Defensive Phenotypes Research Articles

Abstract 5438: Circulating tumor cells exhibit a tumor stemness defense phenotype in Head and Neck cancer subjects undergoing cisplatin based chemotherapy

Abstract Introduction: We have proposed that tumor has its own defense mechanism similar to an organism having its own defense at the population level. We also proposed that the center of this tumor defense is the hypoxic niche of cancer stem cells (CSCs). Our lab has previously characterized the hypoxic niche of CSCs, and demonstrated that these CSCs reprogram to a highly inflammatory, aggressive and embryonic stemness phenotype during oxidative stress (1). These reprogrammed CSCs, the tumor stemness defense (TSD) phenotype is transient, and exert altruistic defense against bacterial invasion in SCC-25 cell line (2). Further, VEGF/VEGFR1 autocrine signaling was found in cisplatin induced TSD phenotype (4) and Myc/Hif2alpha stemness pathway was found in TSD phenotype of a leukemia stem cell model (3). We hypothesize that the platinum-induced stress may also activate TSD phenotype in the dormant CSC fraction of head and neck squamous cell carcinoma (HNSCC), which may mobilize to the distant tissue via circulation as circulating tumor cells (CTCs). Methods: To test the hypothesis, we isolated EpCAM+/ABCG2+ CTCs (blood, n=14) and EpCAM+/ABCG2+ CSCs (tumor tissue, n=6/14) of HNSCC subjects under cisplatin treatment. CTCs were collected from peripheral blood mononuclear cells (PBMNC) by the immunomagnetic sorting of EpCAM+ cells, and then cells were expanded in a defined serum free media that we used to maintain the hypoxic CSCs (1). We evaluated the EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs for TSD phenotype by performing qPCR gene expression, Boyden chamber assay of invasion, clonogenic assay and serial transplantation assay in NOD/SCID mice. Data was compared with the EpCAM+/ABCG2- cell population. We also developed a pre-clinical model of cisplatin-induced reprogramming of HNSCC derived CSCs to understand the molecular mechanisms. Results: In 8/14 patient derived EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs showed TSD phenotype associated gene expression, high migration/invasion, self sufficiency (ability to form spheroid when grown in serum free media without growth factors), enhanced secretion of protumorigenic growth factors; VEGF, SDF-1, PIGF, and HMGB1. These CTCs and CSCs have shown tumor formation with significant CSC frequency, when injected to NOD/SCID mice. Finally, using a SCC-25 cell line derived pre-clinical model, we demonstrated that cisplatin therapy reprogram CSCs to TSD phenotype. Conclusion: Our work indicates that CTCs with TSD phenotype may indicate the interaction between cisplatin and CSC niche defense. Using the pre-clinical model, the TSD phenotype based CSC niche defense mechanism may be further explored to develop markers for therapy response, metastasis or recurrence in HNSCC.

Abstract 122: Fusobacterium nucleatum in saliva of oral cancer patients induces tumor stemness defense phenotype in cancer stem cells

