Modulating tumoral exosomes and fibroblast phenotype using nanoliposomes augments cancer immunotherapy.

Cancer cells program fibroblasts into cancer associated fibroblasts (CAFs) in a two-step manner. First, cancer cells secrete exosomes to program quiescent fibroblasts into activated CAFs. Second, cancer cells maintain the CAF phenotype via activation of signal transduction pathways. We rationalized that inhibiting this two-step process can normalize CAFs into quiescent fibroblasts and augment the efficacy of immunotherapy. We show that cancer cell-targeted nanoliposomes that inhibit sequential steps of exosome biogenesis and release from lung cancer cells block the differentiation of lung fibroblasts into CAFs. In parallel, we demonstrate that CAF-targeted nanoliposomes that block two distinct nodes in fibroblast growth factor receptor (FGFR)-Wnt/ß-catenin signaling pathway can reverse activate CAFs into quiescent fibroblasts. Co-administration of both nanoliposomes significantly improves the infiltration of cytotoxic T cells and enhances the antitumor efficacy of αPD-L1 in immunocompetent lung cancer-bearing mice. Simultaneously blocking the tumoral exosome-mediated activation of fibroblasts and FGFR-Wnt/ß-catenin signaling constitutes a promising approach to augment immunotherapy.

Population screening shows risk of inherited cancer and familial hypercholesterolemia in Oregon.

The Healthy Oregon Project (HOP) is a statewide effort that aims to build a large research repository and influence the health of Oregonians through providing no-cost genetic screening to participants for a next-generation sequencing 32-gene panel comprising genes related to inherited cancers and familial hypercholesterolemia. This type of unbiased population screening can detect at-risk individuals who may otherwise be missed by conventional medical approaches. However, challenges exist for this type of high-throughput testing in an academic setting, including developing a low-cost high-efficiency test and scaling up the clinical laboratory for processing large numbers of samples. Modifications to our academic clinical laboratory including efficient test design, robotics, and a streamlined analysis approach increased our ability to test more than 1,000 samples per month for HOP using only one dedicated HOP laboratory technologist. Additionally, enrollment using a HIPAA-compliant smartphone app and sample collection using mouthwash increased efficiency and reduced cost. Here, we present our experience three years into HOP and discuss the lessons learned, including our successes, challenges, opportunities, and future directions, as well as the genetic screening results for the first 13,670 participants tested. Overall, we have identified 730 pathogenic/likely pathogenic variants in 710 participants in 24 of the 32 genes on the panel. The carrier rate for pathogenic/likely pathogenic variants in the inherited cancer genes on the panel for an unselected population was 5.0% and for familial hypercholesterolemia was 0.3%. Our laboratory experience described here may provide a useful model for population screening projects in other states.

oskar acts with the transcription factor Creb to regulate long-term memory in crickets.

Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into preexisting regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene oskar, was first identified based on its role in establishing the Drosophila melanogaster germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a neural role for oskar. We show that oskar is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket Gryllus bimaculatus. In these stem cells, called neuroblasts, oskar is required together with the ancient animal transcription factor Creb to regulate long-term (but not short-term) olfactory memory. We provide evidence that oskar positively regulates Creb, which plays a conserved role in long-term memory across animals, and that oskar in turn may be a direct target of Creb. Together with previous reports of a role for oskar in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that oskar's original somatic role may have been in the insect nervous system. Moreover, its colocalization and functional cooperation with the conserved pluripotency gene piwi in the nervous system may have facilitated oskar's later co-option to the germ line in holometabolous insects.

Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells.

Cancer progresses by evading the immune system. Elucidating diverse immune evasion strategies is a critical step in the search for next-generation immunotherapies for cancer. Here we report that cancer cells can hijack the mitochondria from immune cells via physical nanotubes. Mitochondria are essential for metabolism and activation of immune cells. By using field-emission scanning electron microscopy, fluorophore-tagged mitochondrial transfer tracing and metabolic quantification, we demonstrate that the nanotube-mediated transfer of mitochondria from immune cells to cancer cells metabolically empowers the cancer cells and depletes the immune cells. Inhibiting the nanotube assembly machinery significantly reduced mitochondrial transfer and prevented the depletion of immune cells. Combining a farnesyltransferase and geranylgeranyltransferase 1 inhibitor, namely, L-778123, which partially inhibited nanotube formation and mitochondrial transfer, with a programmed cell death protein 1 immune checkpoint inhibitor improved the antitumour outcomes in an aggressive immunocompetent breast cancer model. Nanotube-mediated mitochondrial hijacking can emerge as a novel target for developing next-generation immunotherapy agents for cancer.

