints of the crowded retina, by featuring several characteristics of amoeboid migration. These include cell and nucleus shape changes, and persistent rearward polarization of stable F-actin, which enable horizontal cells to successfully move through the crowded retina. Interference with the organization of the developing retina by changing nuclear properties or overall tissue architecture, hampers efficient horizontal cell migration and layer formation. Thus, cell-tissue interplay is crucial for efficient migration of horizontal cells in the retina. In view of high proportion of multipolar neurons, the here uncovered ameboid-like neuronal migration mode might also be crucial in other areas of the developing brain.


title: Long-term evolution of antibiotic persistence in P. aeruginosa lung infections

abstract: Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as persistence. While a variety of resistance mechanisms and underlying genetics are well characterised in vitro and in vivo, the evolution of persistence, and how it interacts with resistance in situ is less well understood. We assayed for persistence and resistance with three clinically relevant antibiotics: meropenem, ciprofloxacin and tobramycin, in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to forty years of evolution. We find evidence that persistence is under positive selection in the lung and that it can particularly act as an evolutionary stepping stone to resistance. However, this pattern is not universal and depends on the bacterial clone type and antibiotic used, indicating an important role for antibiotic mode of action.

category: evolutionary biology


title: In situ architecture of the ciliary base reveals the stepwise assembly of IFT trains

abstract:
The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. Here, we use in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base. We combine this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which IFT-A, then dynein-1b, and finally kinesin-2 sequentially bind before entry into the cilium.

One Sentence SummaryNative molecular structure of the ciliary transition zone and hierarchical order of IFT assembly visualized within Chlamydomonas cells.


title: Identification of multi-omics molecular signature of acute radiation injury using nonhuman primate model

abstract: The availability of validated biomarkers to assess radiation exposure and to assist in developing medical countermeasures remains an unmet need. We used a cobalt-60 gamma-irradiated nonhuman primate (NHP) model to delineate a multi-omics-based serum probability index of radiation exposure. Both male and female NHPs were irradiated with different doses ranging from 6.0 to 8.5 Gy, with 0.5 Gy increments between doses. We leveraged high resolution mass spectrometry for analysis of metabolites, lipids, and proteins at 1,2, and 6 days post-irradiation in NHP serum. A logistic regression model was implemented to develop a 4-analyte panel to stratify irradiated NHPs from unirradiated with high accuracy that was agnostic for all doses of {gamma}-rays tested in the study, up to six days after exposure. This panel was comprised of Serpin Family A9, acetylcarnitine, PC (16:0/22:6), and suberylglycine, which showed 2 - 4-fold elevation in serum abundance upon irradiation in NHPs, and can potentially be translated for human use following larger validation studies. Taken together, this study, for the first time, demonstrates the utility of a combinatorial molecular characterization approach using an NHP model for developing minimally invasive assays from small volumes of blood that can be effectively used for radiation exposure assessments.

category: biochemistry


title: Ab initio phasing macromolecular structures using electron-counted MicroED data

abstract: Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that was collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion-beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous rotation data were collected using an ultra-low exposure rate on a Falcon 4 direct electron detector in electron-counting mode. For the first sample, triclinic lysozyme extending to 0.87 [A] resolution, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a modest 1.5 [A] resolution. These results demonstrate that macromolecules can be determined to sub-[A]ngstrom resolution by MicroED and that ab initio phasing can be successfully applied to counting data collected on a direct electron detector.


title: Supercharged Cellulases Show Reduced Non-Productive Binding, But Enhanced Activity, on Pretreated Lignocellulosic Biomass

abstract: Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydrate-binding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose deconstruction by increasing targeted substrate-bound CD concentration but often at the cost of increased non-productive enzyme binding. Here, we demonstrate how a computational protein design strategy can be applied to a model endocellulase enzyme (Cel5A) from Thermobifida fusca to allow fine-tuning its CBM surface charge, which led to increased hydrolytic activity towards pretreated lignocellulosic biomass (e.g., corn stover) by up to [~]330% versus the wild-type Cel5A control. We established that the mechanistic basis for this improvement arises from reduced non-productive binding of supercharged Cel5A mutants to cell wall components such as crystalline cellulose (up to 1.7-fold) and lignin (up to 1.8-fold). Interestingly, supercharged Cel5A mutants that showed improved activity on various forms of pretreated corn stover showed increased reversible binding to lignin (up to 2.2-fold) while showing no change in overall thermal stability remarkably. In general, negative supercharging led to increased hydrolytic activity towards both pretreated lignocellulosic biomass and crystalline cellulose whereas positive supercharging led to a reduction of hydrolytic activity. Overall, selective supercharging of protein surfaces was shown to be an effective strategy for improving hydrolytic performance of cellulolytic enzymes for saccharification of real-world pretreated lignocellulosic biomass substrates. Future work should address the implications of supercharging cellulases from various families on inter-enzyme interactions and synergism.