Abstract Background: Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer. Cancer Stem Cells (CSCs) having cell surface markers CD44, ALDH, and EpCAM+ have been identified in OSCC. However, the host pathogen interaction between CSCs and oral microbiome has not yet been studied. A number of studies have recently demonstrated the presence of specific oral bacteria populations and their lipopolysaccharides (LPS) in the tumor microenvironment (1). We previously reported pathogenic bacterial internalization in the CSCs (2). Based on the findings, we have developed an in-vitro model to investigate how oral microbiota may integrate into the tumor microenvironment's CSC population and control its activity. Here, we investigated the host/pathogen interaction between CSCs and FN. Methods: Oral bacteria FN was selectively isolated using FEA agar plates from oral saliva of oral cancer patients (n=10) at stage 3 or 4. The isolated bacteria was co cultured with SCC 25 cells at MOI of 1:50. After co culturing the stemness genes such as SOX 2, OCT 4, Nanog,LPS and ABCG2+ cancer stem cell markers were analysed. ABCG2+ CSCs and ABCG2- cells were immunomagnetically sorted and lysed followed by RNA extraction and RT QPCR was performed to observe if FN has selectively internalised in the CSCs. Also ABCG2+ and ABCG 2- cells were used to perform clonogenic assay. Results: Coculture of FN with SCC 25 cell line induced high expression of stemness genes SOX, OCT 4 and Nanog along with CSC markers ABCG2+. Moreover high expression of LPS was observed indicating the presence of FN in the coculture. Importantly live bacteria was found internalized in ABCG2+ cells but not in ABCG2- cells. High clonogenicity was observed in ABCG2+ CSCs compared to ABCG2- cells. Notably, we found that live bacteria and their LPS, mostly Fusobacterium nucleatum isolated (FN) from clinical subjects, were capable of invading CSCs in the in-vitro setting. Post the host-pathogen interaction; it enabled the activation of a niche modulatory tumor stemness defense (TSD) phenotype in the CSCs. These aggressive CSCs with the TSD phenotype have been found to have a critical role in the progression and relapse of oral cancer. Conclusion: Expression of stemness genes infers the niche defence mechanism of the CSCs of the tumour microenvironment due to infection of FN. Importantly presence of live bacteria in ABCG2+ CSCs indicates that FN selectively infects ABCG2+ CSCs but not ABCG2- cells. High clonogenicity of ABCG2+ CSCs confirms the TSD phenotypic attributes of the CSCs.

Iron chelation and supplementation: A comparison in the management of inflammatory bowel disease using drosophila

AimsInflammatory Bowel Disease (IBD) is associated with systemic iron deficiency and has been managed with iron supplements which cause adverse side effects. Conversely, some reports highlight iron depletion to ameliorate IBD. The underlying intestinal response and comparative benefit of iron depletion and supplementation in IBD is unknown. The aims of this work were to characterize and compare the effects of iron supplementation and iron depletion in IBD. Main methodsIBD was induced in Drosophila melanogaster using 3 % dextran sodium sulfate (DSS) in diet for 7 days. Using this model, we investigated the impacts of acute iron depletion (using bathophenanthroline disulfonate, BPS) and supplementation (using ferrous sulphate, FS), before and after IBD induction, on gut iron homeostasis, cell death, gut permeability, inflammation, antioxidant defence, antimicrobial response and several fly phenotypes. Key findingsDSS decreased fly mass (p < 0.001), increased gut permeability (p < 0.001) and shortened lifespan (p = 0.035) compared to control. The DSS-fed flies also showed significantly elevated lipid peroxidation (p < 0.001), and the upregulated expression of apoptotic marker- drice (p < 0.001), tight junction protein – bbg (p < 0.001), antimicrobial peptide – dpta (p = 0.002) and proinflammatory cytokine – upd2 (p < 0.001). BPS significantly (p < 0.05) increased fly mass and lifespan, decreased gut permeability, decreased lipid peroxidation and decreased levels of drice, bbg, dpta and upd2 in IBD flies. This iron chelation (using BPS) showed better protection from DSS-induced IBD than iron supplementation (using FS). Preventive and curative interventions, by BPS or FS, also differed in outcomes. SignificanceThis may inform precise management strategies aimed at tackling IBD and its recurrence.

An amphibian toxin phenotype is sexually dimorphic and shows seasonal concordant change between sexes

Sexually dimorphic phenotypes are consequential to animal survival, and this is especially apparent with defense phenotypes. Amphibians have poison glands, and several lineages maintain a neurotoxin, tetrodotoxin (TTX), which is largely considered a derived chemical defense trait. However, production pathways are unclear, and, as such, whether males and females differentially produce or appropriate toxin concentrations is not known. We evaluated the relationship between TTX concentrations, sex, and morphology by collecting trait data from more than 850 wild newts of the genus Taricha and tested for sex-based differences, potential sex-based changes to the phenotype between breeding seasons, and factors influencing toxicity. Females, regardless of reproductive status, exhibited greater concentrations than males, and temporal patterns indicate male and female trait values tend to fluctuate concordantly at short time scales, with females showing greater change over time. Tree regression results underscored the importance of size and sex to the phenotype. Sexual dimorphism and plasticity of toxins broaden the ecological roles of animal poisons beyond defense only and should recast how we model the evolution of tetrodotoxin.

