title: Oligodendrocyte-lineage cell exocytosis and L-type prostaglandin D synthase 1 promote oligodendrocyte development and myelination

abstract: In the developing central nervous system, oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes, which form myelin around axons. Oligodendrocytes and myelin are essential for the function of the central nervous system, as evidenced by the severe neurological symptoms that arise in demyelinating diseases such as multiple sclerosis and leukodystrophy. Although many cell-intrinsic mechanisms that regulate oligodendrocyte development and myelination have been reported, it remains unclear whether interactions among oligodendrocyte-lineage cells (OPCs and oligodendrocytes) affect oligodendrocyte development and myelination. Here, we show that blocking vesicle-associated membrane protein (VAMP) 1/2/3-dependent exocytosis from oligodendrocyte-lineage cells impairs oligodendrocyte development, myelination, and motor behavior in mice. Adding oligodendrocyte-lineage cell-secreted molecules to secretion-deficient OPC cultures partially restores the morphological maturation of oligodendrocytes. Moreover, we identified L-type prostaglandin D synthase as an oligodendrocyte-lineage cell-secreted protein that promotes oligodendrocyte development and myelination in vivo. These findings reveal a novel autocrine/paracrine loop model for the regulation of oligodendrocyte and myelin development.


title: Optical Control of Membrane Fluidity Modulates Protein Secretion

abstract: The lipid composition of cellular membranes is dynamic and undergoes remodelling affecting biophysical properties, such as membrane fluidity, which are critical to biological function. Here, we introduce an optical approach to manipulate membrane fluidity based on exogenous synthetic fatty acid with an azobenzene photoswitch, termed FAAzo4. Cells rapidly incorporate FAAzo4 into phosphatidylcholine (PC), the major phospholipid in mammalian cells, in a concentration- and cell type-dependent manner. This generates photoswitchable PC analogs (AzoPC), which are predominantly located in the endoplasmic reticulum (ER). Irradiation causes a rapid photoisomerization that increases membrane fluidity with high spatiotemporal precision. We use these  PhotoCells to study the impact of membrane mechanics on protein export from the ER and demonstrate that this two-step process has distinct membrane fluidity requirements. Our approach represents an unprecedented way of manipulating membrane fluidity in cellulo and opens novel avenues to probe roles of fluidity in a wide variety of biological processes.

category: cell biology


title: Daam1 negatively regulates USP10 activity

abstract: The differentiation of fibroblasts into pathological myofibroblasts during wound healing is in part characterized by increased cell surface expression of v-integrins. Our previous studies found that the deubiquitinase (DUB) USP10 removes ubiquitin from v-integrins, leading to cell surface integrin accumulation, subsequent TGF{beta}1 activation, and myofibroblast differentiation. In this study, a yeast-two hybrid screen elucidated a novel binding partner for USP10, the formin, Daam1. The USP10/Daam1 interaction was also supported by proximity ligation assay (PLA) activity. Treatment with TGF{beta}1 significantly increased USP10 and Daam1 protein expression, PLA signal, and co-localization to actin stress fibers. Furthermore, Daam1 siRNA knockdown significantly reduced a) co-precipitation of USP10 and Daam1 on purified actin stress fibers, and b) {beta}1- and {beta}5-integrin ubiquitination resulting in increased v-, {beta}1-, and {beta}5-integrin total protein levels, v integrin recycling to the cell surface, and extracellular fibronectin (FN) organization. Together, our data suggest that Daam1 negatively regulates USP10s DUB activity and subsequently maintains integrin protein homeostasis.


title: Probing protein ubiquitination in live cells

abstract: The reversible attachment of ubiquitin governs the interaction, activity and degradation of proteins whereby the type and target of this conjugation determine the biological response. The investigation of this complex and multi-faceted protein ubiquitination mostly relies on painstaking biochemical analyses. Here, we employ recombinant binding domains to probe the ubiquitination of proteins in living cells. We immobilize GFP-fused proteins of interest at a distinct cellular structure and detect their ubiquitination state with red fluorescent ubiquitin binders. With this ubiquitin fluorescent three-hybrid (ubiF3H) assay we identified HP1{beta} as a novel ubiquitination target of UHRF1. The use of linkage specific ubiquitin binding domains enabled the discrimination of K48 and K63 linked protein ubiquitination. To enhance signal-to-noise ratio, we implemented fluorescence complementation (ubiF3Hc) with split YFP. Using in addition a cell cycle marker we could show that HP1{beta} is mostly ubiquitinated by UHRF1 during S phase and deubiquitinated by the protease USP7. With this complementation assay we could also directly detect the ubiquitination of the tumor suppressor p53 and monitor its inhibition by the anti-cancer drug Nutlin-3. Altogether, we demonstrate the utility of the ubiF3H assay to probe the ubiquitination of specific proteins and to screen for ligases, proteases and small molecules controlling this posttranslational modification.


title: The integrated stress response remodels the microtubule organizing center to clear unfolded proteins following proteotoxic stress

abstract: When cells encounter stressful situations, they activate the integrated stress response (ISR), which limits total protein synthesis and redirects translation to proteins that help the cells to cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely the S51 phosphorylation site of eukaryotic translation initiation factor eIF2 and genetically replaced eIF2 with eIF2-S51A in squamous cell carcinoma (SCC) stem cells. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during stress, the ISR also functions in promoting cellular recovery once the stress has subsided. This finding exposes a vulnerability to SCC stem cells that could be exploited therapeutically.


title: RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval

abstract:
Highly conserved intraflagellar transport (IFT) trains and certain small GTPases coordinate to direct ciliation and to maintain the ciliary dynamics of signaling molecules via the IFT cargo adaptor BBSome. Unlike murine Rab-like 2 (RABL2) GTPase that enters cilia to drive outward transition zone (TZ) passage of the BBSome, human orthologue fails to enter cilia but resides at the ciliary base, indispensable for ciliation. However, mechanisms underlying how RABL2 regulates ciliation and BBSome barrier passage remain elusive. Here, we show that Chlamydomonas RABL2 regulates basal body targeting of the IFT-B1 subcomplex component of IFT trains as a RABL2-specific effector, mediating ciliation via controlling IFT-B1 basal body amount available for assembling anterograde IFT trains. RABL2GTP binds IFT-B1 to perform IFT; sheds from retrograde IFT trains at the proximal ciliary region right above the TZ; and converts to RABL2GDP rapidly. Next, RABL2GDP activates the ciliary membrane anchored Arf-like 3 (ARL3) GTPase (ARL3GDP) as a ARL3-specific guanine nucleotide exchange factor. Upon detaching from the ciliary membrane, the active ARL3GTP recruits its BBSome effector, autonomous of retrograde IFT train association, to move cross the TZ for ciliary retrieval. This ensures proper BBSome ciliary turnover for maintaining phototactic response of Chlamydomonas cells. For finishing RABL2 ciliary cycle, RABL2GDP passes the TZ for ciliary retrieval by loading onto the ARL3GTP/BBSome as a BBSome cargo. Our data thus propose that RABL2 mediates ciliation and BBSome ciliary retrieval simultaneously but via distinct molecular pathways.

Significance statementIntraflagellar transport (IFT) and its cargo adaptor BBSome are indispensable for ciliation and ciliary singling. Rab-like 2 (RABL2) GTPase mediates ciliation and outward transition zone (TZ) passage of BBSomes with mechanisms yet to be determined. Here, we report that RABL2 decides ciliation by controlling the basal body amount of its effector IFT-B1 available for the assembly of anterograde IFT trains. RABL2GTP cycles through cilia as an IFT-B1 cargo; sheds from IFT at the ciliary base; and undergoes nucleotide exchange for activating ARL3 as an ARL3-specfic guanine nucleotide exchange factor. ARLGTP recruits IFT-shed BBSomes to pass the TZ for ciliary retrieval. RABL2GDP exists cilia via ARL3GTP/BBSome as a BBSome cargo. Therefore, RABL2 functions both outside and inside cilia for initiating IFT and BBSome ciliary retrieval, respectively.


title: Syncollin secreted by activated human neutrophils targets bacteria

abstract: Syncollin is a 16-kDa protein that was originally isolated from the pancreatic zymogen granule. Syncollin is also found in human neutrophils and is secreted from promyelocytic HL-60 cells upon stimulation. Recently, we reported that syncollin is able to bind to bacterial peptidoglycan, to damage the bacterial envelope and to restrict bacterial growth. Here, we show that syncollin is secreted from activated primary human neutrophils, and interacts with extracellular neutrophil traps (NETs) in a similar manner to the well-characterized granular protein myeloperoxidase. In addition, secreted syncollin is able to coat the surface of E. coli in co-cultures of neutrophils and bacteria. On the basis of our findings, we suggest that syncollin plays a role in host defence in the blood.


title: SMN promotes mitochondrial metabolic maturation during myogenesis by regulating the MYOD-miRNA axis.

abstract:
Spinal muscular atrophy (SMA) is a congenital neuromuscular disease caused by the mutation or deletion of survival motor neuron 1 (SMN1) gene. Although the primary cause of progressive muscle atrophy in SMA has classically been considered the degeneration of motor neurons, recent studies have indicated a skeletal muscle-specific pathological phenotype such as impaired mitochondrial function and enhanced cell death. Here we found that the downregulation of SMN causes mitochondrial dysfunction and subsequent cell death in in vitro models of skeletal myogenesis with both a murine C2C12 cell line and human induced pluripotent stem cells. During myogenesis, SMN binds to the genome upstream of the transcriptional start sites of MYOD1 and microRNA (miR)-1 and -206. Accordingly, the loss of SMN downregulates these miRs, whereas supplementation of the miRs recovers the mitochondrial function, cell survival and myotube formation of SMN-deficient C2C12, indicating the SMN-miR axis is essential for myogenic metabolic maturation. Additionally, introduction of the miRs into ex vivo muscle stem cells derived from {Delta}7-SMA mice caused myotube formation and muscle contraction. In conclusion, our data revealed novel transcriptional roles of SMN during myogenesis, providing an alternative muscle-oriented therapeutic strategy for SMA patients.