Artificial intelligence (AI)-assisted exome reanalysis greatly aids in the identification of new positive cases and reduces analysis time in a clinical diagnostic laboratory.

PURPOSE: Artificial intelligence (AI) and variant prioritization tools for genomic variant analysis are being rapidly developed for use in clinical diagnostic testing. However, their clinical utility and reliability are currently limited. Therefore, we performed a validation of a commercial AI tool (Moon) and a comprehensive reanalysis of previously collected clinical exome sequencing cases using an open-source variant prioritization tool (Exomiser) and the now-validated AI tool to test their feasibility in clinical diagnostics. METHODS: A validation study of Moon was performed with 29 positive cases determined by previous manual analysis. After validation, reanalysis was performed on 80 previously manually analyzed nondiagnostic exome cases using Moon. Finally, a comparison between Moon and Exomiser was completed regarding their ability to identify previously completed positive cases and to identify new positive cases. RESULTS: Moon correctly selected the causal variant(s) in 97% of manually analyzed positive cases and identified 7 new positive cases. Exomiser correctly identified the causal gene in 85% of positive cases and agreed with Moon by ranking the new gene in its top 10 list 43% of the time. CONCLUSION: The use of AI in diagnostic laboratories greatly enhances exome sequencing analysis by reducing analysis time and increasing the diagnostic rate.

Boosting Natural Killer Cell Therapies in Glioblastoma Multiforme Using Supramolecular Cationic Inhibitors of Heat Shock Protein 90.

Allogeneic natural killer (aNK) cell adoptive therapy has the potential to dramatically impact clinical outcomes of glioblastoma multiforme (GBM). However, in order to exert therapeutic activity, NK cells require tumor expression of ligands for activating receptors, such as MHC Class I peptide A/B (MICA/B) and ULBPs. Here, we describe the use of a blood-brain barrier (BBB) permissive supramolecular cationic drug vehicle comprising an inhibitor of the chaperone heat shock protein 90 (Hsp90), which sustains a cytotoxic effect on GBM cells, boosts the expression of MICA/B and ULBPs on the residual population, and augments the activity of clinical-grade aNK cells (GTA002). First, we identify Hsp90 mRNA transcription and gain of function as significantly upregulated in GBM compared to other central nervous system tumors. Through a rational chemical design, we optimize a radicicol supramolecular prodrug containing cationic excipients, SCI-101, which displays >2-fold increase in relative BBB penetration compared to less cationic formulations in organoids, in vitro. Using 2D and 3D biological models, we confirm SCI-101 sustains GBM cytotoxicity 72 h after drug removal and induces cell surface MICA/B protein and ULBP mRNA up to 200% in residual tumor cells compared to the naked drug alone without augmenting the shedding of MICA/B, in vitro. Finally, we generate and test the sequential administration of SCI-101 with a clinical aNK cell therapy, GTA002, differentiated and expanded from healthy umbilical cord blood CD34+ hematopoietic stem cells. Using a longitudinal in vitro model, we demonstrate >350% relative cell killing is achieved in SCI-101-treated cell lines compared to vehicle controls. In summary, these data provide a first-of-its-kind BBB-penetrating, long-acting inhibitor of Hsp90 with monotherapy efficacy, which improves response to aNK cells and thus may rapidly alter the treatment paradigm for patients with GBM.

Evolutionary dynamics of sex-biased genes expressed in cricket brains and gonads.

Sex-biased gene expression, particularly sex-biased expression in the gonad, has been linked to rates of protein sequence evolution (nonsynonymous to synonymous substitutions, dN/dS) in animals. However, in insects, sex-biased expression studies remain centred on a few holometabolous species. Moreover, other major tissue types such as the brain remain underexplored. Here, we studied sex-biased gene expression and protein evolution in a hemimetabolous insect, the cricket Gryllus bimaculatus. We generated novel male and female RNA-seq data for two sexual tissue types, the gonad and somatic reproductive system, and for two core components of the nervous system, the brain and ventral nerve cord. From a genome-wide analysis, we report several core findings. Firstly, testis-biased genes had accelerated evolution, as compared to ovary-biased and unbiased genes, which was associated with positive selection events. Secondly, although sex-biased brain genes were much less common than for the gonad, they exhibited a striking tendency for rapid protein sequence evolution, an effect that was stronger for the female than male brain. Further, some sex-biased brain genes were linked to sexual functions and mating behaviours, which we suggest may have accelerated their evolution via sexual selection. Thirdly, a tendency for narrow cross-tissue expression breadth, suggesting low pleiotropy, was observed for sex-biased brain genes, suggesting relaxed purifying selection, which we speculate may allow enhanced freedom to evolve adaptive protein functional changes. The findings of rapid evolution of testis-biased genes and male and female-biased brain genes are discussed with respect to pleiotropy, positive selection and the mating biology of this cricket.