Evolutionary signatures of a trade-off in direct and indirect defenses across the wild grape genus Vitis.

Evolutionary correlations between chemical defense and protection by mutualist bodyguards have been long predicted, but tests of these pattern remain rare. We use a phylogenetic framework to test for evolutionary correlations indicative of trade-offs or synergisms between direct defense in the form of plant secondary metabolism, and indirect defense in the form of leaf domatia, across 33 species in the wild grape genus, Vitis. We also performed a bioassay with a generalist herbivore to associate our chemical phenotypes with herbivore palatability. Finally, we tested whether defensive traits correlate with the average abiotic characteristics of each species' contemporary range and whether these correlations were consistent with plant defense theory. We found a negative evolutionary correlation between domatia size and the diversity of secondary metabolites in Vitis leaf tissue across the genus, and also that leaves with a higher diversity and richness of secondary metabolites were less palatable to a generalist herbivore, consistent with a trade-off in chemical and mutualistic defense investment. Predictions from plant defense theory were not supported by associations between investment in defense phenotypes and abiotic variables. Our work demonstrates an evolutionary pattern indicative of a trade-off between indirect and direct defense strategies across the Vitis genus.

One of these things is not like the other: Mixed predator cues result in lopsided phenotypic responses in a Neotropical tadpole.

Many organisms have evolved to produce different phenotypes in response to environmental variation. Dendropsophus ebraccatus tadpoles develop opposing shifts in morphology and coloration when they are exposed to invertebrate vs vertebrate predators. Each of these alternate phenotypes are adaptive, conferring a survival advantage against the predator with which tadpoles were reared but imposing a survival cost with the mismatched predator. Here, we measured the phenotypic response of tadpoles to graded cues and mixed cues of both fish and dragonfly nymphs. Prey species like D. ebraccatus commonly co-occur with both of these types of predators, amongst many others as well. In our first experiment, tadpoles increased investment in defensive phenotypes in response to increasing concentrations of predator cues. Whereas morphology only differed in the strongest predation cue, tail spot coloration differed even at the lowest cue concentration. In our second experiment, tadpoles reared with cues from both predators developed an intermediate yet skewed phenotype that was most similar to the fish-induced phenotype. Previous studies have shown that fish are more lethal than dragonfly larvae; thus tadpoles responded most strongly to the more dangerous predator, even though the number of prey consumed by each predator was the same. This may be due to D. ebraccatus having evolved a stronger response to fish or because fish produce more kairomones than do dragonflies for a given amount of food. We demonstrate that not only do tadpoles assess predation risk via the concentration of predation cues in the water, they produce a stronger response to a more lethal predator even when the strength of cues is presumed to be identical.

The toxins of vertically transmitted Spiroplasma.

Vertically transmitted (VT) microbial symbionts play a vital role in the evolution of their insect hosts. A longstanding question in symbiont research is what genes help promote long-term stability of vertically transmitted lifestyles. Symbiont success in insect hosts is due in part to expression of beneficial or manipulative phenotypes that favor symbiont persistence in host populations. In Spiroplasma, these phenotypes have been linked to toxin and virulence domains among a few related strains. However, these domains also appear frequently in phylogenetically distant Spiroplasma, and little is known about their distribution across the Spiroplasma genus. In this study, we present the complete genome sequence of the Spiroplasma symbiont of Drosophila atripex, a non-manipulating member of the Ixodetis clade of Spiroplasma, for which genomic data are still limited. We perform a genus-wide comparative analysis of toxin domains implicated in defensive and reproductive phenotypes. From 12 VT and 31 non-VT Spiroplasma genomes, ribosome-inactivating proteins (RIPs), OTU-like cysteine proteases (OTUs), ankyrins, and ETX/MTX2 domains show high propensity for VT Spiroplasma compared to non-VT Spiroplasma. Specifically, OTU and ankyrin domains can be found only in VT-Spiroplasma, and RIP domains are found in all VT Spiroplasma and three non-VT Spiroplasma. These domains are frequently associated with Spiroplasma plasmids, suggesting a possible mechanism for dispersal and maintenance among heritable strains. Searching insect genome assemblies available on public databases uncovered uncharacterized Spiroplasma genomes from which we identified several spaid-like genes encoding RIP, OTU, and ankyrin domains, suggesting functional interactions among those domain types. Our results suggest a conserved core of symbiont domains play an important role in the evolution and persistence of VT Spiroplasma in insects.

