title: Macrophages regulate gastrointestinal motility through complement component 1q

abstract: Peristaltic movement of the intestine propels food down the length of the gastrointestinal tract to promote nutrient absorption. Interactions between intestinal macrophages and the enteric nervous system regulate gastrointestinal motility, yet we have an incomplete understanding of the molecular mediators of this crosstalk. Here we identify complement component 1q (C1q) as a macrophage product that regulates gut motility. Macrophages were the predominant source of C1q in the mouse intestine and most extraintestinal tissues. Although C1q mediates complement-mediated killing of bacteria in the bloodstream, we found that C1q was not essential for immune defense of the intestine. Instead, C1q-expressing macrophages were localized to the intestinal submucosal plexus where they closely associated with enteric neurons and expressed surface markers characteristic of nerve-adjacent macrophages in other tissues. Mice with a macrophage-specific deletion of C1qa showed changes in enteric neuronal gene expression, increased peristaltic activity, and accelerated intestinal transit. Our findings identify C1q as a key regulator of gastrointestinal motility and provide enhanced insight into the crosstalk between macrophages and the enteric nervous system.


title: T-lymphocyte Tyrosine Hydroxylase Regulates TH	17 T-lymphocytes during Repeated Social Defeat Stress

abstract: Posttraumatic stress disorder (PTSD) is a debilitating psychiatric disorder which results in deleterious changes to psychological and physical health. Patients with PTSD are especially susceptible to co-morbid inflammation-driven pathologies, such as autoimmunity, while also demonstrating increased T-helper 17 (TH17) lymphocyte-driven inflammation. While the exact mechanism of this increased inflammation is unknown, overactivity of the sympathetic nervous system is a hallmark of PTSD. Neurotransmitters of the sympathetic nervous system (i.e., catecholamines) can alter T-lymphocyte function, which we have previously demonstrated to be partially mitochondrial redox-mediated. Furthermore, we have previously elucidated that T-lymphocytes generate their own catecholamines, and strong associations exist between tyrosine hydroxylase (TH; the rate-limiting enzyme in the synthesis of catecholamines) and pro-inflammatory interleukin 17A (IL-17A) expression within purified T-lymphocytes in a preclinical rodent model of PTSD. Therefore, we hypothesized that T-lymphocyte-generated catecholamines drive TH17 T-lymphocyte polarization through a mitochondrial superoxide-dependent mechanism during psychological trauma. To test this, T-lymphocyte-specific TH knockout mice (THT-KO) were subjected to repeated social defeat stress (RSDS). RSDS characteristically increased tumor necrosis factor- (TNF), IL-6, IL-17A, and IL-22, however, IL-17A and IL-22 (TH17 produced cytokines) were selectively attenuated in circulation and in T-lymphocytes of THT-KO animals. When activated ex vivo, secretion of IL-17A and IL-22 by THT-KO T-lymphocytes was also found to be reduced, but could be partially rescued with supplementation of norepinephrine specifically. Interestingly, THT-KO T-lymphocytes were still able to polarize to TH17 under exogenous polarizing conditions. Last, contrary to our hypothesis, we found RSDS-exposed THT-KO T-lymphocytes still displayed elevated mitochondrial superoxide, suggesting increased mitochondrial superoxide is upstream of T-lymphocyte TH induction, activity, and TH17 regulation. Overall, these data demonstrate TH in T-lymphocytes plays a critical role in RSDS-induced TH17 T-lymphocytes and offer a previously undescribed regulator of inflammation in RSDS.

category: immunology


title: Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity

abstract: Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an enzyme that mediates N-terminal modification of several subtrates, including the monocyte-chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody blockade of the receptor CSF1R, which more broadly eliminates tissue macrophages, reversed tumor growth inhibition in Qpctl-/- mice, and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.


title: Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

abstract:
RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splicing junctions from RNA-seq identified hundreds of novel viral RNA splicing events, including the splicing events spanning large parts of the viral genome and production of vIRF4 circRNAs. We found that the 57KO alters the viral RNA splicing efficiency of affected RNA splicing events. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with multiple exons and introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF70-K3 RNA bears two introns, of which the first intron is positioned in the ORF70 coding region as an alternative intron and the second intron in the intergenic region as a constitutive intron. In WT cells expressing ORF57, most ORF70-K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out without affecting the splicing efficiency of the second intron. Using a minigene comprising of ORF70-K3 locus, we further confirmed ORF57 regulation of ORF70-K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70-K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.

Author summaryLike other herpesviruses, the expression of KSHV genes is in a tightly regulated manner during viral infection. The contribution of RNA splicing in the regulation of KSHV gene expression was extensively investigated in this report. We found that approximately one-third of all viral gene transcripts are spliced, with many undergoing RNA alternative splicing. We identified a subset of viral RNA splicing events susceptible to the regulation of viral RNA-binding protein ORF57. This led to the discovery of ORF57-mediated regulation of the bicistronic ORF70-K3 RNA splicing and production of viral thymidylate synthase ORF70 and viral E3-ubiquitin ligase K3. Such regulation allows timely but mutually exclusive expression of these two viral proteins to execute their unique roles in immune evasion and viral genome replication during the KSHV lytic cycle. Thus, these data provide new insight into KSHV biology. Finally, our study represents a blueprint for RNA splicing studies of other pathogens in infected cells.

category: microbiology


title: PfD123 modulates K13-mediated survival and recovery after artemisinin exposure.

abstract: Recent advances in curbing the deadly toll of malaria have been threatened by the emergence of parasites resistant to the front-line antimalarial artemisinin. Resistance is mediated by point-mutations in the parasite protein Kelch13, but the mechanism of resistance is multi-factorial and only partially understood. Resistance-conferring Kelch13 mutations have been shown to lead to low-level activation of the parasites integrated stress response (ISR) which has a protective effect against artemisinin through an unclear mechanism. Furthermore, only a subpopulation of resistant parasites ever survives drug exposure, implying an underlying heterogeneity. By applying scRNAseq to the resistance-relevant early ring stage, we found expansion of a subpopulation in Kelch13 mutant parasites that is chiefly characterized by transcription of the putative positive translational regulator D123, while we conversely observed reduced D123 protein levels at the same stage. Analogous inverse changes in D123 expression are produced by experimental activation of the ISR, and genetically manipulating D123 expression modulates sensitivity to artemisinin, establishing it as a stress-responsive gene that contributes to artemisinin resistance in Kelch13-mutant malaria parasites.


title: DNA uptake by cell wall-deficient bacteria reveals a putative ancient macromolecule uptake mechanism

abstract: Horizontal gene transfer in bacteria is widely believed to occur via three main mechanisms: conjugation, transduction and transformation. These mechanisms facilitate the passage of DNA across the protective cell wall using sophisticated machinery. We present here a new mechanism of DNA uptake that is independent of canonical DNA uptake machineries and is used by bacteria that live without a cell wall. We show that the cell wall-deficient bacteria engulf extracellular material, whereby intracellular vesicles are formed, and DNA is internalized. This mechanism is not specific to DNA, and allows uptake of other macromolecules and even 125 nm lipid nanoparticles (LNPs). Uptake was prevented by molecules known to inhibit eukaryotic endocytosis, suggesting this to be an energy-dependent process. Given that cell wall-deficient bacteria are considered a model for early life forms, our work provides a possible mechanism for primordial cells to acquire new genetic material or food before invention of the bacterial cell wall.


title: Transcriptomic and phenomic investigations reveal elements in biofilm repression and formation in the cyanobacterium Synechococcus elongatus PCC 7942

abstract: Biofilm formation by photosynthetic organisms is a complex behavior that serves multiple functions in the environment. Biofilm formation in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 is regulated in part by a set of small secreted proteins that promotes biofilm formation and a self-suppression mechanism that prevents their expression. Little is known about the regulatory and structural components of the biofilms in PCC 7942, or response to the suppressor signal(s). We performed transcriptomics (RNA-Seq) and phenomics (RB-TnSeq) screens that identified four genes involved in biofilm formation and regulation, more than 25 additional candidates that may impact biofilm formation, and revealed the transcriptomic adaptation to the biofilm state. In so doing, we compared the effectiveness of these two approaches for gene discovery.


title: Fe(III)-dependent anaerobic methane-oxidizing activity in a deep underground borehole demonstrated by in-situ pressure groundwater incubation

abstract: The family Methanoperedenaceae archaea mediate anaerobic oxidation of methane (AOM) in various terrestrial environments. In this study, we newly developed a high-pressure laboratory incubation system by controlling hydraulic pressure ranging from ambient to 5 MPa. Using the system, we investigated groundwater from 214- and 249-m deep boreholes at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Methanoperedenaceae archaea have been revealed by genome-resolved metagenomics, respectively. We incubated the groundwater samples amended with or without amorphous Fe(III) as an electron acceptor and 13C-labelled methane at an in-situ pressure of 1.6 MPa. After three to seven-day incubation, AOM activities were not detected from the 249-m deep groundwater but from the 214-m deep groundwater. The AOM rates were 93.7 {+/-} 40.6 and 27.7 {+/-} 37.5 nM/day with and without Fe(III) amendment. To clarify the differences in AOM activity between the 214- and 249-m deep groundwater samples, we characterized Fe(III) contents in suspended particulates collected by filtration. The particulates were not visible in the 249-m deep groundwater on the filter, while they were abundant and contained Fe(III)-bearing phyllosilicates in the 214-m deep groundwater. These results support the in-situ activity of Fe(III)-dependent AOM in the deep subsurface borehole.