Adaptation of codon and amino acid use for translational functions in highly expressed cricket genes.

BACKGROUND: For multicellular organisms, much remains unknown about the dynamics of synonymous codon and amino acid use in highly expressed genes, including whether their use varies with expression in different tissue types and sexes. Moreover, specific codons and amino acids may have translational functions in highly transcribed genes, that largely depend on their relationships to tRNA gene copies in the genome. However, these relationships and putative functions are poorly understood, particularly in multicellular systems. RESULTS: Here, we studied codon and amino acid use in highly expressed genes from reproductive and nervous system tissues (male and female gonad, somatic reproductive system, brain and ventral nerve cord, and male accessory glands) in the cricket Gryllus bimaculatus. We report an optimal codon, defined as the codon preferentially used in highly expressed genes, for each of the 18 amino acids with synonymous codons in this organism. The optimal codons were mostly shared among tissue types and both sexes. However, the frequency of optimal codons was highest in gonadal genes. Concordant with translational selection, a majority of the optimal codons had abundant matching tRNA gene copies in the genome, but sometimes obligately required wobble tRNAs. We suggest the latter may comprise a mechanism for slowing translation of abundant transcripts, particularly for cell-cycle genes. Non-optimal codons, defined as those least commonly used in highly transcribed genes, intriguingly often had abundant tRNAs, and had elevated use in a subset of genes with specialized functions (gametic and apoptosis genes), suggesting their use promotes the translational upregulation of particular mRNAs. In terms of amino acids, we found evidence suggesting that amino acid frequency, tRNA gene copy number, and amino acid biosynthetic costs (size/complexity) had all interdependently evolved in this insect model, potentially for translational optimization. CONCLUSIONS: Collectively, the results suggest a model whereby codon use in highly expressed genes, including optimal, wobble, and non-optimal codons, and their tRNA abundances, as well as amino acid use, have been influenced by adaptation for various functional roles in translation within this cricket. The effects of expression in different tissue types and the two sexes are discussed.

Absence of a Faster-X Effect in Beetles (Tribolium, Coleoptera).

The faster-X effect, namely the rapid evolution of protein-coding genes on the X chromosome, has been widely reported in metazoans. However, the prevalence of this phenomenon across diverse systems and its potential causes remain largely unresolved. Analysis of sex-biased genes may elucidate its possible mechanisms: for example, in systems with X/Y males a more pronounced faster-X effect in male-biased genes than in female-biased or unbiased genes may suggest fixation of recessive beneficial mutations rather than genetic drift. Further, theory predicts that the faster-X effect should be promoted by X chromosome dosage compensation. Here, we asked whether we could detect a faster-X effect in genes of the beetle Tribolium castaneum (and T. freemani orthologs), which has X/Y sex-determination and heterogametic males. Our comparison of protein sequence divergence (dN/dS) on the X chromosome vs. autosomes indicated a rarely observed absence of a faster-X effect in this organism. Further, analyses of sex-biased gene expression revealed that the X chromosome was particularly highly enriched for ovary-biased genes, which evolved slowly. In addition, an evaluation of male X chromosome dosage compensation in the gonads and in non-gonadal somatic tissues indicated a striking lack of compensation in the testis. This under-expression in testis may limit fixation of recessive beneficial X-linked mutations in genes transcribed in these male sex organs. Taken together, these beetles provide an example of the absence of a faster-X effect on protein evolution in a metazoan, that may result from two plausible factors, strong constraint on abundant X-linked ovary-biased genes and a lack of gonadal dosage compensation.

The Cricket Gryllus bimaculatus: Techniques for Quantitative and Functional Genetic Analyses of Cricket Biology.