A G-type Lectin Receptor Kinase Negatively Regulates Arabidopsis Immunity Against Root-Knot Nematodes.

Root-knot nematodes (Meloidogyne spp., RKN) are responsible for extensive crop losses worldwide. During infection, they penetrate plant roots, migrate between plant cells, and establish feeding sites, known as giant cells, near the root vasculature. Previously, we found that nematode perception and early responses in plants were similar to those of microbial pathogens and required the BRI1-ASSOCIATED KINASE1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 (BAK1/SERK3) coreceptor in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum). Here, we implemented a reverse genetic screen for resistance or sensitivity to RKN using Arabidopsis T-DNA alleles of genes encoding transmembrane receptor-like kinases to identify additional receptors involved in this process. This screen identified a pair of allelic mutations with enhanced resistance to RKN in a gene we named ENHANCED RESISTANCE TO NEMATODES1 (ERN1). ERN1 encodes a G-type lectin receptor kinase (G-LecRK) with a single-pass transmembrane domain. Further characterization showed that ern1 mutants displayed stronger activation of MAP kinases, elevated levels of the defense marker MYB51, and enhanced H2O2 accumulation in roots upon RKN elicitor treatments. Elevated MYB51 expression and ROS bursts were also observed in leaves of ern1 mutants upon flg22 treatment. Complementation of ern1.1 with 35S- or native promotor-driven ERN1 rescued the RKN infection and enhanced defense phenotypes. Our results indicate that ERN1 is an important negative regulator of immunity.

Abstract 648: Oral microbiome reprograms oral cancer stem cells to the highly immunosuppressive tumor stemness defense (TSD) phenotype