title: Development of the First Tractable Genetic System for Parvimonas micra, a Ubiquitous Pathobiont in Human Dysbiotic Disease

abstract: Parvimonas micra is a Gram-positive obligate anaerobe and a typical member of the human microbiome. P. micra is among the most highly enriched species at numerous sites of mucosal dysbiotic disease and is closely associated with the development of multiple types of malignant tumors. Despite its strong association with disease, surprisingly little is known about P. micra pathobiology, which is directly attributable to its longstanding genetic intractability. To address this problem, we directly isolated a collection of P. micra strains from odontogenic abscess clinical specimens and then screened these isolates for natural competence. Amazingly, all of the P. micra clinical isolates exhibited various levels of natural competence, including the reference strain ATCC 33270. By exploiting this ability, we were able to employ cloning-independent methodologies to engineer and complement a variety of targeted chromosomal genetic mutations directly within low passage clinical isolates. To create the first P. micra genetic system, we employed renilla-based bioluminescence for highly sensitive reporter studies. This reporter system was then applied for the development of the novel Theo+ theophylline-inducible riboswitch for tunable gene expression studies over a broad dynamic range. Finally, we demonstrate the feasibility of generating Mariner-based Tn-seq libraries for forward genetic screening in P. micra. With the availability of a highly efficient transformation protocol and the current suite of genetic tools, P. micra should now be considered as a fully genetically tractable organism suitable for molecular genetic research. The methods presented here provide a clear path to investigate the understudied role of P. micra in polymicrobial infections and tumorigenesis.


title: Cross-linked natural IntegroPectin films from Citrus biowaste with intrinsic antimicrobial activity

abstract: Pectin recovered via hydrodynamic cavitation (IntegroPectin) from lemon and grapefruit agri-food waste intrinsically containing antimicrobial bioactive substances (flavonoids, phenolic acids, terpenes, and terpenoids) was used to generate innovative and eco-compatible films that efficiently inhibit the growth of Gram-negative pathogens. Extensive characterization of films confirmed the presence of these substances, which differently interact with the polysaccharide polymer (pectin), plasticizer (glycerol), surfactant (Tween 60), and cross-linker (Ca2+), conferring to these films a unique structure. Besides, IntegroPectin-based films constitute sustained, controlled, and slow-release (up to 72h) systems for bioactive substances in an aqueous environment. This feature is crucial for the good in vitro antimicrobial activity exerted by IntegroPectin films against three Gram-negative bacteria (two indicator pathogen strains Pseudomonas aeruginosa ATCC 10145, P. aeruginosa PAO1, and the clinical isolate Klebsiella pneumoniae) that are involved in the global emergence of the antimicrobial resistance.


title: Micro-colony observation: A rapid and simple approach to count bacterial colony forming units

abstract: For enumerating viable bacteria, traditional dilution plating to count colony forming units (CFU) has always been the preferred method in microbiology owing to its simplicity, albeit laborious and time-consuming. Similar CFU counts can be obtained by quantifying growing microcolonies in conjunction with the perks of a microscope. Here, we employed a simple method of five microliter spotting of differently diluted bacterial culture multiples times on a single petri plate followed by finding out CFU by counting microcolonies using phase contrast microscope. In this method within four-hour period CFU of an Escherichia coli culture can be found out. Further, within ten-hour period, CFU in a culture of Ralstonia solanacearum, a bacterium with generation time around 3 h, can be estimated. The CFU number determined by microcolonies observed is comparable with that obtained by the dilution plating method. Microcolonies number observed in the early hours of growth (2 h in case of E. coli and 8 h in case of R. solanacearum) were found to remain consistent at later h, though there was noticeable increase in size of the colonies. It suggested that microcolonies observed in the early hours indeed represent the bacterial number in the culture. Though manual, the method is less labor intensive apart from being simple, easy, economical and rapid.


title: Interconversion between Serum Amyloid A native and fibril conformations

abstract: Overexpression of Serum Amyloid A (SAA) can lead to a form of amyloidosis where the fibrils are made of SAA fragments, most often SAA1-76. Using Replica-Exchange-with-Tunneling, we study the conversion of a SAA1-76 chain between a the folded conformation and a fibril conformation. We find that the basins in the free energy landscape corresponding to the two motifs are separated by barriers of only about 2-3 kBT. Crucial for the assembly into the fibril structure is the salt bridge 26E-34K that provides a scaffold for forming the fibril conformation.

category: molecular biology


title: Integrated pretraining with evolutionary information to improve RNA secondary structure prediction

abstract: AO_SCPLOWBSTRACTC_SCPLOWRNA secondary structure prediction is a fundamental task in computational and molecular biology. While machine learning approaches in this area have been shown to improve upon traditional RNA folding algorithms, performance remains limited for several reasons such as the small number of experimentally determined RNA structures and suboptimal use of evolutionary information. To address these challenges, we introduce a practical and effective pretraining strategy that enables learning from a larger set of RNA sequences with computationally predicted structures and in the meantime, tapping into the rich evolutionary information available in databases such as Rfam. Coupled with a flexible and scalable neural architecture that can navigate different learning scenarios while providing ease of integrating evolutionary information, our approach significantly improves upon state-of-the-art across a range of benchmarks, including both single sequence and alignment based structure prediction tasks, with particularly notable benefits on new, less well-studied RNA families.


title: Sex-Specific Genes Identified in Sea Urchin Gonads are Expressed Prior to Metamorphosis

abstract:
A great collaboration between the germline and somatic cells of an organism is the creation of a functional gonad. Here we begin to test this mechanism in a sea urchin by use of RNA-seq, and quantitative mRNA measurements throughout the life cycle of the sea urchin, Lytechinus variegatus (Lv). We found through de novo analyses that the ovary and testis of this echinoderm contains the many transcripts predicted for gamete morphology and function, but we also discovered many uncharacterized gene products unique to each gonad. We found that transcripts involved in glycolysis are highly enriched in the ovary compared to the testis, and that Lv has an expansion of nanos genes not observed in other sequenced echinoderms. We developed a pipeline integrating timepoint RNA-seq data throughout development to identify hallmark gene expression in gonads. We found activation of meiotic genes surprisingly early in development, and observed that candidate genes involved in sex determination are first expressed during larval growth, well before metamorphosis. We further discovered that individual larvae express varying levels of male- or female-hallmark genes before metamorphosis, including factors for the germ line, oocyte, sperm, and meiosis related genes. Together these data support the hypothesis that sex determination in the sea urchin is initiated prior to metamorphosis, from a shared profile of male and female factors, and that the many uncharacterized genes unique to the gonads may reveal unique pathways and mechanisms in echinoderm reproduction.

HighlightO_LITestis and Ovary of the sea urchin, Lytechinus variegatus, have distinct transcriptome profiles
C_LIO_LIL. variegatus gonad RNA-seq reveals the expression of 9 nanos genes
C_LIO_LIThe ovary in the sea urchin uniquely expresses genes of the glycolytic pathway, including an ovary-specific transcript of GAPDH
C_LIO_LIGenes involved in meiosis and in sex determination are expressed early in larval development
C_LIO_LIFrom gene expression profiles we conclude here that sex determination is initiated prior to metamorphosis in this echinoderm
C_LI

category: developmental biology


title: Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda)

abstract: Biological aging is connected to life history variation across ecological scales, as well as informing a basic understanding of age-related declines to organismal function. Altered DNA methylation dynamics are a conserved aspect of biological aging and have recently been modeled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic aging, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe aging in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 8% of its lifespan. Although patterns of CHH methylation showed little association with age, both CpG and CHG methylation contexts were strongly associated with aging, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological aging in plants.


title: KLF5-regulated extracellular matrix remodeling secures biliary epithelial tissue integrity against cholestatic liver injury

abstract: Abstract / Introductory paragraphTubular epithelial tissues in the body play fundamental roles as infrastructure constituting conduits to transport various types of biological fluids, for which contiguous and integrated epithelial tissue structures should be maintained continuously and even under stressed conditions. Compared to tissue morphological processes that take place during ontogeny, the mechanisms whereby tubular epithelial tissues maintain their structural integrity in adulthood remains largely unclear. Here, we show that the transcription factor Klf5 is crucial for maintaining the biliary epithelial integrity in tissue remodeling processes induced under cholestatic injury conditions in the adult liver. Loss of Klf5 in the biliary epithelia led to tissue collapse in vivo in injured mouse livers, as well as in vitro in bile ductular organoids in a tissue-autonomous manner and independent of cell proliferation. Klf5 regulated cell junction organization and cell adhesion, along with extracellular matrix remodeling around the expanding biliary epithelia through deposition of Lamb3-containing laminin complexes. Targeting the Lamb3 expression in biliary epithelia in mice recapitulated the tissue collapse phenotype. Together, our results highlight a novel mechanism whereby the epithelial tissue maintains its integrity while undergoing unstable structural transformation.