category: bioengineering


title: RNA Helix Thermodynamics: The End Game

abstract: Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated [Formula] separately from other [Formula] stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37{degrees} C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.


title: 14-3-3ζ constrains insulin secretion in pancreatic β-cells by regulating mitochondrial function

abstract: While critical for neurotransmitter synthesis in the brain, members of the 14-3-3 protein family are often assumed to have redundant, over-lapping roles due to their high sequence homology and ubiquitous expression. Despite this assumption, various mammalian 14-3-3 isoforms have now been implicated in regulating cellular and organismal metabolism; however, these functions were primarily observed in cell lines or from systemic knockout mouse models. To date, we have begun to define the contributions of 14-3-3{zeta} in adipocytes, but whether 14-3-3{zeta} has additional metabolic roles in other cell types, such as the pancreatic {beta}-cell, is unclear. We previously documented a pro-survival role of 14-3-3{zeta} in MIN6 insulinoma cells, as depletion of 14-3-3{zeta} induced cell death, but paradoxically, whole-body deletion of 14-3-3{zeta} in mice resulted in significantly enlarged {beta}-cell area with no effects on insulin secretion. To better understand the role of 14-3-3{zeta} in {beta}-cells, we generated {beta}-cell-specific 14-3-3{zeta} knockout ({beta}14-3-3{zeta}KO) mice, and while no differences in {beta}-cell mass were observed, {beta}14-3-3{zeta}KO mice displayed potentiated insulin secretion due to enhanced mitochondrial function and ATP synthesis. Deletion of 14-3-3{zeta} led to profound changes to the {beta}-cell transcriptome, where pathways associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute treatment of mouse islets and human islets with pan-14-3-3 inhibitors recapitulated the potentiation in glucose-stimulated insulin secretion (GSIS) and mitochondrial function, suggesting that 14-3-3{zeta} is a critical isoform in {beta}-cells that regulates GSIS. In dysfunctional db/db islets and islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3{zeta}, was inversely associated with insulin secretory capacity, and pan-14-3-3 protein inhibition was capable of enhancing GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3{zeta} and its related isoforms in insulin secretion and mitochondrial function in {beta}-cells. A deeper understanding of how 14-3-3{zeta} influences {beta}-cell function will further advance our knowledge of how insulin secretion from {beta}-cells is regulated.


title: 14-3-3ζ constrains insulin secretion by regulating mitochondrial function in pancreatic β-cells


title: Synaptotagmin-7 enhances phasic dopamine release

abstract: Dopamine released from substantia nigra pars compacta (SNc) neurons modulates movement, motivation, and reward. In addition to their tonic firing pattern, dopamine neurons also fire high-frequency bursts that cause superlinear increases in dopamine release. To study this poorly understood form of short-term plasticity, we used the fluorescent dopamine sensor dLight1.3b to examine the role of the calcium-binding protein synaptotagmin-7 (SYT7). We report that SYT7 mediates a hidden component of facilitation, which was unmasked by lowering initial release probability, or by low-frequency stimulation of nerve terminals. In Syt7 KO neurons, there was profound synaptic depression that significantly reduced release during stimulations that mimic in vivo firing patterns of SNc neurons. D2-mediated inhibitory postsynaptic currents in the SNc revealed a similar role for SYT7 in somatodendritic release. Our results indicate that SYT7 drives short-term facilitation of release from dopamine neurons, which likely underlies frequency-dependence of dopamine signaling in vivo.


title: TRPV1-lineage somatosensory fibers communicate with taste neurons in the mouse parabrachial nucleus

abstract:
Trigeminal neurons supply somatosensation to craniofacial tissues. In mouse brain, ascending projections from medullary trigeminal neurons arrive at taste neurons in the autonomic parabrachial nucleus, suggesting taste neurons participate in somatosensory processing. However, the genetic cell types that support this convergence were undefined. Using Cre-directed optogenetics and in vivo neurophysiology in anesthetized mice of both sexes, here we studied whether TRPV1-lineage nociceptive and thermosensory fibers are primary neurons that drive trigeminal circuits reaching parabrachial taste cells. We monitored spiking activity in individual parabrachial neurons during photoexcitation of the terminals of TRPV1-lineage fibers that arrived at the dorsal spinal trigeminal nucleus pars caudalis, which relays orofacial somatosensory messages to the parabrachial area. Parabrachial neural responses to oral delivery of taste, chemesthetic, and thermal stimuli were also recorded. We found that optical excitation of TRPV1-lineage fibers frequently stimulated traditionally defined taste neurons in lateral parabrachial nuclei. The tuning of neurons across diverse tastes associated with their sensitivity to excitation of TRPV1-lineage fibers, which only sparingly engaged neurons oriented to preferred tastes like sucrose. Moreover, neurons that responded to photostimulation of TRPV1-lineage afferents showed strong responses to temperature including noxious heat, which predominantly excited parabrachial bitter taste cells. Multivariate analyses revealed the parabrachial confluence of TRPV1-lineage signals with taste captured sensory valence information shared across aversive gustatory, nociceptive, and thermal stimuli. Our results reveal that trigeminal fibers with defined roles in thermosensation and pain communicate with parabrachial taste neurons.