All extant species are an outcome of nature's "experiments" during evolution, and hence multiple species need to be studied and compared to gain a thorough understanding of evolutionary processes. The field of evolutionary developmental biology (evo-devo) aspires to expand the number of species studied, because most functional genetic studies in animals have been limited to a small number of "traditional" model organisms, many of which belong to the same phylum (Chordata). The phylum Arthropoda, and particularly its component class Insecta, possesses many important characteristics that are considered favorable and attractive for evo-devo research, including an astonishing diversity of extant species and a wide disparity in body plans. The development of the most thoroughly investigated insect genetic model system to date, the fruit fly Drosophila melanogaster (a holometabolous insect), appears highly derived with respect to other insects and indeed with respect to most arthropods. In comparison, crickets (a basally branching hemimetabolous insect lineage compared to the Holometabola) are thought to embody many developmental features that make them more representative of insects. Here we focus on crickets as emerging models to study problems in a wide range of biological areas and summarize the currently available molecular, genomic, forward and reverse genetic, imaging and computational tool kit that has been established or adapted for cricket research. With an emphasis on the cricket species Gryllus bimaculatus, we highlight recent efforts made by the scientific community in establishing this species as a laboratory model for cellular biology and developmental genetics. This broad toolkit has the potential to accelerate many traditional areas of cricket research, including studies of adaptation, evolution, neuroethology, physiology, endocrinology, regeneration, and reproductive behavior. It may also help to establish newer areas, for example, the use of crickets as animal infection model systems and human food sources.

Evidence of multifaceted functions of codon usage in translation within the model beetle Tribolium castaneum.

Synonymous codon use is non-random. Codons most used in highly transcribed genes, often called optimal codons, typically have high gene counts of matching tRNA genes (tRNA abundance) and promote accurate and/or efficient translation. Non-optimal codons, those least used in highly expressed genes, may also affect translation. In multicellular organisms, codon optimality may vary among tissues. At present, however, tissue specificity of codon use remains poorly understood. Here, we studied codon usage of genes highly transcribed in germ line (testis and ovary) and somatic tissues (gonadectomized males and females) of the beetle Tribolium castaneum. The results demonstrate that: (i) the majority of optimal codons were organism-wide, the same in all tissues, and had numerous matching tRNA gene copies (Opt-codon↑tRNAs), consistent with translational selection; (ii) some optimal codons varied among tissues, suggesting tissue-specific tRNA populations; (iii) wobble tRNA were required for translation of certain optimal codons (Opt-codonwobble), possibly allowing precise translation and/or protein folding; and (iv) remarkably, some non-optimal codons had abundant tRNA genes (Nonopt-codon↑tRNAs), and genes using those codons were tightly linked to ribosomal and stress-response functions. Thus, Nonopt-codon↑tRNAs codons may regulate translation of specific genes. Together, the evidence suggests that codon use and tRNA genes regulate multiple translational processes in T. castaneum.

Convergent evolution of germ granule nucleators: A hypothesis.

Germ cells have been considered "the ultimate stem cell" because they alone, during normal development of sexually reproducing organisms, are able to give rise to all organismal cell types. Morphological descriptions of a specialized cytoplasm termed 'germ plasm' and associated electron dense ribonucleoprotein (RNP) structures called 'germ granules' within germ cells date back as early as the 1800s. Both germ plasm and germ granules are implicated in germ line specification across metazoans. However, at a molecular level, little is currently understood about the molecular mechanisms that assemble these entities in germ cells. The discovery that in some animals, the gene products of a small number of lineage-specific genes initiate the assembly (also termed nucleation) of germ granules and/or germ plasm is the first step towards facilitating a better understanding of these complex biological processes. Here, we draw on research spanning over 100years that supports the hypothesis that these nucleator genes may have evolved convergently, allowing them to perform analogous roles across animal lineages.

The genomic basis of parasitism in the Strongyloides clade of nematodes.

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families--families encoding astacin-like and SCP/TAPS proteins--is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

Differential chromatin amplification and chromosome complements in the germline of Strongyloididae (Nematoda).