HighlightsO_LIReduced SMN causes mitochondrial dysregulation in myogenic cells.
C_LIO_LIReduced SMN downregulates miR-1 and miR-206 expression in myogenic cells.
C_LIO_LISMN protein binds to the genome upstream of MYOD1, miR-1 and miR-206.
C_LIO_LImiR-1 and miR-206 are sufficient to improve skeletal muscle function in an SMA model.
C_LI


title: Functional diversity and evolution of the Drosophila sperm proteome

abstract: Given the central role fertilization plays in the health and fitness of sexually reproducing organisms and the well-known evolutionary consequences of sexual selection and sperm competition, knowledge gained by a deeper understanding of sperm (and associated reproductive tissues) proteomes has proven critical to the fields advancement. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. We identified 2563 proteins, with label-free quantitation (LFQ) for 2125 proteins. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins. The top 20 most abundant proteins contained the structural elements - and {beta}-tubulins and sperm leucyl-aminopeptidases (S-Laps). Both gene content and protein abundance were significantly reduced on the X chromosome, a finding consistent with prior genomic studies of the X chromosome gene content and evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. LFQ measured significant levels for 75/83 ribosomal proteins (RPs) we identified, including a number of core constituents. The role of this unique subset of RPs in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins found predominantly in the accessory glands. The possibility of tissue contamination from seminal vesicle or other reproductive tissues was addressed using concentrated salt and detergent treatments. Salt treatment had little effect on sperm proteome composition suggesting only minor contamination during sperm isolation while a significant fraction of Sfps remained associated with sperm following detergent treatment suggesting Sfps may arise within, and have additional functions, in sperm per se.


title: Spindle reorientation in response to mechanical stress is an emergent property of the spindle positioning mechanisms

abstract: Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we have used a combination of optogenetics and computational modelling to better understand how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we find that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the torque balance exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.


title: The Yeast Lifespan Machine: a microfluidic platform for automated replicative lifespan measurements

abstract: The budding yeast, Saccharomyces cerevisiae, has emerged as a model system for studying the aging processes in eukaryotic cells. However, the full complement of tools available in this organism has not been fully applied, in part because of limitations in throughput that restrict the ability to carry out detailed analyses. Recent advances in microfluidics have provided direct longitudinal observation of entire yeast lifespans, but have not yet achieved the normal scale of operation possible in this model system. Here we present a microfluidic platform, called the Yeast Lifespan Machine, where we combine improvements in microfluidics, image acquisition, and image analysis tools to increase robustness and throughput of lifespan measurements in aging yeast cells. We demonstrate the platforms ability to measure the lifespan of large populations of cells and distinguish long- and short-lived mutants, all with minimal involvement of the experimenter. We also show that environmental pH is capable of significantly modulating lifespan depending on the growth media, highlighting how microfluidic technologies reveal determinants of lifespan that are otherwise difficult to ascertain.


title: Molecular dynamics simulations of the Spike trimeric ectodomain of the SARS-CoV-2 Omicron variant: structural relationships with infectivity, evasion to immune system and transmissibility

abstract:
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is replacing Delta, the most prevalent variant worldwide from the beginning of 2021 until early 2022. The Omicron variant is highly transmissible and responsible for a new worldwide COVID-19 wave. Herein, we calculated molecular dynamics simulations of the SARS-CoV-2 trimeric spike protein of Wuhan-Hu-1 strain (wild type, WT) and the Omicron variant of concern. Structural analyses reveal that the SpikeOmicron presents more conformational flexibility than SpikeWT, mainly in the N-terminal domain (NTD) and receptor-binding domain (RBD). Such flexibility results in a broader spectrum of different conformations for SpikeOmicron, whereby the RBD can more easily visit an up-conformational state. We reported how the mutations in this variant may influence the intra- and inter-protomer contacts caused by conformational flexibility of the NTD. Based on our analysis, we suggest that the differences in conformational flexibility between SpikeOmicron and SpikeWT may explain the observed gains in infectivity, immune system evasion and transmissibility in this novel variant.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: biophysics


title: Remodeling of human colon plasma cell repertoire in ulcerative colitis

abstract: Plasma cells (PCs) constitute a significant fraction of cells in colonic mucosa and contribute to inflammatory lymphocytic infiltrates in ulcerative colitis (UC). While gut PCs secrete 3-5 g of immunoglobulins daily, including IgA antibodies that target colitogenic bacteria, their role in UC is not known. Here, we combined B cell sorting with single-cell VDJ- and RNA-seq and monoclonal antibody (mAb) testing to characterize the colonic PC repertoire in healthy individuals and patients with UC. We show that a large fraction of B cell clones is shared between different colon regions and that inflammation in UC disrupts this landscape, causing clonal expansion and isotype skewing from IgA1 and IgA2 to IgG1. mAbs produced from expanded PC clones show low polyreactivity and autoreactivity and target specific bacterial strains. Expression profiles of individual PCs from inflamed and non-inflamed colon regions indicate that inflammation is associated with up-regulation of the unfolded protein response (UPR) and antigen presentation genes. Together, our results characterize the microbiome-specific PC response in the colon, its disruption in UC and how PCs might contribute to inflammation in UC.

category: immunology


title: Distinct upper airway epithelium interferon-stimulated and profibrotic gene expression between adult and infant rhesus macaques infected with SARS-CoV-2

abstract: The global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for respiratory viral diseases. To elucidate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam-infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analysis of the respiratory tract and evaluated systemic cytokine and antibody responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam-infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum while CXCL10 was induced in all animals. Both groups mounted neutralizing antibody (nAb) responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal tissue isolated at day 14 post-infection revealed significant upregulation of multiple interferon-stimulated genes in infants compared to adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix (ECM) composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared to infants. Our observations suggest age-dependent differential airway responses to SARS-CoV-2 infection that could explain the distinction in pathogenesis between infants and adults.


title: Next-generation intranasal Covid-19 vaccine: a polymersome-based protein subunit formulation that provides robust protection against multiple variants of concern and early reduction in viral load of the upper airway in the golden Syrian hamster model

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (Covid-19), an ongoing global public health emergency. Despite the availability of safe and efficacious vaccines, achieving herd immunity remains a challenge due in part to rapid viral evolution. Multiple variants of concern (VOCs) have emerged, the latest being the heavily mutated Omicron, which exhibits the highest resistance to neutralizing antibodies from past vaccination or infection. Currently approved vaccines generate robust systemic immunity, yet poor immunity at the respiratory tract. We have demonstrated that a polymersome-based protein subunit vaccine with wild type (WT) spike protein and CpG adjuvant induces robust systemic immunity (humoral and T cell responses) in mice. Both antigen and adjuvant are encapsulated in artificial cell membrane (ACM) polymersomes - synthetic, nanoscale vesicles that substantially enhance the immune response through efficient delivery to dendritic cells. In the present study, we have formulated a vaccine candidate with the spike protein from Beta variant and assessed its immunogenicity in golden Syrian hamsters. Two doses of ACM-Beta spike vaccine administered via intramuscular (IM) injection evoke modest serum neutralizing titers that are equally efficacious towards WT and Beta viruses. In contrast, the ACM-WT spike vaccine induces a predominantly WT-specific serum neutralizing response with pronounced reduction in potency towards the Beta variant. Remarkably, immunogenicity of the ACM-Beta spike vaccine is greatly enhanced through intranasal (IN) administration. Following IN challenge with the Beta variant, IM-immunized hamsters are fully protected from disease but not infection, displaying similar peak viral RNA loads in oral swabs as non-vaccinated controls. In contrast, hamsters IN vaccinated with ACM-Beta spike vaccine are protected from disease and infection, exhibiting a [~]100-fold drop in total and subgenomic RNA load as early as day 2 post challenge. We further demonstrate that nasal washes from IN-but not IM-immunized animals possess virus neutralizing activity that is broadly efficacious towards Delta and Omicron variants. Altogether, our results show IN administration of ACM-Beta spike vaccine to evoke systemic and mucosal antibodies that cross-neutralize multiple SARS-CoV-2 VOCs. Our work supports IN administration of ACM-Beta spike vaccine for a next-generation vaccination strategy that not only protects against disease but also an infection of the respiratory tract, thus potentially preventing asymptomatic transmission.