title: Modeling Pulse Dynamics of Juvenile Fish Enables the Short-term Forecasting of Population Dynamics in Japanese Pufferfish: A Latent Variable Approach

abstract: The time lag between data collection and management implementation is a source of uncertainty and bias in the calculation of acceptable biological catch. Here, we developed a method to use small-scale survey data for juvenile Japanese pufferfish (Takifugu rubripes) to shorten this time lag and achieve accurate short-term forecasting. A survey of juvenile fish at a local sandy beach in Ise-Mikawa Bay, Japan provides data for the strength of year classes before fisheries recruitment; however, it is difficult to use the raw data owing to the small sample size and large observation errors. We found that a random-effect model overcame these problems and more accurately predicted pulse patterns of catch rates to derive a standardized recruitment index compared with a fixed-effect model. We then showed that a stock assessment model using the standardized recruitment index from the random-effect model outperformed models without the standardized recruitment index with respect to retrospective forecasting ability. This study highlights the applicability of a latent-variable approach for standardizing small-scale survey data and thereby for unbiased forecasting of short-term fish dynamics.

category: ecology


title: When host populations move north, but disease moves south: counter-intuitive impacts of climate warming on disease spread

abstract: Empirical observations and mathematical models show that climate warming can lead to the northern (or, more generally, poleward) spread of host species ranges and their corresponding diseases. Here, we explore an unexpected possibility whereby climate warming induces disease spread in the opposite direction to the directional shift in the host species range. To test our hypothesis, we formulate a reaction-diffusion equation model with a Susceptible-Infected (SI) epidemiological structure for two host species, both susceptible to a disease, but spatially isolated due to distinct thermal niches, and where prior to climate warming the disease is endemic in the northern species only. Previous theoretical results show that species distributions can lag behind species thermal niches when climate warming occurs. As such, we find that climate warming, by shifting both species niches forward, may increase the overlap between northern and southern host species ranges, due to the northern species lagging behind its thermal tolerance limit, thus facilitating a southern disease spread. As our model is general, our findings may apply to viral, bacterial, and prion diseases that do not have thermal tolerance limits and are inextricably linked to their hosts distributions, such as the spread of rabies from arctic to red foxes.


title: Eco-evolutionary feedbacks in the human gut microbiome

abstract: Gut microbiota can evolve within their hosts on human-relevant timescales. Yet little is known about how these changes influence (or are influenced by) the composition of their surrounding community. Here, by combining ecological and evolutionary analyses of a large cohort of human gut metagenomes, we show that the short-term evolution of the microbiota is linked with shifts in its ecological structure. These correlations are not simply explained by expansions of the evolving species, and often involve additional fluctuations in distantly related taxa. We show that similar feedbacks naturally emerge in simple resource competition models, even in the absence of cross-feeding or predation. These results suggest that the structure and function of host microbiota may be shaped by their local evolutionary history, with potential implications for personalized medicine and disease.

category: evolutionary biology


title: Sex-specific transcription and DNA methylation landscapes of the Asian citrus psyllid, a vector of huanglongbing pathogens.

abstract: The relationship of DNA methylation and sex-biased gene expression is of high interest, it allows research into mechanisms of sexual dimorphism and the development of potential novel strategies for insect pest control. The Asian citrus psyllid, Diaphorina citri Kuwayama, is a major vector for the causative agents of Huanglongbing (HLB), which presents an unparalleled challenge to citrus production worldwide. Here, we identify the X chromosome of D. citri and investigate differences in the transcription and DNA methylation landscapes between adult virgin males and females. We find a large number of male-biased genes on the autosomes and a depletion of such on the X chromosome. We have also characterised the methylome of D. citri, finding low genome-wide levels, which is unusual for an hemipteran species, as well as evidence for both promoter and TE methylation. Overall, DNA methylation profiles are similar between the sexes but with a small number of differentially methylated genes found to be involved in sex differentiation. There also appears to be no direct relationship between differential DNA methylation and differential gene expression. Our findings lay the groundwork for the development of novel epigenetic-based pest control methods, and given the similarity of the D. citri methylome to other insect species, these methods could be applicable across agricultural insect pests.


title: Convergent recruitment of Amh as the sex determination gene in two lineages of stickleback fish

abstract: Sex chromosomes vary greatly in their age and levels of differentiation across the tree of life. This variation is largely due to the rates of sex chromosome turnover in different lineages; however, we still lack an explanation for why sex chromosomes are so conserved in some lineages (e.g. Mammals, Birds) but so labile in others (e.g. Fish, Amphibians). Here we add to the information on sex chromosomes in stickleback, a valuable model lineage for the study of sex chromosome evolution, by identifying the sex chromosome and a strong candidate for the master sex determination gene in the brook stickleback, Culaea inconstans. Using whole genome sequencing of wild-caught samples and a lab cross, we identify AmhY, a male specific duplication of the gene Amh, as the candidate master sex determination gene. AmhY resides on Chromosome 20 in C. inconstans and is likely a recent duplication, as both AmhY and the sex linked region of Chromosome 20 show little sequence divergence. Importantly, this duplicate AmhY represents the second independent duplication and recruitment of Amh as the sex determination gene in stickleback and the eighth example now known across teleosts. We discuss this convergence in the context of sex chromosome turnovers and the role that the Amh/AmhrII pathway, which is crucial for sex determination, may play in the evolution of sex chromosomes in teleosts.


title: Recombination, selection and the evolution of tandem gene arrays

abstract: Multi-gene families - immunity genes or sensory receptors, for instance - are often subject to diversifying selection. Allelic diversity may be favoured not only through balancing or frequency dependent selection at individual loci, but also by associating different alleles in multi copy gene families. Using a combination of analytical calculations and simulations, we explored a population genetic model of epistatic selection and unequal recombination, where a trade-off exists between the benefit of allelic diversity and the cost of copy abundance. Starting from the neutral case, where we showed that gene copy number is Gamma-distributed at equilibrium, we derived also mean and shape of the limiting distribution under selection. Considering a more general model which includes variable population size and population substructure, we explored by simulations mean fitness and some summary statistics of the copy number distribution. We determined the relative effects of selection, recombination and demographic parameters in maintaining allelic diversity and shaping mean fitness of a population. One way to control the variance of copy number is by lowering the rate of unequal recombination. Indeed, when encoding recombination by a rate modifier locus, we observe exactly this prediction. Finally, we analyzed the empirical copy number distribution of three genes in human and estimated recombination and selection parameters of our model.


title: Neanderthals did not likely consume Physcomitrium patens - a model moss species

abstract: This manuscript is discussing the method of Weyrich et al., 2017, "Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus". When studying the dietary profile of a Neanderthal specimen from El Sidron cave (Spain) by sequencing ancient DNA present in calcified dental plaque (calculus) the authors identified a wide-range of potential food sources, including woolly rhinoceros, mushrooms, pine nuts, and moss - namely the less-than-abundant model species Physcomitrium patens. We doubted that Neanderthals were actually eating P. patens. By analyzing the ancient DNA reads using different mapping methods, we show likely a misinterpretation based on the previously used methods. The probability of Neanderthals eating P. patens is the same as eating rice or tomato. However, neither crop was grown in Europe at the time Neanderthals thrived.


title: Phenotypic and transcriptomic responses of the sun- and shade-loving plants to sunlight and dim-light conditions

abstract:
Elucidating how plant species respond to variable light conditions is important to understanding the ecological adaptation to heterogeneous natural habitats. However, the phenotypic responses and gene regulatory network of shade-loving plants under distinct light conditions have remained largely unclear. In this study, we assessed the differences in phenotypic and transcriptomic responses between Arabidopsis (sun-loving) and Panax ginseng (shade-loving) to sunlight and dim-light conditions. Our results showed that, compared to the Arabidopsis, ginseng plants not only exhibited a lower degree of phenotypic plasticity in response to distinct light conditions, but also showed higher photosynthetic efficiency under dim-light conditions. Further time-course transcriptome profiling and gene structural analyses revealed that differentially transcriptional regulation together with increased copy number of the photosynthesis-related genes (i.e., electron transfer and carbon fixation) may improve the photosynthetic efficiency of ginseng plants under dim-light conditions. In contrast, the loss-function and inactivation of phytochrome-interacting factors are potentially associated with the observed low degree of phenotypic plasticity of ginseng plants under the changing light conditions. Our study provides new insights on how shade-loving plants respond to variable light conditions. Candidate genes related to shade adaptation in ginseng provide valuable genetic resources for future molecular breeding of high-density planting crops.