This multisensory convergence supports dependencies between gustatory and somatosensory hedonic representations in the brain.


title: Type IV pilus shapes a "bubble-jet" pattern opposing spatial intermixing of two interacting bacterial populations

abstract:
Microbes are social organisms that commonly live in sessile biofilms. Spatial patterns of populations within biofilms can be an important determinant of community-level properties. The best-studied characteristics of spatial patterns is spatial intermixing of different populations. The specific levels of spatial intermixing critically contribute to how the dynamics and functioning of such communities are governed. However, the precise factors that determine spatial patterns and intermixing remain unclear. Here, we investigated the spatial patterning and intermixing of an engineered synthetic consortium composed of two Pseudomonas stutzeri strains that degrade salicylate via metabolic cross-feeding. We found that the consortium self-organizes across space to form a previously unreported spatial pattern (referred to here as a  bubble-jet pattern) that exhibits a low level of intermixing. Interestingly, when the genes encoding for type IV pili were deleted from both strains, a highly intermixed spatial pattern developed and increased the productivity of the entire community. The intermixed pattern was maintained in a robust manner across a wide range of initial ratios between the two strains. Our findings show that the type IV pilus plays a role in mitigating spatial intermixing of different populations in surface-attached microbial communities, with consequences for governing community-level properties. These insights provide tangible clues for the engineering of synthetic microbial systems that perform highly in spatially structured environments.

ImportanceWhen growing on surfaces, multi-species microbial communities form biofilms that exhibit intriguing spatial patterns. These patterns can significantly affect the overall properties of the community, such as enabling otherwise impermissible metabolic functions to occur, as well as driving the evolutionary and ecological processes acting on communities. The development of these patterns is affected by several drivers, including cell-cell interactions, nutrient levels, density of founding cells and surface properties. The type IV pilus is commonly found to mediate surface-associated behaviors of microorganism, but its role on pattern formation within microbial communities is unclear. Here we report that in a cross-feeding consortium, the type IV pilus affects the spatial intermixing of interacting populations involved in pattern formation, and ultimately influences overall community productivity and robustness. This novel insight assists our understanding of the ecological processes of surface-attached microbial communities and suggests a potential strategy to engineer high-performance synthetic microbial communities.

category: microbiology


title: An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins

abstract: There is an unmet need for delivery platforms that realize the full potential of next-generation therapeutic and vaccine technologies, especially those that require intracellular delivery of nucleic acids. The in vivo usefulness of the current state-of-the-art delivery systems is limited by numerous intrinsic weaknesses, including lack of targeting specificity, inefficient entry and endosomal escape into target cells, undesirable immune activation, off-target effects, a small therapeutic window, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we present our characterization of a delivery platform based on the use of engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli strain SVC1) for intracellular cargo delivery. The SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to escape the endosome upon intracellularization, and to have minimal immunogenicity. Here we report findings on key features of this system. First, we demonstrated that bacterial delivery of a short hairpin RNA (shRNA) can target and silence a gene in an in vitro mammalian respiratory cell model. Next, we used an in vivo mouse model to demonstrate that SVC1 bacteria are invasive to epithelial cells of various tissues and organs (eye, nose, mouth, stomach, vagina, skeletal muscle, and lungs) via local administration. We also showed that repeat dosing of SVC1 bacteria to the lungs is minimally immunogenic and that it does not have adverse effects on tissue homeostasis. Finally, to validate the potential of SVC1 bacteria in therapeutic applications, we demonstrated that bacterial delivery of influenza-targeting shRNAs to the respiratory tissues can mitigate viral replication in a mouse model of influenza infection. Our ongoing work is focused on further refining this platform for efficient delivery of nucleic acids, gene editing machinery, and therapeutic proteins, and we expect that this platform technology will enable a wide range of advanced therapeutic approaches.


title: Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii

abstract:
Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the importance of the QS regulation in the bacteria-yeast interactions.