title: Microbial dysbiosis and metabolic disorders promote rheumatoid arthritis progression across successive stages

abstract: Rheumatoid arthritis (RA) is a chronic inflammatory disease that typically progresses through four stages, from mild stiffness to severe disability. The gut-joint axis is critical in RA pathogenesis, while its dynamic patterns and regulatory mechanisms throughout RA progression remain unknown. Here, we performed fecal metagenomic and serum metabolomic studies on 76 patients from four RA stages, 19 patients with osteoarthritis, and 27 healthy individuals. We demonstrated dynamics of microbial dysbiosis and metabolomic disorders and their crucial roles in RA pathogenesis across successive stages, which were in line with clinical RA progression. First, elevated Collinsella aerofaciens and depleted Parabacteroides distasonis increased gut permeability in the first stage. Second, the depletion of Bacteroides uniformis and Bacteroides plebeius led to decreased glycosaminoglycan metabolism, continuously hurting articular cartilage. Third, elevated Escherichia coli prompted an increase in ascorbate degradation particularly in the second and third stages, promoting inflammation and bone destruction. Fourth, abnormally high levels of serum methoxyacetic acid and cysteine-S-sulfate might inhibit osteoblasts in the second stage and enhance osteoclasts in the third stage, respectively, thus promoting bone erosion. Moreover, metagenomic and metabolomic features were identified for different RA stages, presenting profound diagnostic potential. Furthermore, combined with analysis of 16S rRNA genes and scanning electron microscopy on joint synovial fluid from another cohort of 271 RA patients, microbial invasion of the joints such as Eggerthella lenta and Bifidobacterium longum was highly likely to happen in the fourth stage. Our findings elucidate the role of microbial dysbiosis and metabolic disorders in successive stages of RA, which open up new avenues for RA prognosis and therapy.

category: microbiology


title: Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

abstract: In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics and live-cell microscopy, we determined the contribution of the pilotin to assembly, function and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In the absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. In line, the majority of export apparatus subcomplexes is mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, the export of late T3SS substrates including the virulence effectors is hardly affected in these bacteria, whereas early T3SS substrates, such as the needle subunits, are only exported to a very low degree. We found that pilotins transiently interact with the secretin and the large export apparatus component SctV, but mostly localize throughout the bacterial membrane, where they form transient mobile clusters, which do not colocalize with assembled injectisomes. In addition, pilotins interact with non-T3SS components, including sugar transporters. Pilotins therefore have additional functions downstream injectisome assembly, which include the regulation of type III secretion and a potential new link to the cellular energy metabolism.


title: Identification of a novel interaction between Theileria Prohibitin (TaPHB-1) and bovine RUVBL-1

abstract:
Bovine tropical theileriosis causes huge economic loss worldwide. It is a tick borne disease of bovine caused by the parasite Theileria annulata. T. annulata is an intracellular parasite that belongs to the phylum Apicomplexa. The sporozoites of T. annulata are released by the tick into the bloodstream of the host during the blood meal that invades bovine B cells, macrophages, or monocytes. This infection leads to the transformation of the host cells and brings cancer-like phenotype in the host cells. The parasite proteins play a vital role in the transformation of the host cell. However, the parasite factors involved in the host cell transformation are not well explored. Previously, TaPIN1, a peptidyl-prolyl isomerase of T. annulata, was shown to be secreted to the host cytosol and play a role in the host cell transformation. The present study was carried out to explore the parasite-host interactions that may play an important role in the host cell transformation. We identified the parasite proteins that are expressed in the schizont stage with a signal peptide. We narrow down our search to a parasite prohibitin. The in silico analysis of T. annulata prohibitin (TA04375, TaPHB-1) showed that TaPHB-1 shares homology with the mammalian prohibitin 1. With the localization experiments, we confirmed that TaPHB-1 is exported to the parasite surface and also to the host cell cytosol. Further, we observed that the localization of host prohibitin differs in the parasite-infected cells and could not be reverted back by the elimination of the parasite in the infected cells. We found through the yeast-two-hybrid studies that bovine RUVBL1 (BoRUVBL-1) interacts with TaPHB-1. The interaction between BoRUVBL1 and TaPHB-1 was predominantly observed on the parasite surface in the infected bovine cells. The interaction was further confirmed with immunoprecipitation and LC-MS/MS analysis. Further, the LC-MS/MS based TaPHB-1 interactome study reveals that it interacts with proteins that regulate actin cytoskeleton organization, protein folding, mRNA processing, and metabolic processes. Our finding suggests that the parasite releases prohibitin protein into the cytoplasm of the host cell where it interacts with the host RUVBL-1. This finding has implications not only in the understanding of Theileria parasite biology in greater depth but also in the cancer biology where previously differential localization of prohibitin proteins was observed but its interaction partner was not known.

Author summaryTheileria annulata, an apicomplexan, is a unique parasite which can transform host leucocytes. This parasite uses this strategy for its own multiplication. The cells infected with this parasite, when treated with buparvaquone, an anti-theilerial drug, cannot survive without the parasite. This observation suggests that the parasite derived factors are required to maintain the cancerous phenotype of the host cell. We mined the parasite proteome to find out the proteins with signal sequence that may be secreted to the host cell cytosol and being expressed in the schizont stage. The parasite prohibitin (TaPHB-1) chosen for this study was found to be secreted to the host cytoplasm and on the parasite surface. Interestingly, we observed a noticeable change in the localization of the host prohibitin in the parasite infected cells. The host prohibitin that is normally localized to the mitochondria in the uninfected cells was observed in the host cell nucleus similar to the cancerous cells. Since the parasite protein is exported to the host cytoplasm we looked for its interacting partner. We performed yeast-two-hybrid screening with TaPHB-1 with in-house prepared the cDNA library of the infected bovine leucocytes. We identified bovine RUVBL1 as the interacting partner of TaPHB-1. Interestingly, the interaction between parasite prohibitin and bovine RUVBL1 was observed on the parasite surface. Further, analysis of the parasite prohibitin interactome in the infected cells shows that it might be involved with those proteins which regulate actin cytoskeleton organization, protein folding, mRNA processing and metabolic process. Since parasite infected cells have cancer like phenotype, the identification of this novel interaction will open up new avenues not only in the arena of parasite biology but also in the domain of cancer biology.


title: A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites

abstract: With the advent of resistance to existing treatments, new drugs are needed to combat apicomplexan parasites such as the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). To identify new inhibitors of the mitochondrial electron transport chain (ETC) in these parasites, we developed a Seahorse XFe96 flux analyzer approach to screen compounds from the Medicines for Malaria Venture  Pathogen Box for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC of T. gondii, five of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. We pinpoint the molecular targets of these inhibitors, demonstrating that all target ETC Complex III, with MMV688853 additionally targeting a kinase with a key role in parasite invasion of host cells. Most of the compounds remain effective inhibitors of parasites that are resistant to the clinically used Complex III inhibitor atovaquone. In sum, we have developed a versatile screening approach to identify and characterize new inhibitors of the ETC in apicomplexan parasites.


title: SARS-CoV-2 Permissive Glioblastoma Cell Line for High Throughput Antiviral Screening

abstract: Despite the great success of the administered vaccines against SARS-CoV-2, the virus can still spread, as evidenced by the current circulation of the highly contagious Omicron variant. This emphasizes the additional need to develop effective antiviral countermeasures. In the context of early preclinical studies for antiviral assessment, robust cellular infection systems are required to screen drug libraries. In this study, we reported the implementation of a human glioblastoma cell line, stably expressing ACE2, in a SARS-CoV-2 cytopathic effect (CPE) reduction assay. These glioblastoma cells, designated as U87.ACE2+, expressed ACE2 and cathepsin B abundantly, but had low cellular levels of TMPRSS2 and cathepsin L. The U87.ACE2+ cells fused highly efficiently and quickly with SARS-CoV-2 spike expressing cells. Furthermore, upon infection with SARS-CoV-2 wild-type virus, the U87.ACE2+ cells displayed rapidly a clear CPE that resulted in complete cell lysis and destruction of the cell monolayer. By means of several readouts we showed that the U87.ACE2+ cells actively replicate SARS-CoV-2. Interestingly, the U87.ACE2+ cells could be successfully implemented in an MTS-based colorimetric CPE reduction assay, providing IC50 values for Remdesivir in the low nanomolar range. Lastly, the U87.ACE2+ cells were consistently permissive to all tested SARS-CoV-2 variants of concern, including the current Omicron variant. Thus, ACE2 expressing glioblastoma cells are highly permissive to SARS-CoV-2 with productive viral replication and with the induction of a strong CPE that can be utilized in high-throughput screening platforms.


title: Biosynthesis of aurodox, a Type III secretion system inhibitor from Streptomyces goldiniensis

abstract:
The global increase in antimicrobial-resistant infections means that there is a need to develop new antimicrobial molecules and strategies to combat the issue. Aurodox is a linear polyketide natural product that is produced by Streptomyces goldiniensis, yet little is known about aurodox biosynthesis or the nature of the biosynthetic gene cluster (BGC) that encodes its production. To gain a deeper understanding of aurodox biosynthesis by S. goldiniensis, the whole genome of the organism was sequenced, revealing the presence of an 87 kb hybrid Polyketide Synthase/Non-Ribosomal Peptide Synthetase (PKS/NRPS) BGC. The aurodox BGC shares significant homology with the kirromycin BGC from S. collinus T{upsilon} 365; however, the genetic organisation of the BGC differs significantly. The candidate aurodox gene cluster was cloned and expressed in a heterologous host to demonstrate that it was responsible for aurodox biosynthesis and disruption of the primary PKS gene (aurAI) abolished aurodox production. These data support a model whereby the initial core biosynthetic reactions involved in aurodox biosynthesis follow that of kirromycin. Cloning aurM* from S. goldiniensis and expressing this in the kirromycin producer S. collinus T{upsilon} 365 enabled methylation of the pyridone group, suggesting this is the last step in biosynthesis. This methylation step is also sufficient to confer the unique Type III Secretion System inhibitory properties to aurodox.