HighlightThe shade-loving species Panax ginseng possesses lower phenotypic plasticity under distinct light conditions and shows high photosynthesis efficiency under dim-light condition.


title: Single mutation makes Escherichia coli an insect mutualist

abstract: We report an experimental system in which Escherichia coli evolves into an insect mutualist. When the essential gut symbiont of the stinkbug Plautia stali was replaced by E. coli, a few survivor insects exhibited specific localization and vertical transmission of E. coli. Through trans-generational maintenance with P. stali, several hyper-mutating E. coli lines independently evolved hosts high adult emergence and improved body color. Such "mutualistic" E. coli lines exhibited independent mutations disrupting the carbon catabolite repression (CCR) global transcriptional regulator. Each of the mutations reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that the single CCR mutations instantly make E. coli an insect mutualist. Our discovery uncovers that evolution of elaborate mutualism can proceed more easily and rapidly than conventionally envisaged.


title: Integrating top-down and bottom-up approaches to understand the genetic architecture of speciation across a monkeyflower hybrid zone

abstract: Understanding the phenotypic and genetic architecture of reproductive isolation is a longstanding goal of speciation research. In many systems, candidate barrier traits and loci have been identified, but causal connections between them are rarely made. In this study, we combine  top-down and  bottom-up approaches with demographic modeling toward an integrated understanding of speciation across a monkeyflower hybrid zone. Previous work in this system suggests that pollinator-mediated reproductive isolation is a primary barrier to gene flow between two divergent red- and yellow-flowered ecotypes of Mimulus aurantiacus. Several candidate floral traits contributing to pollinator isolation have been identified, including a difference in flower color, which is caused primarily by a single large-effect locus (MaMyb2). Other anonymous SNP loci, potentially contributing to pollinator isolation, also have been identified, but their causal relationships remain untested. Here, we performed demographic analyses, which indicate that this hybrid zone formed by secondary contact, but that subsequent gene flow was restricted in a large fraction of the genome by barrier loci. Using a cline-based genome scan (our bottom-up approach), we demonstrate that candidate barrier loci are broadly distributed across the genome, rather than mapping to one or a few  islands of speciation. A QTL analysis (our top-down approach) revealed most floral traits are highly polygenic, with little evidence that QTL co-localize, indicating that most traits are largely genetically independent. Finally, we find little convincing evidence for the overlap of QTL and candidate barrier loci, suggesting that some loci contribute to other forms of reproductive isolation. Our findings highlight the challenges of understanding the genetic architecture of reproductive isolation and reveal that barriers to gene flow aside from pollinator isolation may play an important role in this system.

category: genetics


title: Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat

abstract: X-linked dystonia-parkinsonism (XDP) is a progressive adult-onset neurodegenerative disorder caused by insertion of a SINE-VNTR-Alu (SVA) retrotransposon in the TAF1 gene. The SVA retrotransposon contains a CCCTCT hexameric repeat tract of variable length, whose length is inversely correlated with age at onset. This places XDP in a broader class of repeat expansion diseases, characterized by the instability of their causative repeat mutations. Here, we observe similar inverse correlations between CCCTCT repeat length with age at onset and age at death and no obvious correlation with disease duration. To gain insight into repeat instability in XDP we performed comprehensive quantitative analyses of somatic instability of the XDP CCCTCT repeat in blood and in seventeen brain regions from affected males. Our findings reveal repeat length-dependent and expansion-based instability of the XDP CCCTCT repeat, with greater levels of expansion in brain than in blood. The brain exhibits regional-specific patterns of instability that are broadly similar across individuals, with cerebellum exhibiting low instability and cortical regions exhibiting relatively high instability. The spectrum of somatic instability in the brain includes a high proportion of moderate repeat length changes of up to 5 repeats, as well as expansions of [~]20->100 repeats and contractions of [~]20-40 repeats at lower frequencies. Comparison with HTT CAG repeat instability in postmortem Huntingtons disease brains reveals similar brain region-specific profiles, indicating common trans-acting factors that contribute to the instability of both repeats. Analyses in XDP brains of expansion of a different SVA-associated CCCTCT located in the LIPG gene, and not known to be disease-associated, reveals repeat length-dependent expansion at overall lower levels relative to the XDP CCCTCT repeat, suggesting that expansion propensity may be modified by local chromatin structure. Together, the data support a role for repeat length-dependent somatic expansion in the process(es) driving the onset of XDP and prompt further investigation into repeat dynamics and the relationship to disease.


title: De novo transcriptome of Taverniera cuneifolia (Roth) Ali.

abstract:
Taverniera cuneifolia has been described as a potent substitute of Licorice in India. It has been used as an expectorant, anti-inflammatory, anti-ulcer, wound healing, blood purifier etc. Glycyrrhizin is one of the most useful bioactive sesquiterpenoid present in this plant. The present study aim to carry out transcriptome analysis in root tissue of Taverniera cuneifolia to identify specific functional genes involved in the biosynthesis of secondary metabolites. The root transcriptome sequencing of Taverniera cuneifolia resulted in a total of ~7.29 Gb of raw data and generated 55,991,233 raw reads. The high quality reads were de novo assembled by Trinity assembler followed through CD-HIT resulted into 35,590 "Unigene" transcripts with an average size of 419 bp. The unigenes were analyzed using BLAST2GO resulted in 27,884 (78.35%) transcript with blast hits, 22,510 (63.25%) transcript with mapping and 21,066 (59.19%) transcript with annotation. Functional annotation was carried out using NCBIs non-redundant and Uniprot databases resulted in the identification of 21,066 (59.19%) annotated transcripts and GO assigned to 24751 (69.54%) transcripts. The gene ontology result shows maximum sequences match with Biological Processes (48%), Molecular Function (27%) and Cellular components (23%). A total of 289 metabolic enriched pathways were identified, which included pathways like Sesquiterpenoid and triterpenoid pathway which were involved in synthesis of secondary metabolite Glycyrrhizin biosynthesis. The enzymes, squalene monooxygenase, farnesyl-diphosphate farnesyltransferase, beta amyrin synthase, beta-amyrin 24-hydroxylase, were identified by functional annotation of transcriptome data. There were several other pathways like terpenoid backbone biosynthesis, steroid biosynthesis, Carotenoid biosynthesis, Flavonoids biosynthesis etc. which have been reported first time from this plant. Transcription factors were predicted by comparison with Plant Transcription Factor Database, and 1557 trancripts belonging to 85 trancription factor families were identified. This transcriptome analysis provided an important resource for future genomic studies in Taverniera cuneifolia, therefore representing basis in further investigation of the plant.

SignificanceLicorice (Glycyrrhiza glabra roots) is used as traditional Chinese herbal medicines in majority of formulations. Licorice is also used in Industries like food, herbal and cosmetics etc. due to its high demand in the market it is imported from foreign countries and is not available locally of superior quality (Liu et al., 2015). In India, Taverniera cuneifolia has been described as a potent substitute of Licorice, it has been quoted in ancient books like Charak Samhita during the Nigandu period (Kamboj, 2000) and Barda dungar ni Vanaspati ane upyog (Thaker 1910). It has been used as an expectorant, anti-inflammatory, anti-ulcer, wound healing, blood purifier etc. Transcriptomic studies will assist in understanding the basic molecular structure, function and organization of information within the genome of Taverniera cuniefolia. This study will help us to identify the key metabolites their expressions and genes responsible for their production.

category: genomics


title: DiSCs: Domains involving SETDB1 and Cohesin are critical regulators of genome topology and stem cell fate

abstract: SETDB1 is a key regulator of lineage-specific genes and endogenous retroviral elements (ERVs) through its deposition of repressive H3K9me3 mark. Apart from its H3K9me3 regulatory role, SETDB1 has seldom been studied in terms of its other potential regulatory roles. To investigate this, a genomic survey of SETDB1 binding in mouse embryonic stem cells across multiple libraries was conducted, leading to the unexpected discovery of regions bereft of common repressive histone marks (H3K9me3, H3K27me3). These regions were enriched with the CTCF motif that is often associated with the topological regulator Cohesin. Further profiling of these non-H3K9me3 regions led to the discovery of a cluster of non-repeat loci that were co-bound by SETDB1 and Cohesin. These regions, which we named DiSCs (Domains involving SETDB1 and Cohesin) were seen to be proximal to the gene promoters involved in embryonic stem cell pluripotency and lineage development. Importantly, it was found that SETDB1-Cohesin co-regulate target gene expression and genome topology at these DiSCs. Depletion of SETDB1 led to localized dysregulation of Cohesin binding thereby locally disrupting topological structures. Dysregulated gene expression trends revealed the importance of this cluster in ES cell maintenance as well as at gene  islands that drive differentiation to other lineages. The  unearthing of the DiSCs thus unravels a unique topological and transcriptional axis of control regulated chiefly by SETDB1.


title: Automated phenol-chloroform extraction of high molecular weight genomic DNA for use in long-read single-molecule sequencing.

abstract: In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institutes Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. We achieved routine extraction of high molecular weight genomic DNA (>100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run can be seen at http://doi.org/10.6084/m9.figshare.17097065


title: Identifying systematic variation at the single-cell level by leveraging low-resolution population-level data

abstract: A major limitation in single-cell genomics is a lack of ability to conduct cost-effective population-level studies. As a result, much of the current research in single-cell genomics focuses on biological processes that are broadly conserved across individuals, such as cellular organization and tissue development. This limitation prevents us from studying the etiology of experimental or clinical conditions that may be inconsistent across individuals owing to molecular variation and a wide range of effects in the population. In order to address this gap, we developed "kernel of integrated single cells" (Keris), a novel model-based framework to inform the analysis of single-cell gene expression data with population-level effects of a condition of interest. By inferring cell-type-specific moments and their variation across conditions using large tissue-level bulk data representing a population, Keris allows us to generate testable hypotheses at the single-cell level that would otherwise require collecting single-cell data from a large number of donors. Within the Keris framework, we show how the combination of low-resolution, large bulk data with small but high-resolution single-cell data enables the identification of changes in cell-subtype compositions and the characterization of subpopulations of cells that are affected by a condition of interest. Using Keris we estimate linear and non-linear age-associated changes in cell-type expression in large bulk peripheral blood mononuclear cells (PBMC) data. Combining with three independent single-cell PBMC datasets, we demonstrate that Keris can identify changes in cell-subtype composition with age and capture cell-type-specific subpopulations of senescent cells. This demonstrates the promise of enhancing single-cell data with population-level information to study compositional changes and to profile condition-affected subpopulations of cells, and provides a potential resource of targets for future clinical interventions.


title: Selective Ablation of 3' RNA ends and Processive RTs Facilitate Direct cDNA Sequencing of Full-length Host cell and Viral Transcripts

abstract: Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read-lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3 RNA ends (termed CASPR) which decreases ribosomal RNA reads and enriches for poly-adenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.


title: Selective Ablation of 3' RNA ends and Processive RTs Facilitate Direct cDNA Sequencing of Full-length Host Cell and Viral Transcripts


title: Age and experience affect the biosynthesis and emission of a Drosophila pheromone

abstract:
The most studied pheromone in Drosophila melanogaster, cis-Vaccenyl Acetate (cVA), is synthesized in the male ejaculatory bulb and transferred to the female during copulation. Combined with other chemicals cVA can modulate fly aggregation, courtship, mating and fighting. It is not detected on the cuticle of isolated males and is only released by males involved in social or sexual interaction. We explored the mechanisms underlying both cVA biosynthesis and emission in males of two wild types and a pheromonal mutant line. The effects of ageing, adult social interaction, and maternally-transmitted cVa and microbes -- both associated with the egg chorion-- on cVA biosynthesis and emission were measured. While ageing and genotype changed both biosynthesis and emission in similar ways, early developmental exposure to maternally-transmitted cVA and microbes strongly decreased cVA emission but not the biosynthesis of this molecule. This indicates that the release -- but not the biosynthesis -- of this sex pheromone strongly depends on early developmental context. The mechanism by which the preimaginal effects occur is unknown, but reinforces the significance of development in determining adult physiology and behaviour.