Abstract Oral squamous cell carcinoma (OSCC) is a global health disease affecting developing countries, mainly the South and South East Asian countries. This cancer type is mostly HPV-16 negative and exhibits aggressiveness and resistance to therapies. Our previous studies have shown an oral microbiota (Fusobacterium nucleatum a gram negative anaerobic bacillus) associated with tumour progression in oral squamous cell carcinoma (1). The enhanced degree of stemness involved may confer the CSCs to be highly aggressive, self-sufficient, transient and niche modulatory, the tumour stemness defense (TSD) phenotype (2). The TSD characteristics are expressed as a part of “stem cell niche defence mechanism” (2). The TSD phenotype in ABCG2+ CSCs are characterized by the activation of the MYC-HIF-2alpha stemness pathway (3), as well as altruistic defense mechanism (4). Here we further studied the role of F.N. in reprograming of oral CSCs to TSD phenotype. Methods: SCC-25 cell line containing EpCAM+/ABCG2+ CSCs were treated with saliva samples of oral cancer patients (n=15). The cells were grown in antibiotic-treated media for 72 hours and then grown in serum-free conditioned media obtained from naïve MSC culture (2). The treated cells were then subjected to immunomagnetic sorting to obtain EPCAM+/ABCG2+ CSCs; TSD phenotype of the sorted CSCs were evaluated as described (2). Some cells were lysed and subjected to bacterial culture to detect F.nucleatum. Next, we evaluated the therapy resistance of the saliva-treated CSCs with chemotherapy, targeted therapy, immunotherapy and small molecular inhibitors of stemness pathways. We also obtained primary oral cancer EpCAM+/ABCG2+ of platinum treated patients (n=12), and lysed these cells for bacterial culture to detect F.nucleatum phenotype. Results: We confirmed the previous findings that F.nucleatum can be recovered from the EpCAM+/ABCG2+ of SCC-25 cells treated with patient derived saliva (processed saliva samples from relapsed cases; n=6/16 live bacteria recovered). These EpCAM+/ABCG2+ CSCs exhibited TSD phenotype, including the activation of MYC-HIF-2alpha stemness pathway, as well as TLR2/4/NFkB pathway. Importantly, the intracellular F.nucleatum exhibited an increase in surface proteins Fap 2, FadA, the two proteins involved in oncogenesis. We confirmed the FN induced TSD phenotype by treating SCC-25 cells with laboratory derived FN (ATCC 25586). Importantly, the primary tumor derived EpCAM+/ABCG2+ CSCs of 6/18 subjects exhibited the presence of F.N. Conclusion: Our findings indicate the potential role of oral microbiome especially the F. nulceatum in oral cancer stem cell reprograming to the highly aggressive TSD phenotype. 1. Cancer research. 2018;78(13 Supplement):3064-. 2. PMID: 362488583. 3. PMID: 33887214.4. PMID: 31266772 Citation Format: Partha Jyoti Saikia, Lekhika Pathak, Shirsajit Mitra, Bikul Das. Oral microbiome reprograms oral cancer stem cells to the highly immunosuppressive tumor stemness defense (TSD) phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 648.

Abstract 2772: The development of immunotherapy agents from the indigenous herbal plants and microbes used by the tantric practitioner of Vedic Jiva Upakara Cikitsha Tantra (Vedic altruism-based medicine system) of Assam, India

Abstract The ancient India's indigenous tantric community had its unique medicinal philosophy as well as clinical practice, distinct from Ayurveda. One such tantric community was Assam's Vedic Jiva Upakarvada practitioners (1). These practitioners developed an unique health care practice in the silk village of Sualkuchi, and the adjoining area, a part of the Vedic Silk Road (the North-South Silk Road stretching from Tibet to South East Asia via India's Assam state). We have been systematically studying the medical practice of these healing practitioners for the last two decades (1). These practitioners used more than 36 different plant-derived products, as well as soil, plus fecal and urine products rich in diverse microbial communities. They postulated that these agents contain good nigudah (1), microscopic life forms that can activate Avatar Kosha in our body (1). Using our pre-clinical in vitro as well as mouse model of cancer stem cells (2-4), we have screened 12 out of 36 plants, as well as microbial-rich soil of a sacred hill area frequented by tantric practitioners to collect herbal plants, animal feces and soils (the Suad muni ashram area of Sualkuchi). Here we report our preliminary results of the screening of these ingredients for their potential immunotherapy uses. Method: These herbal plants/soils/animal feces items were dissolved in the RPMI culture media using a traditional method using water, as well as special plant-derived juices. The conditioned RPMI culture media was then used to culture CSCs of tumor stemness defense (TSD) phenotype (2). Results: Our preliminary results show that while some of the agents induce apoptosis of TSD, other agents are capable of inducing TSD phenotypes to CSCs of diverse tumors including oral cancer (SCC-25 cell line), breast cancer (MCF-7) and colon cancer cell line (LOVO). Conclusion: The traditional healer used products may have unique anti-cancer including immuno-activating properties that need further exploration. We are now using our mouse models to find out if some of these agents including unique microbes present in these agents may transform cold tumors into hot or immunogenic tumors. 1. https://www.biorxiv.org/content/10.1101/2020.11.14.382572v1.full.pdf 2.PMID: 36248858 3. PMID: 28884113. 3. PMID: 31387932. 4. PMID: 31266772 Citation Format: Lekhika Pathak, Tutumoni Baishya, Partha Saikia, Nihar Ranjan Das, Sailen Baishya, Rupam Das, Pragya Dutta, Chayanika Das, Mallika Maral, Gakul Das, Bikul Das. The development of immunotherapy agents from the indigenous herbal plants and microbes used by the tantric practitioner of Vedic Jiva Upakara Cikitsha Tantra (Vedic altruism-based medicine system) of Assam, India [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2772.