HighlightsThe avirulent A. tumefaciens 6N2 has two replicons and a complex QS architecture

The profile of QS-regulated proteins is modified in dual cultures with Pa. laurentii

The bacterial QS activity alters the proteome of the yeast Pa. laurentii


title: The ESX-1 secretion system senses bacterial contact and prepares mycobacteria for environmental adaptation

abstract: The ESX-1 system (6-kDa early secretory antigenic target (ESAT-6) secretion system-1) is essential for Mycobacterium tuberculosis pathogenesis and conjugal transfer in Mycobacterium smegmatis, yet little is known about how its function is regulated. Live-cell fluorescence microscopy showed natively expressed ESX-1 was organized into distinct foci predominantly observed at cell-cell contacts. These foci formed when two cells touched and required a fully assembled ESX-1 system in both bacteria, suggesting the generation of an ESX-1 megacomplex across multiple membranes. The emergence of ESX-1 foci and ESX-1 secretion was environmentally dependent: foci formed in low nitrogen environments in which secretion was suppressed, yet with increasing concentrations of nitrogen, ESX-1 systems diffused along the plasma membrane and secretion was activated. Genome-wide transcriptional profiling revealed ESX-1 dependent induction of genes required for the SOS response and error prone DNA replication in high nitrogen. Based on these findings, we propose a new model of ESX-1 function where ESX-1 localization and secretion are responsive to nitrogen levels and form an integral node in the mycobacterial response to neighboring cells and environmental adaptation.


title: Tuberin levels during cellular differentiation in brain development

abstract:
Tuberin is a member of a large protein complex, Tuberous Sclerosis Complex, and acts as a sensor for nutrient status regulating protein synthesis and cell cycle progression. Mutations in the Tuberin gene, TSC2, lead to the formation of tumors and developmental defects in many organ systems, including the central nervous system. Tuberin is expressed in the brain throughout development and levels of Tuberin have been found to decrease during neuronal differentiation in cell lines in vitro. Our current work investigates the levels of Tuberin at two stages of embryonic development in vivo, and we study the mRNA and protein levels during a time course using immortalized cell lines in vitro. Our results show that Tuberin levels remain stable in the olfactory bulb but decrease in the Purkinje cell layer during embryonic mouse brain development. We show here that Tuberin levels are higher when cells are cultured as neurospheres, and knockdown of Tuberin results in a reduction in the number of neurospheres. These data provide support for the hypothesis that Tuberin is an important regulator of stemness and the reduction of Tuberin levels might support functional differentiation in the central nervous system. Understanding how Tuberin expression is regulated throughout neural development is essential to fully comprehend the role of this protein in several developmental and neural pathologies.

HIGHLIGHTSO_LITuberin protein levels are decreased in the Purkinje cell layer in later stages of embryonic development.
C_LIO_LITuberin protein and mRNA levels decrease as cells undergo neuronal differentiation.
C_LIO_LIDownregulation of Tuberin impairs neurosphere formation.
C_LIO_LITuberin is implicated in the maintenance of stemness in the developing brain.
C_LI


title: Combining metabolomics and experimental evolution reveals key mechanisms underlying longevity differences in laboratory evolved Drosophila melanogaster populations

abstract: Experimental evolution with Drosophila melanogaster has been used extensively for decades to study aging and longevity. In recent years, the addition of DNA and RNA sequencing to this framework has allowed researchers to leverage the statistical power inherent to experimental evolution study the genetic basis of longevity itself. Here we incorporated metabolomic data into to this framework to generate even deeper insights into the physiological and genetic mechanisms underlying longevity differences in three groups of experimentally evolved D. melanogaster populations with different aging and longevity patterns. Our metabolomic analysis found that aging alters mitochondrial metabolism through increased consumption of NAD+ and increased usage of the TCA cycle. Combining our genomic and metabolomic data produced a list of biologically relevant candidate genes. Among these candidates, we found significant enrichment for genes and pathways associated with neurological development and function, and carbohydrate metabolism. While we do not explicitly find enrichment for aging canonical genes, neurological dysregulation and carbohydrate metabolism are both known to be associated with accelerated aging and reduced longevity. Taken together, our results provide plausible genetic mechanisms for what might be driving longevity differences in this experimental system. More broadly, our findings demonstrate the value of combining multiple types of omic data with experimental evolution when attempting to dissect mechanisms underlying complex and highly polygenic traits like aging.


title: Hair phenotype diversity across Indriidae lemurs

abstract:
ObjectivesHair (i.e., pelage/fur) is a salient feature of primate (including human) diversity and evolution-- serving functions tied to thermoregulation, protection, camouflage, and signaling--but wild primate pelage evolution remains relatively understudied. Specifically, assessing multiple hypotheses across distinct phylogenetic scales is essential but is rarely conducted. We examine whole body hair color and density variation across Indriidae (Avahi, Indri, Propithecus)--a lineage that, like humans, exhibits vertical posture (i.e., their whole bodies are vertical to the sun).