Summary statementWe show how the biosynthesis and release of a key Drosophila pheromone is affected by ageing, by early exposure to this pheromone and to microbes, and by social context.

category: animal behavior and cognition


title: Elucidating Heterogeneous Iron Biomineralization Patterns in a Denitrifying As(III)-Oxidizing Bacterium: Implications for Arsenic Immobilization

abstract: Anaerobic nitrate-dependent iron(II) oxidation is a process common to many bacterial species, which promotes the formation of Fe(III) minerals that can influence the fate of soil and groundwater pollutants, such as arsenic. Herein, we investigated simultaneous nitrate-dependent Fe(II) and As(III) oxidation by Acidovorax sp. strain ST3 with the aim of studying the Fe biominerals formed, their As immobilization capabilities and the metabolic effect on cells. X-ray powder diffraction (XRD) and scanning transmission electron microscopy (STEM) nanodiffraction were applied for biomineral characterization in bulk and at the nanoscale, respectively. NanoSIMS (nanoscale secondary ion mass spectrometry) was used to map the intra and extracellular As and Fe distribution at the single-cell level and to trace metabolically active cells, by incorporation of a 13C-labeled substrate (acetate). Metabolic heterogeneity among bacterial cells was detected, with periplasmic Fe mineral encrustation deleterious to cell metabolism. Interestingly, Fe and As were not co-localized in all cells, indicating delocalized sites of As(III) and Fe(II) oxidation. The Fe(III) minerals lepidocrocite and goethite were identified in XRD, although only lepidocrocite was identified via STEM nanodiffraction. Extracellular amorphous nanoparticles were formed earlier and retained more As(III/V) than crystalline "flakes" of lepidocrocite, indicating that longer incubation periods promote the formation of more crystalline minerals with lower As retention capabilities. Thus, the addition of nitrate promotes Fe(II) oxidation and formation of Fe(III) biominerals by ST3 cells which retain As(III/V), and although this process was metabolically detrimental to some cells, it warrants further examination as a viable mechanism for As removal in anoxic environments by biostimulation with nitrate.

category: biochemistry


title: Protein Refolding Guided by High-Throughput Differential Scanning Fluorimetry: a case study of an HtrA-Family Bacterial Protease

abstract:
The high temperature requirement A (HtrA) serine protease family presents an attractive target class with recent interest for antibacterial development. These proteins contain numerous binding sites and regulatory loops and display diverse oligomerization patterns that depend on substrate occupancy and type. HtrA proteins that are purified natively often coelute with peptides and activating species, shifting oligomerization and protein structure to different activated populations. Here, a redesigned approach to HtrA production by purifying the protein from inclusion bodies under denaturing conditions, and screening for optimal refolding buffer composition using biophysical techniques that are function-agnostic and dont rely on any target-specific readouts or assays is described. This systematic workflow will translate to the production of HtrA-family proteins in higher quantities and of purer composition than the current literature standard.

HighlightsO_LIBacterial HtrA proteins represent an attractive target for next-generation antibiotics discovery.
C_LIO_LINatively purified HtrA proteins coelute with activating peptides and other contaminants, shifting the tertiary and quaternary structure and impacting activation of the protein.
C_LIO_LIA denaturing purification of HtrA from inclusion bodies offers substantially improved yields while stripping protein of these contaminating species.
C_LIO_LIOur extended screening method for optimal refolding conditions using a commercially available buffer screen and high-throughput differential scanning fluorimetry assay allowed us to generate HtrA protein with high purity and high yield.
C_LI

Graphical Abstract

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title: RTEL1 and MCM10 overcome topological stress during vertebrate replication termination

abstract: Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress in Xenopus egg extracts. We found that the helicase RTEL1 and the replisome protein MCM10 were highly enriched on DNA under these conditions. We show that RTEL1 normally plays a minor role during fork convergence while the role of MCM10 is normally negligible. However, RTEL1 and MCM10 both become crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 exert non-additive effects on fork convergence and physically interact, suggesting that they function together. Furthermore, RTEL1 and MCM10 do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 appear to play a general role in promoting progression of stalled forks, including when forks stall during termination. Overall, our data identify an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.


title: Rapid deep widefield neuron finder driven by virtual calcium imaging data

abstract: Widefield microscope provides optical access to multi-millimeter fields of view and thousands of neurons in mammalian brains at video rate. However, calcium imaging at cellular resolution has been mostly contaminated by tissue scattering and background signals, making neuronal activities extraction challenging and time-consuming. Here we present a deep widefield neuron finder (DeepWonder), which is fueled by simulated calcium recordings but effectively works on experimental data with an order of magnitude faster speed and improved inference accuracy than traditional approaches. The efficient DeepWonder accomplished fifty-fold signal-to-background ratio enhancement in processing terabytes-scale cortex-wide recording, with over 14000 neurons extracted in 17 hours in workstation-grade computing resources compared to nearly week-long processing time with previous methods. DeepWonder circumvented the numerous computational resources and could serve as a guideline to massive data processing in widefield neuronal imaging.

category: bioengineering


title: Methacrylate-Modified Gold Nanoparticles Enable Non-Invasive Monitoring of Photocrosslinked Hydrogel Scaffolds

abstract: Photocrosslinked hydrogels, such as methacrylate-modified gelatin (gelMA) and hyaluronic acid (HAMA), are widely utilized as tissue engineering scaffolds and/or drug delivery vehicles, but lack a suitable means for non-invasive, longitudinal monitoring of surgical placement, biodegradation, and drug release. Therefore, we developed a novel photopolymerizable X-ray contrast agent, methacrylate-modified gold nanoparticles (AuMA NPs), to enable covalent-linking to methacrylate-modified hydrogels (gelMA and HAMA) in one-step during photocrosslinking and non-invasive monitoring by X-ray micro-computed tomography (micro-CT). Hydrogels exhibited a linear increase in X-ray attenuation with increased Au NP concentration to enable quantitative imaging by contrast-enhanced micro-CT. The enzymatic and hydrolytic degradation kinetics of gelMA-Au NP hydrogels were longitudinally monitored by micro-CT for up to one month in vitro, yielding results that were consistent with concurrent measurements by optical spectroscopy and gravimetric analysis. Importantly, AuMA NPs did not disrupt the hydrogel network, rheology, mechanical properties, and hydrolytic stability compared with gelMA alone. GelMA-Au NP hydrogels were thus able to be bioprinted into well-defined three-dimensional architectures supporting endothelial cell viability and growth. Overall, AuMA NPs enabled the preparation of both conventional photopolymerized hydrogels and bioprinted scaffolds with tunable X-ray contrast for noninvasive, longitudinal monitoring of placement, degradation, and NP release by micro-CT.


title: Power analysis for spatial omics

abstract: As spatially-resolved multiplex profiling of RNA and proteins becomes more prominent, it is increasingly important to understand the statistical power available to test specific hypotheses when designing and interpreting such experiments. Ideally, it would be possible to create an oracle that predicts sampling requirements for generalized spatial experiments. However, the unknown number of relevant spatial features and the complexity of spatial data analysis makes this challenging. Here, we enumerate multiple parameters of interest that should be considered in the design of a properly powered spatial omics. We introduce a method for tunable in silico tissue generation, and use it with spatial profiling datasets to construct an exploratory computational framework for single cell spatial power analysis. Finally, we demonstrate that our framework can be applied across diverse spatial data modalities and tissues of interest.

category: bioinformatics


title: The computational models of AlphaFold2 and RoseTTAfold carry protein foldability information

abstract:
Protein folding has been a "holy grail" problem of biology for fifty years, and the recent breakthrough from AlphaFold2 and RoseTTAfold set a profound milestone for solving this problem. AlphaFold2 and RoseTTAfold were successful in predicting protein structures from peptide sequences with high accuracy. Meanwhile, although the protein folding problem also cares about the kinetic pathways of protein folding, AlphaFold2 and RoseTTAfold were not trained with this functionality. Considering that their training sets contain proteins from foldable sequences and sequence evolutionary information, we wondered if the computational models from AlphaFold2 and RoseTTAfold might carry protein foldability information. To test this idea, we systematically predicted the structural models of 149 circular permutants and 148 alanine insertion mutants of the 149-residue dihydrofolate reductase of Escherichia coli with AlphaFold2 and RoseTTAfold. Our data showed that although AlphaFold2 and RoseTTAfold could not directly identify unfoldable proteins, the structural variations of computational models are correlated with protein foldability. Furthermore, this correlation is independent of secondary structures. Most importantly, the structural variations of computational models are quantitatively correlated with protein foldability but not protein function. Our work could be of great value to the design of circular permutants, the design of fragment complementary proteins, the design of novel proteins, and the development of computational tools for predicting protein folding kinetics.