ImportanceEnterohaemorrhagic Escherichia coli (EHEC) is a significant global pathogen for which traditional antibiotic treatment is not recommended. Aurodox inhibits the ability of EHEC to establish infection in the host gut through the specific targeting of the Type III Secretion System, whilst circumventing the induction of toxin production associated with traditional antibiotics. These properties suggest aurodox could be a promising anti-virulence compound for EHEC, which merits further investigation. Here, we have characterised the aurodox biosynthetic gene cluster from Streptomyces goldiniensis and have established the key enzymatic steps of aurodox biosynthesis that give rise to the unique anti-virulence activity. These data provide the basis for future chemical and genetic approaches to produce aurodox derivatives with increased efficacy and the potential to engineer novel elfamycins.


title: Vector competence and transcriptional response of Aedes aegypti for Ebinur Lake virus, a newly mosquito-borne orthobunyavirus

abstract: The global impact of mosquito-borne diseases is increasing in the last decades. The newly classified orthobunyavirus, Ebinur Lake virus (EBIV) has been verified with highly virulent pathogenic to adult laboratory mice, and antibodies against EBIV have been detected in humans. As a potential emerging virus, it is necessary to assess the vector capacity of mosquitoes for EBIV to predicting its risk to public health. Herein, Aedes aegypti, the gradually important vector in China, was used as a model to evaluate the vector competence for EBIV. It was showed that EBIV can be transmitted by Ae. aegypti through oral feeding and the transmission rates could get to 11.8% at 14 days post infection (dpi). The highest infection rate, dissemination rate and ovary infection rate were 70%, 42.9%, and 29.4%, respectively. Through intrathoracic infection, Ae. aegypti was highly susceptible to EBIV and the transmission rates could get to 90% at 10 dpi. Moreover, the infection rate, dissemination rate and ovary infection rate were all 100%. Transcriptome analysis demonstrated EBIV can alter expressions of mosquito genes related to immune-related process and metabolism-related process. Defensin-C and chitinase 10 had been continuously down-regulated in the mosquitoes infected by intrathoracic inoculation. Our studies made the comprehensive analysis of the vector competence and transcriptional response of Ae. aegypti for EBIV, which implied the potential risk of EBIV to public health. Moreover, these findings indicated a complex interplay between EBIV and the mosquito immune system to affect the vector transmission capability.

category: molecular biology


title: Omicron Spike protein has a positive electrostatic surface that promotes ACE2 recognition and antibody escape

abstract: High transmissibility is a hallmark of the Omicron variant of SARS-CoV-2. Understanding the molecular determinants of Omicrons transmissibility will impact development of intervention strategies. Here we map the electrostatic potential surface of the Spike protein to show that major SARS-CoV-2 variants have accumulated positive charges in solvent-exposed regions of the Spike protein, especially its ACE2-binding interface. Significantly, the Omicron Spike-ACE2 complex has complementary electrostatic surfaces. In contrast, interfaces between Omicron and neutralizing antibodies tend to have similar positively charged surfaces. Structural modeling demonstrates that the electrostatic property of Omicrons Spike receptor binding domain (S RBD) plays a role in enhancing ACE2 recognition and destabilizing Spike-antibody complexes. Collectively, our structural analysis implies that Omicron S RBD interaction interfaces have been optimized to simultaneously promote access to human ACE2 receptors and evade antibodies. These findings suggest that electrostatic interactions are a major contributing factor for increased Omicron transmissibility relative to other variants.


title: DE-NOVO HEMATOPOIESIS FROM THE FETAL LUNG

abstract: Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial to hematopoietic transition (EHT) to give rise to hematopoietic progenitors. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs), but lung HECs have never been described. Using explant cultures of murine and human fetal lungs, we demonstrate that the fetal lung is a source of HECs that have the functional capacity to undergo EHT to produce de-novo HSPCs. Flow cytometric and functional assessment of fetal lung explants showed the production of HSPCs that expressed key EHT and pre-HSPC markers. scRNA-Seq and small molecule modulation demonstrated that fetal lung EHT is reliant on canonical EHT signaling pathways. These findings suggest that functional HECs are present in the fetal lung, thus establishing this location as a potential extramedullary site of de-novo hematopoiesis.

category: developmental biology


title: Oligodendrocyte origin and development in the zebrafish visual system

abstract: Oligodendrocytes are the myelinating cells in the central nervous system (CNS). Their developmental origin and specification are well known in birds and mammals but remains unclear in fish. To fulfill this gap, we have studied their early progression during zebrafish visual morphogenesis using the transgenic line Olig2:GFP. We have tracked the Olig2+ cells in the visual system from 48 hours post fertilization (hpf) until 11 days post fertilization (dpf). We have also analyzed the differential expression of the Sox2 and Sox10 transcription factors in this cellular line. Oligodendrocyte progenitor cells (OPCs) originate at 48hpf in regions close to the preoptic area, near to the ventral hypothalamus. Then, at 5 dpf, they migrate to the optic chiasm, where they invade the optic nerve, extending towards the retina. While OPCs in the retina also express Sox2, in the the optic tectum they express Sox10. Within the optic nerve tract, they express both. We have also observed that these Olig2:GFP line do not colocalize with the expression of Mbpa, a myelin marker, but are intimately intertwined. Our data matches with other animal models, where OPCs are specified in the preoptic area and migrate to the optic nerve through the optic chiasm. We revealed that oligodendrocyte is a complex population of cells expressing different transcription factors.


title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

abstract: High-density lipoprotein (HDL) particles suppress inflammation-induced tissue injury via vascular and myeloid cell-dependent mechanisms. As such, HDL-associated bioactive lipids such as sphingosine 1-phosphate (S1P) and prostacyclin (PGI2) signal via their respective G protein-coupled receptors on target cells to promote vascular endothelial function and suppress platelet and myeloid-dependent pathophysiology. Here we have constructed a fusion protein of apolipoprotein A1 (ApoA1) and apolipoprotein M (ApoM) (A1M) that forms HDL-like particles and chaperones S1P and Iloprost, stable PGI2 analog. The A1M/S1P complex activates S1P receptor-1 (S1PR1) as a Gi-biased agonist and attenuates the inflammation-induced NF{kappa}B pathway while A1M/Iloprost acts via IP receptor to inhibit platelet aggregation and promote endothelial barrier function. In addition to enhancing the endothelial barrier, A1M/S1P suppresses neutrophil influx, oxidative burst and inflammatory mediator secretion in a sterile inflammation model. We propose that A1M could be useful as a therapeutic to induce S1P and PGI2-dependent anti-inflammatory functions and suppress collateral tissue injury.

category: biochemistry


title: The impact of the soluble protein fraction and kernel hardness on wheat flour starch digestibility

abstract: Wheat is the staple crop for 35% of the worlds population, providing a major source of calories for much of the worlds population. Starch is the main source of energy in wheat flour, but the digestibility of wheat starch varies greatly between different flours and wheat products. This has relevance from a health perspective because wheat starch products that are rapidly digested and elicit large post-prandial glucose peaks are associated with a host of cardiac and metabolic disorders. In this study, we investigate the impact of protein on starch digestion in three commercially sourced flours with different grain hardness. Grain hardness impacted on flour particle size, but not significantly on starch digestion. A soluble extract of wheat proteins was found to dramatically reduce starch digestion, even following gastric proteolysis. Proteomic analysis revealed that this soluble extract was enriched in proteinaceous -amylase inhibitors which were partially degraded during gastric proteolysis. Therefore, we conclude that the soluble proteins of wheat flour have a significant contribution towards retarding starch digestion, even following gastric digestion.


title: Characterisation and sequence mapping of large RNA and mRNA therapeutics using mass spectrometry

abstract: Large RNA including messenger RNA (mRNA) has emerged as an important new class of therapeutic. Recently this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study we have developed an automated, high throughput workflow for the rapid characterisation and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilised on magnetic particles was performed in conjunction with high resolution liquid chromatography mass spectrometry analysis. Sequence mapping was performed using automated oligoribonucleotide annotation and identifications based on MS/MS spectra. Using this approach >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS Co-V2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation can be completed within 90 minutes. The ability to rapidly identify, characterise and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation and impurity analysis.


title: Development of methylation-based biomarkers for breast cancer detection by model training and validation in synthetic cell-free DNA

abstract: Circulating tumour-derived DNA (ctDNA) carries the genetic and epigenetic characteristics of the tumour from which it is derived and can give information about the biology and tissue origins of the underlying tumour. DNA methylation is an epigenetic mark that is specific to individual tissues and, as methylation profiles are disrupted in tumours, they can indicate the tissue of origin and cancer type of ctDNA. We have developed a set of methylation biomarkers for detecting breast cancer in plasma cell-free DNA (cfDNA). First, we mined publicly available methylation datasets to create synthetic methylation profiles that were modelled to reflect cfDNA from healthy subjects and cancer patients. These profiles were restricted to the most differentially methylated CpGs between breast tumour samples and haematopoietic cells. Regularised logistic regression models were trained using 10-fold cross-validation on synthetic cfDNA datasets with distinct fractions of breast tumour DNA spiked in silico into healthy cfDNA with the addition of 10% of a mix of different tissues. Initial validation with synthetic cfDNA permitted detection of breast cancer-derived DNA with as little as 0.25% tumour DNA spiked in silico into healthy subject cfDNA with an area under ROC curve (AUC) of 0.63. Performances of classifiers increased with increased fractions of spike-in tumour DNA (AUCs 0.77 and 0.93 at tumour DNA fractions 0.5% and 1% respectively). We then combined the most discriminative CpG markers from our models with methylation markers of breast cancer that had already been published to obtain a single marker set. In vitro testing of MCF-7 breast cancer cell line DNA spiked into leukocyte DNA showed highly significant correlation for individual markers between laboratory-measured and published methylation data for MCF-7 and leukocytes (R > 0.89, P < 2.2 x 10-16). These preliminary data indicate promising results for detection of breast cancer cell line DNA using this methylation marker set, which now require testing in cfDNA from breast cancer patients and healthy controls.

category: cancer biology


title: Protein undernutrition reduces the efficacy of praziquantel in a murine model of Schistosoma mansoni infection

abstract:
BackgroundUndernutrition and schistosomiasis are public health problems and often occur in low and middle-income countries. Protein undernutrition can alter the host-parasite environment system and aggravate the course of schistosomiasis. This study aimed to assess the impact of a protein-deficient diet on the efficacy of praziquantel.