Nematodes of the genus Strongyloides are intestinal parasites of vertebrates including man. Currently, Strongyloides and its sister genus Parastrongyloides are being developed as models for translational and basic biological research. Strongyloides spp. alternate between parthenogenetic parasitic and single free-living sexual generations, with the latter giving rise to all female parasitic progeny. Parastrongyloides trichosuri always reproduces sexually and may form many consecutive free-living generations. Although the free-living adults of both these species share a superficial similarity in overall appearance when compared to Caenorhabditis elegans, there are dramatic differences between them, in particular with respect to the organization of the germline. Here we address two such differences, which have puzzled investigators for several generations. First, we characterize a population of non-dividing giant nuclei in the distal gonad, the region that in C. elegans is populated by mitotically dividing germline stem cells and early meiotic cells. We show that in these nuclei, autosomes are present in higher copy numbers than X chromosomes. Consistently, autosomal genes are expressed at higher levels than X chromosomal ones, suggesting that these worms use differential chromatin amplification for controlling gene expression. Second, we address the lack of males in the progeny of free-living Strongyloides spp. We find that male-determining (nullo-X) sperm are present in P. trichosuri, a species known to produce male progeny, and absent in Strongyloides papillosus, which is consistent for a species that does not. Surprisingly, nullo-X sperm appears to be present in Strongyloides ratti, even though this species does not produce male progeny. This suggests that different species of Strongyloides employ various strategies to prevent the formation of males in the all-parasitic progeny of the free-living generation.

Germline organization in Strongyloides nematodes reveals alternative differentiation and regulation mechanisms.

Nematodes of the genus Strongyloides are important parasites of vertebrates including man. Currently, little is known about their germline organization or reproductive biology and how this influences their parasitic life strategies. Here, we analyze the structure of the germline in several Strongyloides and closely related species and uncover striking differences in the development, germline organization, and fluid dynamics compared to the model organism Caenorhabditis elegans. With a focus on Strongyloides ratti, we reveal that the proliferation of germ cells is restricted to early and mid-larval development, thus limiting the number of progeny. In order to understand key germline events (specifically germ cell progression and the transcriptional status of the germline), we monitored conserved histone modifications, in particular H3Pser10 and H3K4me3. The evolutionary significance of these events is subsequently highlighted through comparisons with six other nematode species, revealing underlying complexities and variations in the development of the germline among nematodes.

Parastrongyloides trichosuri suggests that XX/XO sex determination is ancestral in Strongyloididae (Nematoda).

The parasitic roundworms Strongyloides stercoralis (in man) and Strongyloides ratti (in rats) employ environmentally controlled XX/XO sex determination with a pair of X chromosomes and two pairs of autosomes. Strongyloides papillosus (in sheep) has only two pairs of chromosomes, one of which combines the genetic material homologous to the S. ratti chromosomes X and I. This species creates males through the elimination of one copy of the portion related to the X chromosome (chromatin diminution). It is not clear which one of these two sex-determining mechanisms is ancestral. We demonstrate that Strongyloides vituli (in cattle) has two pairs of chromosomes like its very close relative S. papillosus whereas Parastrongyloides trichosuri, a closely related out-group to Strongyloides spp. in Australian brushtail possums, has three chromosome pairs and employs XX/XO sex determination. The X chromosome of P. trichosuri is homologous to the X chromosome of S. ratti. Our data strongly suggest that the last common ancestor of Strongyloides spp. and Parastrongyloides spp. had two pairs of autosomes along with two or one X chromosome in females and males, respectively. The situation with two pairs of chromosomes is likely derived and occurred through the fusion of the X chromosome with an autosome.

Herpes simplex virus 1 glycoprotein B and US3 collaborate to inhibit CD1d antigen presentation and NKT cell function.

Herpes simplex viruses (HSVs) are prevalent human pathogens that establish latency in human neuronal cells and efficiently evade the immune system. It has been a major medical challenge to eradicate them and, despite intensive efforts, an effective vaccine is not available. We previously showed that upon infection of antigen-presenting cells, HSV type 1 (HSV-1) rapidly and efficiently downregulates the major histocompatibility complex class I-like antigen-presenting molecule, CD1d, and potently inhibits its recognition by CD1d-restricted natural killer T (NKT) cells. It suppresses CD1d expression primarily by inhibiting its recycling to the cell surface after endocytosis. We identify here the viral glycoprotein B (gB) as the predominant CD1d-interacting protein. gB initiates the interaction with CD1d in the endoplasmic reticulum and stably associates with it throughout CD1d trafficking. However, an additional HSV-1 component, the serine-threonine kinase US3, is required for optimal CD1d downregulation. US3 expression in infected cells leads to gB enrichment in the trans-Golgi network (TGN) and enhances the relocalization of both gB and CD1d to this compartment, suggesting that following internalization CD1d is translocated from the endocytic pathway to the TGN by its association with gB. Importantly, both US3 and gB are required for efficient inhibition of CD1d antigen presentation and NKT cell activation. In summary, our results suggest that HSV-1 uses gB and US3 to rapidly inhibit NKT cell function in the initial antiviral response.