Materials and MethodsOur analyses consider multiple phylogenetic scales (family-level, genus-level) and hypotheses (e.g., Glogers rule, the body cooling hypotheses). We obtain hair color and density from museum and/or wild animals, opsin genotypes from wild animals, and climate data from WorldClim. To analyze our data, we use phylogenetic generalized linear mixed models (PGLMM) using Markov chain Monte Carlo algorithms.

ResultsOur results show that across the Indriidae family, darker hair is typical in wetter regions. However, within Propithecus, dark black hair is common in colder forest regions. Results also show pelage redness increases in populations exhibiting enhanced color vision. Lastly, we find follicle density on the crown and limbs increases in dry and open environments.

DiscussionThis study highlights how different selective pressures across distinct phylogenetic scales have likely acted on primate hair evolution. Specifically, our data across Propithecus may implicate thermoregulation and is the first empirical evidence of Bogerts rule in mammals. Our study also provides rare empirical evidence supporting an early hypothesis on hominin hair evolution.


title: Wild-type Caenorhabditis elegans Isolates Exhibit Distinct Gene Expression Profiles in Response to Microbial Infection

abstract: The soil-dwelling nematode Caenorhabditis elegans serves as a model system to study innate immunity against microbial pathogens. C. elegans have been collected from around the world, where they, presumably, adapted to regional microbial ecologies. Here we use survival assays and RNA- sequencing to better understand how two isolates from disparate climates respond to pathogenic bacteria. We found that, relative to N2 (originally isolated in Bristol, UK), CB4856 (isolated in Hawaii), was more susceptible to the Gram-positive pathogen, Staphylococcus epidermidis, but equally susceptible to Staphylococcus aureus as well as two Gram-negative pathogens, Providencia rettgeri and Pseudomonas aeruginosa. We performed transcriptome analysis of infected worms and found gene- expression profiles were considerably different in an isolate-specific and pathogen-specific manner. We performed GO term analysis to categorize differential gene expression in response to S. epidermidis. In N2, genes that encoded detoxification enzymes and extracellular matrix proteins were significantly enriched, while in CB4856, genes that encoded detoxification enzymes, C-type lectins, and lipid metabolism proteins were enriched, suggesting they have different responses to these bacterial pathogens, despite being the same species. Overall, discerning gene expression signatures in an isolate by pathogen manner can help us to understand the different possibilities for the evolution of immune responses within organisms.


title: Loss of wings induces the expression of the worker-like phenotype in queens of a ponerine ant

abstract:
Many highly-eusocial insect species are characterized by morphological differences between females. This is especially pronounced in ants where queens usually possess a fully developed thorax with wings and are specialized for reproduction while workers have a reduced thorax without wings and show various levels of reproductive degeneration that is associated with their helper role in the colony. Despite their morphological differentiation, queens and workers still show some plasticity leading to overlapping behavioral and physiological phenotypes. We investigated the level of queen plasticity and the factor that induces a worker-like phenotype in the ant species Harpegnathos saltator that has limited queen-worker dimorphism and workers that can assume the reproductive role of a queen in the colony. By comparing alate and dealate young queens, so-called gynes, we found that the loss of wings initiated the expression of behavioral and physiological characteristics of ant workers. In contrast to alate gynes, dealate gynes displayed higher frequencies of worker-like behaviors. In addition, dealate gynes showed a worker-like range of reproductive states unlike alate gynes. Like workers, dealate gynes lost the chemical signaling that is characteristic of alate gynes. Since gynes can activate this worker-like phenotype after wing loss, the essential difference between the ant queens and workers in this species with limited queen-worker dimorphism is a dispersal polyphenism. If the phenotypic plasticity observed in H. saltator is representative of the early stages of ant eusociality, an emerging dispersal dimorphism rather than a distinct reproductive dimorphism might represent one of the first steps in ant evolution.