Methodology/principal findingsThirty-day-old mice were fed with a protein-deficient diet, and 40 days later, they were individually infected with fifty Schistosoma mansoni cercariae. A 28-day-treatment with praziquantel at 100 mg/kg for five consecutive days followed by distilled water begins at the 36th day post-infection. Mice were sacrificed on the 64th day post-infection. We determined the parasitological burden, liver and intestine histomorphometry, liver injury, and immunomodulation parameters. Praziquantel treatment of infected mice fed with a standard diet (IN-PZQ) resulted in a significant reduction of worm and egg burdens and a normalization of iron and calcium levels. The therapy also improved schistosomiasis-induced hepatopathy and oxidative stress. The anti-inflammatory and immunomodulatory activities of praziquantel were also significant in these mice. When infected mice receiving the protein-deficient diet were treated with praziquantel (ILP-PZQ), the body weight loss and hepatomegaly were not alleviated, and the worm and liver egg burdens were significantly higher than those of IN-PZQ mice (P < 0.001). The treatment did not reduce the increased activities of ALT and {gamma}-GGT, the high malondialdehyde concentration, and the liver granuloma volume. The iron and calcium levels were not ameliorated and differed from those of IN-PZQ mice (P < 0.001 and P < 0.05). Moreover, in these mice, praziquantel treatment did not reverse the high level of IL-5 and the low mRNA expression of CCL3/MIP-1 and CXCL-10/IP-10 induced by S. mansoni infection.

Conclusion/significanceThese results demonstrated that a protein-deficient diet reduced the schistosomicidal, antioxidant, anti-inflammatory, and immunomodulatory activities of praziquantel.

Authors summaryAlmost 90% of people requiring schistosomiasis preventive chemotherapy in 2018 lived in sub-Saharan Africa. Besides, 205.3 million children under five years suffer and die of undernutrition in low- and middle-income countries. The physiopathology of schistosomiasis mansoni involves liver damage, oxidative stress, and perturbation of the immune response. These disturbances are intensified by undernutrition. Praziquantel is used to treat schistosomiasis, but its efficacy on the comorbidity of S. mansoni infection and undernutrition has not been investigated. We conducted this study to assess the effectiveness of praziquantel on S. mansoni infection in mice fed with a protein-deficient diet. We recorded growth retardation, hepatomegaly, and high worm and egg burdens in mice fed with a protein-deficient diet and treated with PZQ. Moreover, the treatment did not reverse the liver function injury, oxidative stress, high iron level, and low calcium level. The proinflammatory cytokine IL-5 was still high, and the gene expression of some macrophage-associated chemokines was reduced. Therefore, this study demonstrated that in a murine model of a protein-deficient diet, the efficacy of praziquantel on S. mansoni infection was reduced. It also underlines the importance of targeting protein deficiency and malnutrition in populations living in schistosomiasis endemic areas for efficient disease control.

category: pathology


title: Arid5a mediates an IL-17-dependent pathway that drives autoimmunity but not antifungal host defense

abstract: IL-17 contributes to the pathogenesis of certain autoimmune diseases, but conversely is essential for host defense against fungi. Antibody-based biologic drugs that neutralize IL-17 are effective in autoimmunity but can be accompanied by adverse side effects. Candida albicans is a commensal fungus that is the primary causative agent of oropharyngeal and disseminated candidiasis. Defects in IL-17 signaling cause susceptibility to candidiasis in mice and humans. A key facet of IL-17 receptor signaling involves RNA binding proteins (RBP), which orchestrate the fate of target mRNA transcripts. In tissue culture models we showed that the RBP AT-rich interacting protein 5a (Arid5a) promotes the stability and/or translation of multiple IL-17-dependent mRNAs. Moreover, during OPC, Arid5a is elevated within the oral mucosa in an IL-17-dependent manner. However, the contribution of Arid5a to IL-17-driven events in vivo is poorly defined. Here, we used CRISPR/Cas9 to generate mice lacking Arid5a. Arid5a-/- mice were fully resistant to experimental autoimmune encephalomyelitis (EAE), an autoimmune setting in which IL-17 signaling drives pathology. Surprisingly, Arid5a-/- mice were resistant to OPC and systemic candidiasis, similar to immunocompetent WT mice and contrasting with mice defective in IL-17 signaling. Therefore, Arid5a-dependent signals mediate pathology in autoimmunity yet are not required for immunity to candidiasis, indicating that selective targeting of IL-17 signaling pathway components may be a viable strategy for development of therapeutics that spare IL-17-driven host defense.


title: Genetic load in incomplete lupus erythematosus

abstract: Incomplete lupus erythematosus (ILE) patients have lupus features but insufficient criteria for systemic lupus erythematosus (SLE) classification. Some ILE patients transition to SLE, but most avoid major organ involvement. This study tested whether the milder disease course in ILE is influenced by reduced SLE-risk allele genetic load. We calculated the genetic load based on 99 SLE-associated risk alleles in European American SLE patients (>4 ACR-1997 criteria, n=171), ILE patients (3 ACR-1997 criteria; n=174), a subset of ILE patients not meeting SLICC classification (ILESLICC, n=119), and healthy controls (n=133). ILE and SLE patients had significantly greater SLE-risk allele genetic load compared to healthy controls, while ILESLICC patients had a trend toward an increased genetic load, although not statistically significant. However, the genetic load did not differ between ILE and SLE. In conclusion, ILE and SLE patients have comparable genetic loads of SLE risk loci, suggesting similar genetic predisposition between these conditions. Phenotypic differences between SLE and ILE may instead be influenced by ILE-specific variants and gene-environment interactions.


title: An arginase 2 promoter transgenic illuminates anti-inflammatory signalling in zebrafish

abstract: The innate immune response to inflammatory stimuli must be finely balanced to produce an appropriate pro-inflammatory response while allowing a subsequent return to homeostasis. In recent years, in vivo transgenic zebrafish models have shed light on the temporal regulation of the pro-inflammatory innate response to immune challenges. However, until now, there have been no zebrafish transgenic models of anti-inflammatory signalling. We compared existing expression data of arginase genes in zebrafish neutrophils and macrophages, strong candidates for an anti-inflammatory marker, and identified that arginase 2 is the most highly expressed Arginase in zebrafish immune cells. We developed an arginase 2 (arg2) bacterial artificial chromosome (BAC) transgenic line, TgBAC(arg2:eGFP)sh571, driving GFP expression under the control of the arg2 promoter. We show that, under resting conditions, arg2:GFP is expressed in ionocytes, matching the in situ hybridisation pattern. Upon immune challenge by injury, bacterial and fungal insults, arg2:GFP is predominantly expressed in neutrophils at early timepoints post-insult. Later in infections, arg2:GFP is expressed in cells associated with foci of infection (including neutrophils and macrophages), alongside liver expression. Our data indicate that arginase 2 is predominantly expressed in neutrophils after immune challenge and suggest that anti-inflammatory signals coincide with pro-inflammatory signals during early wound and infection responses.


title: Bile Acid Restrained T Cell Activation Explains Cholestasis Aggravated Hepatitis B Virus Infection

abstract: Cholestasis is a common complication of Hepatitis B Virus (HBV) infection, characterized by increased intrahepatic and plasma bile acid levels. Cholestasis was found negatively associated with hepatitis outcome, however; the exact mechanism by which cholestasis impact on anti-viral immunity and impede HBV clearance remains elusive. Here, we found that cholestatic mice are featured with dysfunctional T cell response, and bile acids inhibit the activation and metabolic reprogramming of CD4+ T cells. Mechanistically, bile acids disrupt intracellular calcium homeostasis via inhibiting mitochondria calcium uptake and elevating cytoplasmic Ca2+ concentration of CD4+ T cells, leading to STIM1 and ORAIL1 decoupling and impaired store-operated Ca2+ entry which is essential for NFAT signaling and T cell activation. Moreover, in a transgenic mouse model of HBV infection, it was confirmed that cholestasis compromised T cells activation resulting in poor viral clearance. Collectively, our results suggest that bile acids play pivotal roles in anti-HBV infection via controlling T cells activation and metabolism, and that targeting regulation of bile acids may be a therapeutic strategy for host virus defense.