HighlightsO_LIAlphaFold2 and RoseTTAfold could not directly identify unfoldable proteins.
C_LIO_LIThe structural variations of computational models are correlated with protein foldability.
C_LIO_LIThis correlation is independent of secondary structures.
C_LIO_LIThe structural variations of computational models are quantitatively correlated with protein foldability but not protein function.
C_LI


title: Accurate strain-level microbiome composition analysis from short reads

abstract: Because bacterial strains can exhibit different biological properties, strain-level composition analysis plays a vital role in understanding the functions and dynamics of microbial communities. Metagenomic sequencing has become the major means for probing the microbial composition in host-associated or environmental samples. Despite a plethora of composition analysis tools, they are not optimized to address the challenges in strain-level analysis: a reference database with highly similar reference strain genomes and the presence of multiple strains under one species in a sample. In this work, we present a new strain-level composition analysis tool named StrainScan that employs a novel tree-based k-mer indexing structure to strike a balance between the strain identification accuracy and the computational complexity. We rigorously tested StrainScan on many simulated and real sequencing data and benchmarked StrainScan with popular strain-level analysis tools including Krakenuniq, StrainSeeker, Pathoscope2, Sigma, StrainGE, and Strainest. The results show that StrainScan has higher accuracy and resolution than the the state-of-the-art tools on strain-level composition analysis. It improves the F1-score by 20% in identifying multiple strains with at least 99.89% average nucleotide identity. StrainScan takes short reads and a set of reference strains as input and its source codes are freely available at https://github.com/liaoherui/strainScan.


title: Design in the DARK: Learning Deep Generative Models for De Novo Protein Design

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe design of novel protein sequences is providing paths towards the development of novel therapeutics and materials. At the forefront is the challenging field of de novo protein design, which looks to design protein sequences unlike those found in nature using general design methodologies. In this work, we develop a tool for de novo design, based on a deep generative sequence model, that rapidly samples novel protein sequences with diverse and ordered structures. To build this tool we develop a framework, called DARK, that trains the underlying generative model on an iteratively expanding set of synthetic sequences. The resulting model generalizes where models trained on natural sequences struggle and greatly improves on the efficiency of comparable sampling-based approaches. We further show how it can generate high quality candidates for de novo design problems and aid in the development of further novel design methods, in all, providing another step, amongst others, towards truly automated and intelligent protein design.


title: Effective generation of tumor-infiltrating lymphocyte products from metastatic non-small-cell lung cancer (NSCLC) lesions irrespective of location and previous treatments.

abstract: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Because current treatment regimens show limited success rates, alternative therapeutic approaches are needed. We recently showed that treatment-naive, stage I/II primary NSCLC tumors contain a high percentage of tumor-reactive T cells, and that these tumor-reactive T cells can be effectively expanded and used for the generation of autologous TIL therapy. Whether these promising findings also hold true for metastatic lesions is unknown yet critical for the translation into the clinic. We here studied the lymphocytic composition and the feasibility to generate tumor-responsive TIL products from metastatic NSCLC lesions that were derived from different locations and from patients who had previously received different treatment regimens, or who were treatment naive. The overall numbers and composition of lymphocyte infiltrates from this various metastatic lesions was by and large comparable to that of early-stage primary NSCLC tumors. With the clinically approved TIL expansion protocol, we effectively expanded TILs from metastatic NSCLC lesions to numbers that were compatible with TIL transfusion, irrespective of the location of the metastasis and of the previous treatment. Importantly, 16 of 21 (76%) tested TIL products displayed anti-tumoral activity, as determined by their production of the key pro-inflammatory cytokines IFN-{gamma}, TNF, IL-2 and of CD137 expression upon incubation with autologous tumor digests by CD8+ and CD4+ T cells. In summary, metastatic NSCLC lesions constitute a viable source for the generation of tumor-reactive TIL products for therapeutic purposes irrespective of their location and the pretreatment regimens.

category: cancer biology


title: Steroidogenic factor-1 lineage origin of skin lesions in Carney complex syndrome

abstract: Carney complex (CNC) is a rare familial multi-neoplastic syndrome predisposing to endocrine and non-endocrine tumors due to inactivating mutations of PRKAR1A leading to perturbations of the cAMP protein kinase A (PKA) signaling pathway. Skin lesions are the most common manifestation of CNC, including lentigines, blue nevi and cutaneous myxomas, in unusual locations such as oral and genital mucosa. Unlike endocrine disorders, the pathogenesis of skin lesions remains unexplained. Here, we show that embryonic invalidation of the Prkar1a gene in Steroidogenic Factor-1-expressing cells, leads to the development of familial skin pigmentation alterations reminiscent of those in patients. Immunohistological and molecular analyses coupled with genetic monitoring of recombinant cell lineages in mouse skin, suggest that familial lentiginosis and myxomas occurs in skin areas specifically enriched in dermal melanocytes. In lentigines and blue nevi-prone areas from mutant mice and patients, Prkar1a/PRKAR1A invalidation occurs in a subset of dermal fibroblasts capable of inducing, under the influence of PKA signaling, the production of pro-melanogenic EDN3 and HGF signals. Our model strongly suggests that the origin of the typical CNC cutaneous lesions is the result of non-cell-autonomous pro-melanogenic activity of a dermal fibroblast population sharing a community of origin with SF-1 lineage.

category: pathology


title: Mindin is essential for cutaneous fibrogenesis in a new mouse model of systemic sclerosis

abstract: Fibrosis is a result of chronically activated fibroblasts leading to the overproduction of extracellular matrix (ECM), causing tissue hardening and loss of organ function. Systemic sclerosis (SSc) is a fibrotic skin disease marked by inflammation, autoimmunity and vasculopathy along with progressive fibrosis of the skin and internal organs. A major bottleneck in understanding the etiology of SSc has been the lack of a holistic animal model that can mimic the human SSc disease. We found that the transcription factor Snail is overexpressed in the epidermis of SSc patients and a transgenic mouse recapitulating this expression pattern is sufficient to induce hallmark clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin in fibrogenesis. Mindin is produced by Snail transgenic skin keratinocytes and aids fibrogenesis by inducing inflammatory cytokine and collagen production in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.


title: Multiple Synaptic Contacts combined with Dendritic Filtering enhance Spatio-Temporal Pattern Recognition capabilities of Single Neurons

abstract: A cortical neuron typically makes multiple synaptic contacts on the dendrites of a post-synaptic target neuron. The functional implications of this apparent redundancy are unclear. The dendritic location of a synaptic contact affects the time-course of the somatic post-synaptic potential (PSP) due to dendritic cable filtering. Consequently, a single pre-synaptic axonal spike results with a PSP composed of multiple temporal profiles. Here, we developed a "filter-and-fire" (F&F) neuron model that captures these features and show that the memory capacity of this neuron is threefold larger than that of a leaky integrate-and-fire (I&F) neuron, when trained to emit precisely timed output spikes for specific input patterns. Furthermore, the F&F neuron can learn to recognize spatio-temporal input patterns, e.g., MNIST digits, where the I&F model completely fails. Multiple synaptic contacts between pairs of cortical neurons are therefore an important feature rather than a bug and can serve to reduce axonal wiring requirements.

category: neuroscience


title: Acute Axon Damage and Demyelination are Mitigated by 4-Aminopyridine (4-AP) Therapy after Experimental Traumatic Brain Injury

abstract:
Damage to long axons in white matter tracts is a major pathology in closed head traumatic brain injury (TBI). Acute TBI treatments are needed that protect against axon damage and promote recovery of axon function to prevent long term symptoms and neurodegeneration. Our prior characterization of axon damage and demyelination after TBI led us to examine repurposing of 4-aminopyridine (4-AP), an FDA-approved inhibitor of voltage-gated potassium (Kv) channels. 4-AP is currently indicated to provide symptomatic relief for patients with chronic stage multiple sclerosis, which involves axon damage and demyelination. We tested clinically relevant dosage of 4-AP as an acute treatment for experimental TBI and found multiple benefits in corpus callosum axons. This randomized, controlled pre-clinical study focused on the first week after TBI, when axons are particularly vulnerable. 4-AP treatment initiated one day post-injury dramatically reduced axon damage detected by intra-axonal fluorescence accumulations in Thy1-YFP mice of both sexes. Detailed electron microscopy in C57BL/6 mice showed that 4-AP reduced pathological features of mitochondrial swelling, cytoskeletal disruption, and demyelination. Furthermore, 4-AP improved the molecular organization of axon nodal regions by restoring disrupted paranode domains and reducing Kv1.2 channel dispersion. 4-AP treatment did not resolve deficits in action potential conduction across the corpus callosum, based on ex vivo electrophysiological recordings at 7 days post-TBI. Thus, this first study of repurposing 4-AP as an acute treatment for TBI demonstrates pre-clinical efficacy in decreasing pathological hallmarks of axon damage. Studies beyond this acute phase are now warranted to assess functional utility and outcome trajectory.