Summary StatementAnt queens who have lost their wings express worker-like behaviors and physiology including the display of dominance behavior during hierarchy establishment, which is normally a worker-only behavior in this species.

category: animal behavior and cognition


title: Structure of the human ATM kinase and mechanism of Nbs1 binding

abstract: DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and ultimately cancer. Central to the sensing of DSBs are ATM (Ataxia-telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, and the MRN (Mre11-Rad50-Nbs1) protein complex that activates ATM. How the MRN complex recruits and activates ATM kinase is poorly understood. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM kinase and is required to retain active ATM at sites of DNA damage. Here, we report the 2.5 [A] resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped autoinhibited structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning the N and C lobes along with relieving the blockage of the substrate-binding site. We show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these interactions with mutagenesis and biochemical assays.


title: Identifying common and novel cell types in single-cell RNA-sequencing data using FR-Match

abstract: Reference cell type atlases powered by single cell transcriptomic profiling technologies have become available to study cellular diversity at a granular level. We present FR-Match for matching query datasets to reference atlases with robust and accurate performance for identifying common and novel cell types and suboptimally clustered cell types in the query data. FR-Match shows excellent performance for cross-platform, cross-sample type, and cross-tissue region cell type matching.


title: Cell type matching in single-cell RNA-sequencing data using FR-Match


title: The ASCC2 CUE domain contacts adjacent ubiquitins to recognize K63-linked polyubiquitin

abstract: Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the ALKBH3-ASCC repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains using its CUE domain, a type of ubiquitin-binding domain that typically binds monoubiquitin and does not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. Whereas the ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, the contacts with the proximal ubiquitin are unique to ASCC2. The N-terminal portion of the ASCC2 1 helix, including residues E467 and S470, contributes to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response.


title: Repurposing Tranexamic Acid as an Anticancer Agent

abstract: Tranexamic Acid (TA) is a clinically used antifibrinolytic that acts as a lysine mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic lysine and arginine, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

category: cancer biology


title: A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis

abstract: How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.


title: Old and ancient trees are life history lottery winners and act as evolutionary buffers against long-term environmental change

abstract: Trees can live many centuries with sustained fecundity and death is largely stochastic. We use a neutral stochastic model to examine the demographic patterns that emerge over time, across a range of population sizes and empirically observed mortality rates. A small proportion of trees ([~]1% at 1.5% mortality) are life-history  lottery winners, achieving ages >10-20x median age. Maximum age increases with bigger populations and lower mortality rates. One quarter of trees ([~]24%) achieve ages that are 3-4 times greater than median age. Three age classes (Mature, Old, and Ancient) contribute unique historical diversity across complex environmental cycles. Ancient trees are an emergent property of forests that requires many centuries to generate. They rradically change generation time variance and population fitness, bridging infrequent environmental cycles. These life-history  lottery winners are vital to future forest dynamics and invaluable data about environmental history and individual longevity. Old-growth forests contain trees that cannot be replaced through restoration or regeneration in the near future. They simply must be protected to preserve their unique diversity.


title: The quantitative contribution of different Photosystem II compartments to non-photochemical quenching in Arabidopsis

abstract: Excess excitation energy in the light-harvesting antenna of Photosystem II (PSII) can cause irreversible damage to the photosynthetic apparatus. In periods of high light intensity, a feedback mechanism known as non-photochemical quenching (NPQ), induces the formation of quenchers which can safely dissipate excess excitation energy as heat. Although quenchers have been identified in more than one compartment of the PSII supercomplex, there is currently no quantitative description of how much NPQ is occurring at each of these locations. Here, we perform time-resolved fluorescence measurements on WT and antenna mutants lacking LHCII (NoL) and all peripheral antenna (Ch1 and Ch1lhcb5). By combining the results with those of steady-state fluorescence experiments we are able to estimate the intrinsic rate of NPQ for each plant and each PSII compartment. It is concluded that 60-70% of quenching occurs in LHCII, 15-20% in the minor antenna and 15-20% in the PSII core.


title: Transcriptional Regulation of Synthetic Polymer Networks

abstract: Individual cells direct non-equilibrium processes through coordinated signal transduction and gene expression, allowing for dynamic control over multicellular, system-wide behavior. This behavior extends to remodeling the extracellular polymer matrix that encases biofilms and tissues, where constituent cells dictate spatiotemporal network properties including stiffness, pattern formation, and transport properties. The majority of synthetic polymer networks cannot recreate these phenomena due to their lack of autonomous centralized actuators (i.e., cells). In addition, non-living polymer networks that perform computation are generally restricted to a few inputs (e.g., light, pH, enzymes), limiting the logical complexity available to a single network chemistry. Toward synergizing the advantages of living and synthetic systems, engineered living materials leverage genetic and metabolic programming to establish control over material-wide properties. Here we demonstrate that a bacterial metal respiration mechanism, extracellular electron transfer (EET), can control metal-catalyzed radical cross-linking of polymer networks. Linking metabolic electron flux to a synthetic redox catalyst allows dynamic, tunable, and predictable control over material formation and bulk polymer network mechanics using genetic circuits. By programming key EET genes with transcriptional Boolean logic, we rationally design computational networks that sense-and-respond to multiple inputs in biological contexts. Finally, we capitalize on the wide reactivity of EET and redox catalyses to predictably control another class of living synthetic materials using copper(I) alkyne-azide cycloaddition click chemistry. Our results demonstrate the utility of EET as a bridge for controlling abiotic materials and how the design rules of synthetic biology can be applied to emulate physiological behavior in polymer networks.