title: The Transcription Factor Bach2 Negatively Regulates Natural Killer Cell Maturation and Function

abstract: BTB domain And CNC Homolog 2 (Bach2) is a transcription repressor that actively participates in T and B lymphocyte development, but it is unknown if Bach2 is also involved in the development of innate immune cells, such as natural killer (NK) cells. Here, we followed the expression of Bach2 during NK cell development, finding that it peaked in CD27+CD11b+ cells and decreased upon further maturation. Bach2 expression positively correlated with that of the transcription factor TCF1 and negatively correlated with genes encoding NK effector molecules as well as genes involved in the cell cycle. Bach2-deficient mice showed increased numbers of terminally differentiated NK cells with increased production of granzymes and cytokines. NK cell-mediated control of tumor metastasis was also augmented in the absence of Bach2. Therefore, Bach2 is a key checkpoint protein regulating NK terminal maturation.


title: Variation in Plasmodium falciparum sexual commitment rates and responses to environmental modification

abstract:
Asexual blood stage malaria parasites must produce sexual progeny in order to infect mosquitoes. It is therefore important to understand the scope of variation in sexual commitment rates of the most important human parasite Plasmodium falciparum, and to assess responses to conditions that may affect this commitment. First, using a genetically modified line with inducible elevated sexual commitment we compared two different methods of measuring the rates, either staining the Pfs16 marker in very early gametocytes or counting gametocytes after further development. The methods showed correlated estimates in evaluation across multiple independent replicate assays, with higher sensitivity and precision being achieved by Pfs16 marker detection in early gametocytes, so this method was used to survey a wide range of P. falciparum lines. A panel of six recently culture-established clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3.3% to 12.2%, with some significant differences among isolates as well as variation among biological replicates of each. Comparing 13 long-term laboratory-adapted lines of diverse origins, eight had sexual commitment rates similar to those of the recent clinical isolates, with means across multiple biological replicates for each line ranging from 4.7% to 14.5%, while four lines had significantly lower rates with means ranging from 0.3 to 1.6%, and one line with a non-functional ap2-g gene always showed zero sexual commitment. Among a subset of parasite lines, adding choline to suppress commitment had quantitatively variable effects, although these were significant in most assays of lines that had relatively high rates. This study indicates the importance of multiple assay replicates and comparisons of diverse isolates to understand natural and culture-induced variation in this key reproductive trait, relevant to investigating potential effects of parasite adaptation on transmission.

Author SummaryOnly sexual malaria parasites are transmitted from humans to mosquitoes, so it is vital to understand variation in sexual commitment rates of blood stage malaria parasites, and responses to conditions that affect this. We compared two different methods of measuring the rates in a Plasmodium falciparum line with engineered sexual commitment variation, demonstrating higher sensitivity and precision by detection of an early differentiation marker, and this method was then used to survey diverse P. falciparum lines in culture with extensive biological replicate measurements. Recent clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3% to 12%, with significant differences among isolates as well as variation among biological replicates. Long-term laboratory-adapted lines of diverse origins had a wide range, most having rates similar to those of the clinical isolates, while a minority consistently had much lower or zero rates. There was quantitative variation among lines in the effects of adding choline to suppress commitment, although most assays of lines that had relatively high rates showed significant effects. Performing multiple biological replicates and comparisons of recent parasite isolates is vital to understand intra-specific variation in this important reproductive trait, and move towards investigating direct effects on disease transmission.


title: Saccharibacteria harness light energy using Type-1 rhodopsins that may rely on retinal sourced from microbial hosts

abstract: Microbial rhodopsins are a family of photoreceptive membrane proteins with a wide distribution across the Tree of Life. Within the Candidate Phyla Radiation (CPR), a diverse group of putatively episymbiotic bacteria, the genetic potential to produce rhodopsins appears to be confined to a small clade of organisms from sunlit environments. Here, we characterize the metabolic context and biophysical features of Saccharibacteria Type-1 rhodopsin sequences derived from metagenomic surveys and show that these proteins function as outward proton pumps. This provides one of the only known mechanisms by which CPR can generate a proton gradient for ATP synthesis. Intriguingly, Saccharibacteria do not encode the genetic machinery to produce all-trans-retinal, the chromophore essential for rhodopsin function, but their rhodopsins are able to rapidly uptake this cofactor when provided in experimental assays. We found consistent evidence for the capacity to produce retinal from {beta}-carotene in organisms co-occurring with Saccharibacteria, and this genetic potential was dominated by Actinobacteria, which are known hosts of Saccharibacteria in other habitats. If Actinobacteria serve as hosts for Saccharibacteria in freshwater environments, exchange of retinal for use by rhodopsin may be a feature of their associations.


title: Comparative miRNA Transcriptomics of Mouse and Macaque Reveals Cytoskeleton is An Inhibitor for C. neoformans Invasion into Brain

abstract: Cryptococcal meningoencephalitis is an emerging infection shifted from primarily ART-naive to being ART-experienced HIV/AIDS patients, COVID-19 patients and also in immune competent individuals, mainly caused by the human opportunistic pathogen Cryptococcus neoformans, yet mechanisms of the brain or CNS dissemination remain to elucidate, which is the deadest process for the disease. Meanwhile, illustrations of clinically relevant responses in cryptococcosis were limited, as the low availabilities of clinical samples. In this study, macaque and mouse infection models were employed and miRNA-mRNA transcriptomes were performed and combined, which revealed cytoskeleton, a major feather in HIV/AIDS patients, was a centric pathway regulated in both two infection models. Notably, assays of clinical immune cells confirmed an enhanced "Trojan Horse" in HIV/AIDS patients, which can be shut down by cytoskeleton inhibitors. Furthermore, we identified a novel enhancer for macrophage "Trojan Horse", myocilin, and an enhanced fungal burden was achieved in brains of MYOC transgenic mice. Taking together, this study reveals fundamental roles of cytoskeleton and MYOC in blocking fungal CNS dissemination, which not only helps to understand the high prevalence of cryptococcal meningitis in HIV/AIDS, but also facilitates the development of novel drugs for therapies of meningoencephalitis caused by C. neoformans and other pathogenic microorganisms.


title: Comparative miRNA Transcriptomics of Mouse and Macaque Reveals MYOC is An Inhibitor for C. neoformans Invasion into Brain


title: Structure and mechanism of the tripartite ATP-independent periplasmic (TRAP) transporter

abstract: In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential carboxylate- and sulfonate-containing nutrients into the cytoplasm. Unlike other secondary active transporters, TRAP transporters cannot receive their substrates directly, but do so indirectly via a secreted soluble substrate-binding protein. How a sodium-driven secondary active transporter is strictly coupled to a passenger-carrying substrate-binding domain is poorly understood. Here, we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 [A] resolution. SiaM has 12-TMs that come together to form a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium-coupled elevator transporter VcINDY. Interestingly, the SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, indicating TRAP transporters may operate as monomers, rather than the typically observed oligomers. We have identified the Na+ and sialic acid binding sites in SiaM and confirmed a strict dependence on the substrate-binding protein SiaP for uptake. We have determined the SiaP crystal structure that, together with co-evolution driven docking studies, provides a molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We conclude that TRAP proteins are conceptually a marriage between an ABC importer and a secondary active transporter, which we describe herein as an  elevator-with-an-operator mechanism.


title: Quantitative fate mapping: Reconstructing progenitor field dynamics via retrospective lineage barcoding

abstract: Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; however, whether these lineage barcodes can capture the dynamics of complex progenitor fields remains unclear. Here, we introduce quantitative fate mapping, an approach to simultaneously map the fate and quantify the commitment time, commitment bias, and population size of multiple progenitor groups during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a generalizable barcoding mutagenesis model. We validate these approaches using realistically-simulated barcoding results as well as experimental results from a barcoding stem cell line. We further establish criteria for the minimum number of cells that must be analyzed for robust quantitative fate mapping. Overall, this work demonstrates how lineage barcodes, natural or synthetic, can be used to obtain quantitative fate maps, thus enabling analysis of progenitor dynamics long after embryonic development in any organism.


title: Nitric oxide signaling controls collective contractions in a colonial choanoflagellate

abstract: Although signaling by the gaseous molecule nitric oxide (NO) regulates key physiological processes in animals, including contractility1-3, immunity4,5, development6-9 and locomotion10,11, the early evolution of animal NO signaling remains unclear. To reconstruct the role of NO in the animal stem lineage, we set out to study NO signaling in choanoflagellates, the closest living relatives of animals12. In animals, NO produced by the nitric oxide synthase (NOS) canonically signals through cGMP by activating soluble guanylate cyclases (sGCs)13,14. We surveyed the distribution of the NO signaling pathway components across the diversity of choanoflagellates and found three species that express NOS, sGCs, and downstream genes previously shown to be involved in the NO/cGMP pathway. One of these, Choanoeca flexa, forms multicellular sheets that undergo collective contractions controlled by cGMP15. We found that treatment with NO induces sustained contractions in C. flexa by signaling through an sGC/cGMP pathway. Biochemical assays show that NO directly binds C. flexa sGC1 and stimulates its cyclase activity. The NO/cGMP pathway acts independently from other inducers of C. flexa contraction, including mechanical stimuli and heat, but sGC activity is required for contractions induced by light-to-dark transitions. The output of NO signaling in C. flexa - contractions resulting in a switch from feeding to swimming - resembles the effect of NO in sponges1-3 and cnidarians11,16,17, where it interrupts feeding and activates contractility. These data provide insights into the biology of the first animals and the evolution of NO signaling.