SIGNIFICANCE STATEMENTTraumatic brain injury (TBI) is an acute injury that, if unresolved, can progress to cause persistent, debilitating symptoms. Currently, no treatments effectively prevent damage to long myelinated axons in white matter tracts, which is a hallmark pathology of TBI. 4-aminopyridine (4-AP) is FDA-approved to treat chronic symptoms in patients with multiple sclerosis, which involves autoimmune damage to myelinated axons. As the first assessment of repurposing 4-AP as an acute treatment for TBI, our randomized, controlled studies tested the hypothesis that low-dose 4-AP initiated one day after experimental TBI will reduce acute axon damage and demyelination. We found that 4-AP treatment significantly reduced the progression of axon pathology and demyelination during the first week after TBI using clinically relevant experimental conditions.


title: Role of posterodorsal medial amygdala kisspeptin and urocortin-3 in pubertal timing in female mice

abstract: Post-traumatic stress disorder impedes pubertal development and disrupts pulsatile LH secretion in humans and rodents. The posterodorsal sub-nucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, pubertal timing, as well as emotional processing and anxiety. Psychosocial stress exposure alters neuronal activity within the MePD increasing the expression of Urocortin3 (Ucn3) and its receptor corticotropin-releasing factor type-2 receptor (CRFR2) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. We test the hypothesis that psychosocial stress, processed by the MePD, is relayed to the hypothalamic GnRH pulse generator to delay puberty in female mice. We exposed C57Bl6/J female mice to the predator odor, 2,4,5-Trimethylthiazole (TMT), during pubertal transition and examined the effect on pubertal timing, pre-pubertal LH pulses and anxiety-like behaviour. Subsequently, we virally infected Ucn3-cre-tdTomato female mice with stimulatory DREADDs targeting MePD Ucn3 neurons and determined the effect on pubertal timing and pre-pubertal LH pulse frequency. Exposure to TMT during pubertal development delayed puberty, suppressed pre-pubertal LH pulsatility and enhanced anxiety-like behaviour, while activation of MePD Ucn3 neurons reduced LH pulse frequency and delayed puberty. Early psychosocial stress exposure decreases GnRH pulse generator frequency delaying puberty while inducing anxiety-behaviour in female mice, an effect potentially involving Ucn3 neurons in the MePD.


title: Role of posterodorsal medial amygdala urocortin-3 in pubertal timing in female mice


title: A Guide for Quantifying and Optimizing Measurement Reliability for the Study of Individual Differences

abstract: Characterizing individual variations is central to interpreting individual differences in neuroscience and clinical studies. While the field has examined multifaceted individual differences in brain functional organization, it is only in recent years that neuroimaging researchers have begun to place a priority on its quantification and optimization. Here, we highlight a potential analytic pitfall that can lead to contaminated estimates of inter-individual differences. We define a two-dimensional individual variation field map to decipher sources of individual variation and their relation to fingerprinting and measures of reliability. We illustrate theoretical gradient flow that represents the most effective direction for optimization when measuring individual differences. We propose to use this general framework for dissecting within- and between-individual variation and provide a supporting online tool for the purposes of guiding optimization efforts in biomarker discovery.


title: Multiscale Entropy Analysis of Retinal Signals Reveals Reduced Complexity in a Mouse Model of Alzheimer's Disease

abstract: Alzheimers disease (AD) is one of the most significant health challenges of our time, affecting a growing number of the elderly population. In recent years, the retina has received increased attention as a candidate for AD biomarkers since it appears to manifest the pathological signatures of the disease. Therefore, its electrical activity may hint at AD-related physiological changes. However, it is unclear how AD affects retinal electrophysiology and what tools are more appropriate to detect these possible changes. In this study, we used entropy tools to estimate the complexity of the dynamics of healthy and diseased retinas at different ages. We recorded microelectroretinogram responses to visual stimuli of different nature from retinas of young and adult, wild-type and 5xFAD -an animal model of AD- mice. To estimate the complexity of signals, we used the multiscale entropy approach, which calculates the entropy at several time scales using a coarse graining procedure. We found that young retinas had more complex responses to different visual stimuli. Further, the responses of young, wild-type retinas to natural-like stimuli exhibited significantly higher complexity than young, 5xFAD retinas. Our findings support a theory of complexity-loss with aging and disease and can have significant implications for early AD diagnosis.


title: Automated Human Habenula Segmentation from T1-weighted Magnetic Resonance Images using V-Net

abstract: The Habenula (Hb), a small bilateral midbrain structure, plays an important role in aversion and reward processing. Despite increasing interest in imaging human Hb structure and function, it is challenging to define the Hb in magnetic resonance imaging (MRI) due to its small size and low anatomical contrast to the surrounding thalamus. In this study, we developed a deep learning-based human Hb segmentation model. This model was trained and tested using about a thousand subjects 3T T1-weighted images with Hb labels from our previous myelin content-based Hb segmentation. The predicted Hb segmentation showed high similarity and small surface distance to the label; the average (across test subjects) dice similarity coefficient, mean distance, and Hausdorff distance were 0.79, 0.22 mm, and 1.63 mm, respectively. We also demonstrated out-of-sample robustness using other 7T and 3T MRI datasets. The proposed Hb segmentation method is automated, objective, fast, robust, and reliable, relieving the requirement of T2-weighted images in the established myelin content-based Hb segmentation to suite a broader range of MRI studies.


title: Unified surface and volumetric projection of physiological imaging data

abstract: Projection of volumetric data onto the cortical surface is an important precursor to performing surface-based analysis. Numerous projection methods have been reported in the literature, many of which make assumptions which tie them to use with specific modalities, notably blood oxygenation level dependent (BOLD) imaging. This means that they may not be appropriate for use with modalities where subcortical tissue contributes a signal of interest. This work details a new projection that provides a number of generalisations and extensions to existing methods. Namely, it may be used to project arbitrary data without making modality-specific assumptions and can produce unified surface and volumetric representations of data (a concept also known as grayordinates space). When constrained to the same assumptions as existing methods, a comparison using simulation data shows that it produces similar outputs. When these assumptions are relaxed to project simulation data containing both cortical and subcortical signals to and from a unified surface and volume space, substantial and statistically significant differences in recovery of ground truth are observed compared to existing methods.


title: Sampling-based Bayesian inference in recurrent circuits of stochastic spiking neurons

abstract: Two facts about cortex are widely accepted: neuronal responses show large spiking variability with near Poisson statistics and cortical circuits feature abundant recurrent connections between neurons. How these spiking and circuit properties combine to support sensory representation and information processing is not well understood. We build a theoretical framework showing that these two ubiquitous features of cortex combine to produce optimal sampling-based Bayesian inference. Recurrent connections store an internal model of the external world, and Poissonian variability of spike responses drives flexible sampling from the posterior stimulus distributions obtained by combining feedforward and recurrent neuronal inputs. We illustrate how this framework for sampling-based inference can be used by cortex to represent latent multivariate stimuli organized either hierarchically or in parallel. A neural signature of such network sampling are internally generated differential correlations whose amplitude is determined by the prior stored in the circuit, which provides an experimentally testable prediction for our framework.


title: In silico transcriptomics identifies FDA-approved drugs and biological pathways for protection against cisplatin-induced hearing loss

abstract:
Acquired hearing loss is a major health problem that affects 5-10% of the world population. However, there are no FDA-approved drugs for the treatment or prevention of hearing loss. Employing the Connectivity Map (CMap) that contains >54,000 compounds, we performed an unbiased in silico screen using the transcriptomic profiles of cisplatin-resistant and -sensitive cancer cell lines. Pathway enrichment analysis identified gene-drug targets for which 30 candidate drugs were selected with potential to confer protection against cisplatin-induced ototoxicity. In parallel, transcriptomic analysis of a cisplatin-treated cochlear-derived cell line identified common enriched pathway targets. We subsequently tested these top 30 candidate compounds, 15 (50%) of which are FDA-approved for other indications, and 26 (87%) of which were validated for their protective effects in either a cochlear-derived cell line or zebrafish lateral line neuromasts, thus confirming our in silico transcriptomic approach. Among these top compounds, niclosamide, a salicyanilide drug approved by the FDA for treating tapeworm infections for decades, protected from cisplatin- and noise-induced hearing loss in mice. Finally, niclosamide and ezetimibe (an Nrf2 agonist) exerted synergistic protection against cisplatin-ototoxicity in zebrafish, validating the Nrf2 pathway as part of niclosamides mechanism of action. Taken together, employing the CMap, we identified multiple pathways and drugs against cisplatin ototoxicity and confirmed that niclosamide can effectively be repurposed as an otoprotectant for future clinical trials against cisplatin- and noise-induced hearing loss.