category: synthetic biology


title: T7Max transcription system

abstract: Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. Here we present a modified T7 RNA polymerase promoter that acts to significantly increase the yields of both transcription and translation within in vitro systems. The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase-based in vitro protein expression system. Unlike other methods of limiting linear template degradation, the T7Max promoter increases transcript concentration in a T7 transcription reaction, providing more mRNA for translation.


title: The genome of the camphor tree and the genetic and climatic relevance of the top-geoherbalism in this medicinal plant

abstract: Camphor tree (Cinnamomum camphora (L.) J. Presl), a species in the magnoliid family Lauraceae, is known for its rich volatile oils and is used as a medical cardiotonic and as a scent in many perfumed hygiene products. Here, we present a high-quality chromosome-scale genome of C. camphora with a scaffold N50 of 64.34 Mb and an assembled genome size of 755.41 Mb. Phylogenetic inference revealed that the magnoliids are a sister group to the clade of eudicots and monocots. Comparative genomic analyses identified two rounds of ancient whole-genome duplication (WGD). Tandem duplicated genes exhibited a higher evolutionary rate, a more recent evolutionary history and a more clustered distribution on chromosomes, contributing to the production of secondary metabolites, especially monoterpenes and sesquiterpenes, which are the principal essential oil components. Three-dimensional analyses of the volatile metabolites, gene expression and climate data of samples with the same genotype grown in different locations showed that low temperature and low precipitation during the cold season modulate the expression of genes in the terpenoid biosynthesis pathways, especially TPS genes, which facilitates the accumulation of volatile compounds. Our study lays a theoretical foundation for policy-making regarding the agroforestry applications of camphor tree.

category: genomics


title: MiR-339-3p aggravates rat vascular inflammation induced by AT1R autoantibodies by down-regulating BKα protein expression

abstract: The abnormality of large-conductance calcium-activated potassium channels (BK channels) is an important factor in inducing vascular inflammation. BK channel agonists can readily recover BK channel function and improve vascular inflammation. However, it is not clear how to improve BK dysfunction caused by downregulation of BK channel protein expression. This study found that angiotensin II-1 receptor autoantibodies (AT1-AA), which are widely present in the body of various types of cardiovascular diseases, can down-regulate the expression of BK channel protein and induce vascular inflammation. Further research found that the elevated neural precursor cells expressed developmentally downregulated 4-like (NEDD4L) protein level is involved in the down-regulation of BK channel  subunit (BK) protein level by AT1-AA. Bioinformatics analysis and experiments have confirmed that miR-339-3p plays an irreplaceable role in the high expression of NEDD4L and the low expression of BK, and aggravates the vascular inflammation induced by AT1-AA. Overall, AT1-AA increased miR-339-3p expression (targeting BK via the miR-339-3p/NEDD4L axis or miR-339-3p alone), reduced BK protein expression in VSMCs, and induced vascular inflammation. The results of the study indicate that miR-339-3p may become a new target for reversing vascular inflammation in AT1-AA-positive patients.

category: molecular biology


title: Comparative genomics of Xylella fastidiosa suggests determinants of host-specificity and expands its mobile genetic elements repertoire