category: evolutionary biology


title: Octopaminergic/tyraminergic Tdc2 neurons regulate biased sperm usage in female Drosophila melanogaster

abstract: In polyandrous internally fertilizing species, a multiply-mated female can use stored sperm from different males in a biased manner to fertilize her eggs. The females ability to assess sperm quality and compatibility is essential for her reproductive success, and represents an important aspect of postcopulatory sexual selection. In Drosophila melanogaster, previous studies demonstrated that the female nervous system plays an active role in influencing progeny paternity proportion, and suggested a role for octopaminergic/tyraminergic Tdc2 neurons in this process. Here, we report that inhibiting Tdc2 neuronal activity causes females to produce a higher-than-normal proportion of first-male progeny. This difference is not due to differences in sperm storage or release, but instead is attributable to the suppression of second-male sperm usage bias that normally occurs in control females. We further show that a subset of Tdc2 neurons innervating the female reproductive tract is largely responsible for the progeny proportion phenotype that is observed when Tdc2 neurons are inhibited globally. On the contrary, overactivation of Tdc2 neurons does not further affect sperm storage and release or progeny proportion. These results suggest that octopaminergic/tyraminergic signaling allows a multiply-mated female to bias sperm usage, and identify a new role for the female nervous system in postcopulatory sexual selection.


title: Genome Enrichment of Rare, Unknown Species from Complicated Microbiome by Nanopore Selective Sequencing

abstract: Rare species are vital members of a microbial community, but retrieving their genomes is difficult due to their low abundance. The ReadUntil (RU) approach allows nanopore devices to sequence specific DNA molecules selectively in real-time, which provides an opportunity for enriching rare species. However, there is still a gap in RU-based enriching of rare and unknown species in environmental samples whose community composition is unclear, and many species lack corresponding reference in public databases. Here we present metaRUpore to overcome this challenge. We applied metaRUpore to a thermophilic anaerobic digester (TAD) community, it successfully redirected the sequencing throughput from high-abundance populations to rare species while facilitating the recovery of 41 high-quality metagenome-assembled genomes (MAGs) at low sequencing effort. The simplicity and robustness of the approach make it accessible for labs with moderate computational resources and hold the potential to become the standard practice in future metagenomic sequencing of complicated microbiomes.

category: bioinformatics


title: Architecture and self-assembly of the jumbo bacteriophage nuclear shell

abstract: Bacteria encode myriad defenses that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR/Cas systems. In response, one family of large bacteriophage employs a nucleus-like compartment to protect their replicating genomes by excluding host defense factors. However, the principle composition and structure of this compartment remain unknown. Here, we find that the bacteriophage nuclear shell assembles primarily from one protein, termed chimallin. Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin cooperatively self-assembles as a flexible sheet into closed micron-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.


title: Single-cell transcriptomic and proteomic analysis of Parkinson's disease Brains

abstract:
Parkinsons disease (PD) is a prevalent neurodegenerative disorder where recent evidence suggests pathogenesis may be mediated by inflammatory processes. The molecular architecture of the disease remains to be fully elucidated. We performed single-nucleus transcriptomics and unbiased proteomics using postmortem tissue obtained from the prefrontal cortex of 12 individuals with late-stage PD and age-matched controls. We analyzed [~]80,000 nuclei and identified eight major cell types, including brain-resident T cells, each with distinct transcriptional changes in line with the known genetics of PD. By analyzing Lewy body pathology in the same postmortem tissue, we found that -synuclein pathology is inversely correlated with chaperone expression in excitatory neurons. Examining cell-cell interactions, we found a selective abatement of neuron-astrocyte interactions and enhanced neuroinflammation. Proteomic analyses of the same brains identified synaptic proteins in prefrontal cortex that were preferentially downregulated in PD. Strikingly, comparing this dataset to a regionally similar published analysis for Alzheimers disease (AD), we found no common differentially expressed genes in neurons, but identified many shared differentially expressed genes in glial cells, suggesting that disease etiology in PD and AD are likely distinct. These data are presented as a resource for interrogating the molecular and cellular basis of PD and other neurodegenerative diseases.

One Sentence SummaryWe provide an extensive single cell analysis profiling nearly 80,000 brain nuclei from prefrontal cortex of late-stage Parkinsons disease brains, demonstrate that -synuclein pathology is inversely correlated with chaperone expression in excitatory neurons, found a selective abatement of neuron-astrocyte interactions with enhanced neuroinflammation, and augmented the study with proteomic analysis and cross-comparisons with Alzheimers disease datasets, providing valuable insights into the pathways of neurodegeneration and a deep definition of the underlying molecular pathology for Parkinsons disease.

category: neuroscience


title: The chemokine receptor CXCR3 promotes CD8+ T cell-dependent lung pathology during influenza pathogenesis

abstract:
While the role of CD8+ T cells in influenza clearance is established, their contribution to pathological lung injury is increasingly appreciated. To explore if protective versus pathological functions can be linked to CD8+ T cell subpopulations, we dissected their responses in influenza-infected murine lungs. Our single-cell RNASeq (scRNAseq) analysis revealed significant diversity in CD8+ T cell subpopulations during peak viral load vs. infection-resolved state. While enrichment of Cxcr3hi CD8+ T effector (Teff) subset was associated with a more robust cytotoxic response, both CD8+ Teff and CD8+ T central memory (TCM) exhibited equally potent effector potential. The scRNAseq analysis identified unique regulons regulating the cytotoxic response in CD8+ T cells. The neutralization of CXCR3 mitigated lung injury without affecting viral clearance. IFN-{gamma} was dispensable to regulate the cytotoxic response of Cxcr3hi CD8+ T cells. Collectively, our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-linked lung injury.

TEASERThe CXCR3 expressing CD8+ T cell subset causes severe lung pathology and exacerbates disease severity without affecting viral clearance during influenza infection


title: STING1 activation in brain endothelium by Plasmodium-sequestered heme leads to lethal cerebral malaria through activation of type I IFN response.

abstract:
Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifn{beta}1 transcription in the brain of mice infected with Plasmodium berghei (Pba). This STING1/IFN{beta}-mediated response increases brain CXCL10 governing the extent of brain leucocyte infiltration and blood-brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN-driven brain inflammation was demonstrated in brain endothelial-specific IFN{beta}-reporter and STING1-deficient Pba-infected mice, which are significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba-infected erythrocytes activated STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFN{beta} in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin triggering brain endothelial inflammatory responses via STING1/IFN{beta}/CXCL10 axis crucial to CM pathogenesis and lethality.

SignificanceCM results from loss of blood-brain endothelial barrier function caused by unrestrained inflammatory response in the natural course of infection by Plasmodium parasites. However, the role of brain endothelium in triggering inflammatory mechanisms is still undetermined. We found that the innate immune sensor STING1 is crucial for production of IFN{beta} in brain endothelial cells in Plasmodium-infected mice. This in turn stimulates CXCL10-mediated recruitment of leukocytes and subsequent brain inflammation and tissue damage. We identified within extracellular particles released from Plasmodium-infected erythrocytes, a fraction containing products of hemoglobin degradation, namely heme, which we show can bind STING1. Our results unravel a new angle of CM pathogenesis: heme contained in particles triggers the STING/IFN{beta}/CXCL10 axis in brain endothelial cells.


title: Brain endothelial STING1 activation by Plasmodium-sequestered heme promotes cerebral malaria via type I IFN response.


title: Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia

abstract: Amniotic fluid surrounding the developing fetus is a complex biological fluid rich in metabolically active bio-factors. The presence of extracellular vesicles (EVs) in amniotic fluid has been mainly related to fetal urine. We here characterized EVs from term amniotic fluid in terms of surface marker expression using different orthogonal techniques. EVs appeared to be a heterogeneous population expressing markers of renal, placental, epithelial and stem cells. Moreover, we compared amniotic fluid EVs from normal pregnancies with those of preeclampsia, a hypertensive disorder affecting up to 8% of pregnancies worldwide. An increase of CD105 (endoglin) expressing EVs was observed in preeclamptic amniotic fluid by bead-based cytofluorimetric analysis, and further confirmed using a chip-based analysis. HLA-G, a typical placental marker, was not co-expressed by the majority of CD105+ EVs, suggesting their origin from amniotic fluid cells. At a functional level, preeclampsia-derived EVs, but not normal pregnancy EVs, showed an antiangiogenic effect, possibly due to the decoy effect of endoglin. In addition, several miRNAs were differentially expressed in preeclampsia-derived EVs and directly related to the modulation of angiogenesis and trophoblast function. Our results provide a characterization of term amniotic fluid-EVs, supporting their origin from fetal and placental cells. In preeclampsia, the observed antiangiogenic characteristics of amniotic fluid-EVs may reflect the hypoxic and antiangiogenic microenvironment and could possibly impact on the developing fetus or on the surrounding fetal membranes.


title: TiDeTree: A Bayesian phylogenetic framework to estimate single-cell trees and population dynamic parameters from genetic lineage tracing data

abstract: The development of a multicellular organism is governed by an elaborate balance between cell division, death, and differentiation. These core developmental processes can be quantified from single-cell phylogenies. Here we present TiDeTree, a Bayesian phylogenetic framework for inference of time-scaled single-cell phylogenies and population dynamic parameters such as cell division, death, and differentiation rates from genetic lineage tracing data. We show that the performance of TiDeTree can be improved by incorporating multiple sources of additional independent information into the inference. Finally, we apply TiDeTree to a lineage tracing dataset to estimate time-scaled phylogenies, cell division, and apoptosis rates. We envision TiDeTree to find wide application in single-cell lineage tracing data analysis which will improve our understanding of cellular processes during development. The source code of TiDeTree is publicly available at https://github.com/seidels/tidetree.


title: Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP

abstract:
Our expanding knowledge of the roles small regulatory RNAs play across numerous areas of biology, coupled with the promise of RNA-targeted therapies and small RNA-based medicines, create an urgent need for tools that can accurately identify and quantify small RNA:target interactions at scale. MicroRNAs (miRNA) are a major class of small RNAs in plants and animals. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments in chimeric CrossLinking and ImmunoPrecipitation (CLIP) provides a direct readout of miRNA targets with high-throughput sequencing. Despite the power of this approach, widespread adoption of chimeric CLIP has been slow due to both methodological technical complexity as well as limited recovery of chimeric molecules (particularly beyond the most abundant miRNAs). Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery. We show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal- to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further demonstrate that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeting by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP-identified targets and indicated that probe-enriched chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and for revealing a deeper picture of regulatory networks for specific miRNAs of biological interest.