Significant StatementEmploying the Connectivity Map as our in silico transcriptomic screening strategy we identified FDA-approved drugs and biological pathways for protection against cisplatin-induced hearing loss.


title: Gnas ablation in CD11c+ cells prevents high-fat diet-induced obesity by elevating adipose tissue catecholamine levels and thermogenesis

abstract: CD11c+ immune cells are a potential therapeutic target for treatment of obesity-related insulin resistance and type 2 diabetes (T2D). In obesity, CD11c+ immune cells are recruited to white adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. In this study, we found that ablation of Gnas, the gene that encodes Gas, in CD11c expressing cells protects mice from high-fat diet-induced obesity, glucose intolerance and insulin resistance. Gnas{Delta}CD11c mice (KO) had increased oxygen consumption, energy expenditure, and beigeing of white adipose tissue (WAT). Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and the absence of T and B cells by crossing the KO to a Rag1-/- background did not alter the phenotype. Notably, we observed elevated norepinephrine and elevated cAMP signaling in the WAT of KO mice. The KO adipose tissue also had reduced expression of catecholamine transport and degradation enzymes. Collectively, our results identified an important role of Gas in CD11c+ cells in whole body metabolism regulation by controlling norepinephrine levels in WAT, modulating catecholamine-induced lipolysis and increasing thermogenesis that together created a lean phenotype.


title: Design and Characterization of Lipid Nanocarriers for Oral Delivery of Immunotherapeutic Peptides

abstract:
The use of therapeutic proteins and peptides is of great interest for the treatment of many diseases, and advances in nanotechnology offer a path toward their stable delivery via preferred routes of administration. In this study, we sought to design and formulate a nanostructured lipid carrier (NLC) containing a nominal antigen (insulin peptide) for oral delivery. We utilized the design of experiments (DOE) statistical method to determine the dependencies of formulation variables on physicochemical particle characteristics including particle size, polydispersity (PDI), melting point, and latent heat of melting. The particles were determined to be non-toxic in vitro, readily taken up by primary immune cells, and found to accumulate in regional lymph nodes following oral administration. We believe that this platform technology could be broadly useful for the treatment of autoimmune diseases by supporting the development of oral delivery-based antigen specific immunotherapies.

Highlights3-5 bullets, 85 char or less O_LIA Design of Experiments method led the formulation of biocompatible nanoparticles
C_LIO_LINLC accumulate into gut-draining lymphatic tissues following oral administration
C_LIO_LINLC protect their antigen cargo and promote its presentation
C_LIO_LINLC formulation is well-suited for oral delivery of immunomodulatory agents
C_LI

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/478027v1_ufig1.gif" ALT="Figure 1">
View larger version (37K):
org.highwire.dtl.DTLVardef@1d18054org.highwire.dtl.DTLVardef@14038ddorg.highwire.dtl.DTLVardef@1555abdorg.highwire.dtl.DTLVardef@b83a42_HPS_FORMAT_FIGEXP  M_FIG The development of nanostructured lipid carriers containing a nominal antigen (insulin peptide) for oral delivery consists on (1) nanoparticle formulation using a statistical method, (2) in-vitro studies to assess cellular toxicity and uptake and T cell activation, and (3) in-vivo studies to assess bio-distribution.

C_FIG


title: Reprogramming Alveolar Macrophage Responses to TGF-β Reveals CCR2+ Monocyte Activity that Promotes Bronchiolitis Obliterans Syndrome

abstract: Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-{beta} bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes are engulfed primarily by alveolar macrophages (AM), which become reprogrammed to become less responsive to TGF-{beta} and reduce TGF-{beta} bioavailability through secretion of the TGF-{beta} antagonist Decorin. In untreated recipients, high airway TGF-{beta} activity stimulates AM to express CCL2 leading to CCR2+ monocyte-driven BOS development. Moreover, we find TGF-{beta} receptor 2-dependent differentiation of CCR2+ monocytes is required for the generation of monocyte-derived AM, which in turn promote BOS by expanding tissue-resident memory CD8+ T cells that inflict airway injury through Blimp-1-mediated Granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-{beta}-dependent network, which couples CCR2+ monocyte recruitment and differentiation to alloimmunity and BOS. Alveolar macrophage plasticity can be harnessed to prevent Bronchiolitis Obliterans Syndrome.


title: Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal anti-tumor immunity

abstract:
Gut microbiota are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anti-cancer immune responses, however, are largely unknown. Here, we find that ICT induces translocation of specific endogenous gut microbiota into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, gut microbiota activated dendritic cells (DCs) traffic a selective subset of gut bacteria to mesenteric lymph nodes (MLN) and promote optimal anti-tumor T-cell responses in both the tumor draining lymph nodes (TDLN) and the primary tumor. Antibiotic treatment resulted in decreased gut microbiota translocation into MLN and TDLN, diminished polyfunctional effector CD8+ T cell responses, and attenuated response to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anti-cancer immunity.

One sentence summaryFollowing immune checkpoint blockade therapy, dendritic cells traffic gut microbiota into secondary lymphoid organs, promoting optimal extraintestinal anti-cancer immunity.


title: PD-L1 blockade restores CAR T cell activity through IFNγ-regulation of CD163+ macrophages

abstract:
BackgroundThe immune suppressive tumor microenvironment (TME) that inhibits T cell infiltration, survival, and anti-tumor activity has posed a major challenge for developing effective immunotherapies for solid tumors. Chimeric antigen receptor (CAR)-engineered T cell therapy has shown unprecedented clinical response in treating patients with hematological malignancies, and intense investigation is underway to achieve similar responses with solid tumors. Immunologically cold tumors, including prostate cancers, are often infiltrated with abundant tumor-associated macrophages (TAMs), and infiltration of CD163+ M2 macrophages correlates with tumor progression and poor responses to immunotherapy. However, the impact of TAMs on CAR T cell activity alone and in combination with TME immunomodulators is unclear.

MethodsTo model this in vitro, we utilized a novel co-culture system with tumor cells, CAR T cells, and polarized M1 or M2 macrophages from CD14+ PBMCs collected from healthy human donors. Tumor cell killing, T cell activation and proliferation, and macrophage phenotypes were evaluated by flow cytometry, cytokine production, RNA sequencing, and functional blockade of signaling pathways using antibodies and small molecule inhibitors. We also evaluated the TME in humanized mice following CAR T cell therapy for validation of our in vitro findings.

ResultsWe observed inhibition of CAR T cell activity with the presence of M2 macrophages, but not M1 macrophages, coinciding with a robust induction of PD-L1 in M2 macrophages. We observed similar PD-L1 expression in TAMs following CAR T cell therapy in the TME of humanized mice. PD-L1, but not PD-1, blockade in combination with CAR T cell therapy altered phenotypes to more M1-like subsets and led to loss of CD163+ M2 macrophages via IFN{gamma} signaling, resulting in improved anti-tumor activity of CAR T cells.

ConclusionThis study reveals an alternative mechanism by which the combination of CAR T cells and immune checkpoint blockade modulates the immune landscape of solid tumors to enhance therapeutic efficacy of CAR T cells.


title: Aeromonas hydrophila induces skin disturbance through mucosal microbiota dysbiosis in Pangasianodon hypophthalmus

abstract:
Bacterial pathogens are well-equipped to adhere to and initiate infection in teleost fish. The fish skin mucus serves as the first barrier against environmental pathogens. The mucus harbors commensal microbes that form the microbiota, impacting the host physiological and immunological regulation. However, how the skin mucosal microbiota responds to the presence of pathogens remains largely unexplored. Thus, little is known about the status of skin mucus prior to the infection with noticeable symptoms. In this study, we aim to investigate the interaction between pathogen and skin mucosal microbiota, as well as the fish skin immune responses in the presence of pathogens. By challenging striped catfish (Pangasianodon hypophthalmus) with different concentrations of bacterial pathogen Aeromonas hydrophila (AH), the skin immune response and the mucosal microbiota were examined by qPCR and 16S rRNA gene sequencing analysis. We found a pathogen concentration able to stimulate the skin immune response associated with the significant mucosal microbiota change and re-confirmed with the ex vivo fish skin model. Further analysis indicated that the change was attributed to the significant increase in opportunistic pathogens over AH. We concluded that the presence and increase of AH results in dysbiosis of mucosal microbiota that can stimulate skin immune response. We believe our work can shed some light on host-pathogen-commensal microbiota interaction and therefore contribute to aquaculture infection prevention.

IMPORTANCEThe fish skin mucosal microbiota is essentially the first barrier in response to the presence of pathogens. This study is the first study to elucidate the interaction between the AH, the skin mucosal microbiota, and the striped catfish skin at the initiation stage of infection. Our study provides a platform to study both the correlation and causation of the interaction between pathogen, the fish skin, and the skin mucosal microbiota. This work provides information that changes in the AH-induced mucosal microbiota result in skin disturbance with immune stimulation.


title: LuminoCell: a versatile and affordable luminometer platform for monitoring in-cell luciferase-based reporters

abstract: Luciferase reporter assays represent a simple and sensitive experimental system in cell and molecular biology to study multiple biological processes. However, the application of these assays is often limited by the costs of conventional luminometer instruments and the versatility of their use in different experimental conditions. Therefore, we aimed to develop a small, affordable luminometer allowing continuous measurement of luciferase activity, designed for inclusion into various kinds of tissue culture incubators. Here we introduce LuminoCell - an open-source platform for the construction of an affordable, sensitive, and portable luminometer capable of realtime monitoring in-cell luciferase activity. The LuminoCell costs $40, requires less than 1 hour to assemble, and it is capable of performing real-time sensitive detection of both magnitude and duration of the activity of major signalling pathways in cell cultures, including receptor tyrosine kinases (EGF, FGF), WNT/{beta}-catenin, and NF-{kappa}B. Additionally, we show that the LuminoCell is suitable to be used in cytotoxicity assays as well as for monitoring periodic circadian gene expression.