abstract:
The Gram-negative bacterium Xylella fastidiosa colonizes plant xylem vessels and is obligately vectored by xylem sap-feeding hemipteran insects. X. fastidiosa causes diseases in many plant species but in a variety of its plant hosts this bacterium behaves as a commensal endophyte. Originally confined to the Americas, infecting mainly grapevine, citrus and coffee plants, X. fastidiosa has spread to several plant species in Europe, causing devastating crop diseases. Although many pathogenicity and virulence factors have been identified in X. fastidiosa which enable the bacterium to successfully establish in the xylem tissue, the mechanisms by which distinct X. fastidiosa strains colonize and cause disease in specific plant hosts have not been fully elucidated. Here we present comparative analyses of 94 publicly available whole-genome sequences of X. fastidiosa strains with the goal of providing insights into plant host specificity determinants for this phytopathogen as well as of expanding the knowledge of its mobile genetic elements (MGE) content, mainly prophages. Our results revealed a pangenome of 4,549 protein coding sequences (CDSs) which is still open. The core- and accessory genomes comprise 954 and 2,219 CDSs, respectively. Phylogenetic tree construction using all core genome CDSs grouped the strains in three major clades of subspecies fastidiosa, multiplex and pauca, with subclades related to the strains sequence type (ST) obtained from multi-locus sequence typing (MLST). The geographic region where the strains were collected showed stronger association with the clades of X. fastidiosa strains rather than the plant species from which they were isolated. Among the CDS related to virulence and pathogenicity found in the core genome, those related to lipopolysaccharide (LPS) synthesis and trimeric autotransporter adhesins (TAA) are somewhat related with the plant host of a given strain according to phylogenetic inference. The X. fastidiosa accessory genome is represented by an abundant and heterogeneous mobilome, which includes a diversity of prophage regions. In summary, the genome comparisons reported here will enable a better understanding of the diversity of phylogenetically close genomes and warrant further investigation of LPS and TAAs as potential X. fastidiosa host-specificity determinants.

Impact statementThe bacterium Xylella fastidiosa is a pathogen that infects many plant species and has caused devastating diseases in grapevine, citrus, coffee, and olive plants. This phytopathogen X. fastidiosa is original from the Americas and has emerged in Europe where it is causing severe economic losses for olive producers, mainly in Italy. Although many pathogenicity and virulence factors have been identified in X. fastidiosa, which enable this bacterium to successfully establish in the xylem vessels network, the mechanisms by which distinct X. fastidiosa strains colonize and cause disease in the different plant host species have not been fully elucidated. The comparative analyses of 94 whole-genome sequences from X. fastidiosa strains from diverse hosts and geographic regions provide insights into host specificity determinants for this phytopathogen as well as expand the knowledge of its mobile genetic elements (MGE) content, mainly prophages. Our results contribute for a better understanding of the diversity of phylogenetically close genomes and warrant further experimental investigation of lipopolysaccharide and trimeric autotransporter adhesins as potential host-specificity determinants for X. fastidiosa.

Data summaryAll genomic sequences were accessed from publicly available GenBank RefSeq database at NCBI (National Center for Biotechnology Information). A full listing of NCBI accession numbers for X. fastidiosa strains described in this paper is available in Table S1 (available in the online version of this article).


title: Distinct phases within large RNP granules of C. elegans oocytes are associated with differential stress responses of RNA binding proteins

abstract: One emerging paradigm of cellular organization of RNA and RNA binding proteins is the formation of membraneless organelles (MLOs). Examples of MLOs include several types of RNP (ribonucleoprotein) granules that form via phase separation. Proper regulation of the phase transitions of RNA binding proteins is critical as dysregulation can lead to disease states. Germ granules are small RNP granules conserved across metazoa. In C. elegans, when oogenesis undergoes an extended meiotic arrest, germ granules assemble into much larger, more complex RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. As a step towards gaining insight into the function of RNP granules, in this report we characterize distinct phases for four RNA binding proteins in arrested oocytes. We find that PGL-1 is dynamic and has liquid-like properties, while MEG-3 has gel-like properties, both similar to their properties in small germ granules of embryos. We also show that MEX-3 exhibits gel-like properties in many regards but is more dynamic than MEG-3. We find CGH-1 is dynamic but does not consistently behave liquid-like, and may be an intermediate phase within RNP granules. We also show that the distinct phases of the RNA binding proteins are associated with differential responses to stress.

category: cell biology


title: Repression of MAPK/Erk signaling by Efnb2-Ephb4-Rasa1 is required for lymphatic valve formation

abstract: The lymphatic vascular system plays important roles in various physiological and pathological processes, and lack of lymphatic or lymphovenous valves always causes lymph or blood reflux, and can lead to lymphedema. However, the molecular mechanism underlying the valve formation is poorly understood. Here we report that the MAPK/Erk signaling needs to be repressed during the valve-forming lymphatic endothelial cells (LECs) fate determination, which differs from its positive role in the LECs specification. Up-regulation of MAPK/Erk signaling in ephb4b, efnb2a;efnb2b and rasa1a;rasa1b mutants leads to lymphatic valve defects, whereas simultaneous loss of Erk1 and Erk2 causes valve hyperplasia. Moreover, valve defects in ephb4b or rasa1a;rasa1b mutants are mitigated in the presence of MEK inhibitors, indicating a new function of Efnb2-Ephb4-Rasa1 cassette in lymphatic valve progenitor cells specification by repressing MAPK/Erk activity. Therefore, our findings provide a mechanistic understanding of the lymphatic valve formation and potential drug targets for related lymphatic diseases.