HighlightsO_LINo-gel chimeric eCLIP improves recovery of miRNA:mRNA interactions by 70-fold
C_LIO_LIProbe- and PCR-enrichment deeply profiles mRNA targets of miRNAs of interest
C_LIO_LIChimeric eCLIP targets experimentally identify non-computationally predicted interactions
C_LIO_LIIncreased depth recovers [~]6 million miRNA:target chimeras in HEK293T
C_LI

category: genomics


title: Genomic profiling of HIV-1 integration in microglia links viral insertions to TAD organization

abstract: HIV-1 persists in anatomically distinct cellular and tissue reservoirs as a stably integrated provirus that is a major barrier to HIV-1 cure. Proviral insertions are largely characterized in blood cells, while HIV-1 integration patterns remain unexplored in microglia, the major brain reservoir. Here, we employ genomics approaches to obtain the first HIV-1 integration site (IS) profiling in microglia and perform in-depth analysis of transcriptome, specific histone signatures and chromatin accessibility on different genomic scales. We show that HIV-1 follows genic insertion patterns into introns of actively transcribed genes, characteristic of blood reservoirs. HIV-1 insertional hotspot analysis by non-negative matrix factorization (NMF)-based approach clusters IS signatures with genic- and super-enhancers. Chromatin accessibility transcription factor (TF) footprints reveal that increased CTCF binding marks latently infected microglia compared to productively infected one. We identify CTCF-enriched topologically associated domain (TAD) borders with signatures of active chromatin as a neighborhood for HIV-1 integration in microglia and CD4+ T cells. Our findings further strengthen the notion that HIV-1 follows the patterns of host cell genome organization to integrate and to establish the silent proviral state and reveal that these principles are largely conserved in different anatomical latent reservoirs.


title: Constitutively active STAT5b feminizes mouse liver gene expression

abstract: STAT5 is an essential transcriptional regulator of the sex-biased actions of growth hormone (GH) in the liver. Delivery of constitutively active STAT5 (STAT5CA) to male mouse liver using an engineered adeno-associated virus with high tropism for the liver is shown to induce widespread feminization of the liver, with extensive induction of female-biased genes and repression of male-biased genes, largely mimicking results obtained when male mice are given GH as a continuous infusion. Many of the gene expression changes observed were associated with STAT5 binding to liver chromatin, supporting the proposed direct role of persistently active STAT5 in continuous GH-induced liver feminization. The feminizing effects of STAT5CA were dose-dependent; moreover, at higher levels, overexpression of STAT5CA resulted in some histopathology not seen in continuous GH-infused male liver, including hepatocyte hyperplasia and distorted liver architecture. These findings establish that the persistent activation of STAT5 by GH that characterizes female liver is by itself sufficient to account for the female-biased expression of a majority of female-biased genes. Moreover, histological changes seen when STAT5CA is overexpressed highlight the importance of carefully evaluating such effects before considering such STAT5 derivatives for therapeutic use in treating liver disease.


title: Genome assembly of the numbat (Myrmecobius fasciatus), the only termitivorous marsupial

abstract: The numbat (Myrmecobius fasciatus) is a critically endangered Australian marsupial, and the last surviving member of the Myrmecobiidae family. The numbat regularly undergoes torpor and is unique amongst marsupials as it is the only diurnal and termitivorous species. Here we sequenced the first draft genome of the numbat using 10x Genomics chromium linked-read technology, resulting in a 3.42 Gbp genome with a scaffold N50 of 223 Kbp. A global transcriptome from liver, lung and tongue was also generated to aid genome annotation with Fgenesh++, identifying 21,465 protein-coding genes and 78.7% complete mammalian BUSCOs. To investigate adaptation to the numbats termitivorous diet and arid/semi-arid range, we interrogated the most highly expressed transcripts within the tongue and manually annotated taste, vomeronasal and aquaporin gene families. Antimicrobial proteins and proteins involved in digestion were highly expressed in the tongue, as expected. Similarly, umami taste receptors were also expressed in the tongue, however sweet taste receptors were not expressed in this tissue. This pattern of taste receptor expression, combined with the putative contraction of the bitter taste receptor gene repertoire in the numbat genome, may indicate a potential evolutionary adaptation to their specialized termitivorous diet. Vomeronasal and aquaporin gene repertoires were similar to other marsupials and have not undergone expansion or contraction. The draft numbat genome is a valuable tool for conservation and can be applied to population genetics/genomics studies and to investigate the unique biology of this interesting species.


title: Climate Change Impacts on Broussenetia papyrifera Pollens - Metabolome Investigations and Prospects of Allergy Prevalence

abstract: Broussonetia papyrifera (B. papyrifera) is an allergenic plant in the mulberry family that grows at varied elevations and climatic conditions worldwide. In northern Pakistan, B. papyrifera is abundant and it produces a substantial amount of pollen that disperses in the air causing allergies in some humans. Climate change affects pollen production. To investigate potential changes in pollens development and potential allergenicity, B. papyrifera pollens were collected in summer and in spring from different regions in Pakistan. Study samples were subjected to morphological analysis, Fourier Transform Infrared Spectroscopy (FTIR) analysis for biochemical differences, and Liquid Chromatography-Mass Spectrometry (LCMS) for metabolome analysis. Morphological studies of the dried pollen by light microscopy showed seasonal and regional differences in pollens size and exine morphology. FTIR analysis showed inter-regional and inter-seasonal differences in the metabolome of the pollen. Differences in lipid and protein functional groups of pollen from different regions showed variation in the FTIR spectra. These differences in FTIR spectra correlated with the changing climatic conditions. Metabolome analysis of targeted pollen samples identified 33 organic compounds of seven different groups. Four unsaturated fatty acids were identified that have a potential role in allergic responses. The findings in this study are unique in demonstrating climatic variables that effect B. papyrifera pollen physiology (FTIR analysis) which also confirms differences in pollen-associated lipid metabolites identified by LCMS analysis. These results demonstrate information that may be used to predict potential changes in allergy risks from pollens of B. papyrifera in the future. The findings may provide a model for predicting variation in pollen structure and associated allergies in response to climate changes for other species.

category: scientific communication and education


title: Functional characterization of a neuropeptide receptor exogenously expressed in Aplysia neurons

abstract: Neuropeptides act mostly on a class of G-protein coupled receptors, and play a fundamental role in the functions of neural circuits underlying behaviors. However, the functions of neuropeptide receptors are poorly understood. Here, we used the mollusc model system Aplysia and microinjected the exogenous neuropeptide receptor apATRPR (Aplysia Allatotropin-related peptide receptor) with an expression vector (pNEX3) into Aplysia neurons that did not express the receptor. Physiological experiments demonstrated that apATRPR could mediate the excitability increase activated by its ligand, apATRP (Aplysia Allatotropin-related peptide), in the Aplysia neurons that now express the receptor. This study provides the first definitive evidence for a physiological function of a neuropeptide receptor in molluscan animals.


title: Acai (Euterpe oleracea Mart) modulates oxidative stress and inflammation by NF-κB inactivation and Nrf2 up-regulation in experimental diabetes

abstract: To evaluate the effects of acai extract (EA) on oxidative stress and inflammation induced by high glucose in cultured mouse immortalized mesangial cells (MiMC) and diabetic rats. MiMC cell viability and proliferation were determined by MTT. Extracellular and intracellular nitric oxide (NO) and intracellular ROS were also measured. The cell proteins were extracted for analysis of catalase, Nrf2, p-Nrf2, SOD-1, SOD-2, iNOS, NF-{kappa}B, p-NF-{kappa}B and TNF- expression. Male, adult Wistar rats were distributed into 3 groups: control (CTL) and diabetic (DM) rats who received water and DMEA and received 1 mL/day EA (200 mg/kg) via gavage for 8 consecutive weeks. After treatment with EA, metabolic profile, renal function and thiobarbituric acid reactives substances (TBARS) levels were evaluated, and kidneys were collected for qualitative histological analysis. EA maintained cell viability above 90% in all groups; it decreased proliferation in the HG group, both significant. NO levels, ROS generation, iNOS, NF-{kappa}B, p-NF-{kappa}B and TNF- expression were reduced significantly after 72 h of EA treatment, with significant increases for all antioxidants studied. DMEA vs DM showed a significant increase in body weight, improved kidney function and reduced TBARS excretion. EA treatment decreased proliferation, oxidative stress and inflammation in MiMC, and although acai did not decrease fasting glucose, it recovered the body weight and delayed the decline of renal function in the diabetic animals, suppressing the signaling of inflammatory mediators via NF-{kappa}B inactivation and increasing all antioxidants studied by upregulating the Nrf2 response pathway.