Aloe barbadensis rinds employ physical and chemical defense mechanisms against insect herbivores with varying success

Plants deter herbivory through a suite of physical and chemical defenses. While most studies have focused on understanding one or the other, efficient plant defense against herbivory is often cumulative. To examine this, we used Aloe barbadensis (Asphodelaceae) as a model, since in natural settings, the species is found to be well-defended against insect herbivores. To start with, we hypothesized that the epicuticular wax potentially plays an important role in herbivore defense as the major physical defense. To test our hypothesis, we conducted bioactivity-guided extraction and polarity-based fractionation of A. barbadensis rinds. We then mixed them into an artificial diet, to determine the effect of these compounds on the growth and development of two species of caterpillars, tobacco hornworm (Manduca sexta; Lepidoptera: Sphingidae) and fall armyworm (Spodoptera frugiperda; Lepidoptera: Noctuidae). Thereafter, we identified and quantified different volatile compounds emitted by the epicuticular waxes to analyze if any herbivore deterrent compounds are emitted and might be responsible for A. barbadensis repelling insect herbivores. Our results show that the surface waxes and volatiles prevented the feeding of the larvae, and the extracts from the rinds differentially affected their growth and development. Our results do not definitively explain the entire defense phenotype; however, they do help to shed light on the combination of physical and chemical mechanisms enacted by the species.

Adverse effects of inbreeding on the transgenerational expression of herbivore-induced defense traits in Solanum carolinense.

In addition to directly inducing physical and chemical defenses, herbivory experienced by plants in one generation can influence the expression of defensive traits in offspring. Plant defense phenotypes can be compromised by inbreeding, and there is some evidence that such adverse effects can extend to the transgenerational expression of induced resistance. We explored how the inbreeding status of maternal Solanum carolinense plants influenced the transgenerational effects of herbivory on the defensive traits and herbivore resistance of offspring. Manduca sexta caterpillars were used to damage inbred and outbred S. carolinense maternal plants and cross pollinations were performed to produced seeds from herbivore-damaged and undamaged, inbred and outbred maternal plants. Seeds were grown in the greenhouse to assess offspring defense-related traits (i.e., leaf trichomes, internode spines, volatile organic compounds) and resistance to herbivores. We found that feeding by M. sexta caterpillars on maternal plants had a positive influence on trichome and spine production in offspring and that caterpillar development on offspring of herbivore-damaged maternal plants was delayed relative to that on offspring of undamaged plants. Offspring of inbred maternal plants had reduced spine production, compared to those of outbred maternal plants, and caterpillars performed better on the offspring of inbred plants. Both herbivory and inbreeding in the maternal generation altered volatile emissions of offspring. In general, maternal plant inbreeding dampened transgenerational effects of herbivory on offspring defensive traits and herbivore resistance. Taken together, this study demonstrates that inducible defenses in S. carolinense can persist across generations and that inbreeding compromises transgenerational resistance in S. carolinense.

Targeting hypoxia-induced tumor stemness by activating pathogen-induced stem cell niche defense.

Tumor hypoxia and oxidative stress reprograms cancer stem cells (CSCs) to a highly aggressive and inflammatory phenotypic state of tumor stemness. Previously, we characterized tumor stemness phenotype in the ATP Binding Cassette Subfamily G Member 2 (ABCG2)–positive migratory side population (SPm) fraction of CSCs exposed to extreme hypoxia followed by reoxygenation. Here, we report that post-hypoxia/reoxygenation SPm+/ABCG2+ CSCs exerts defense against pathogen invasion that involves bystander apoptosis of non-infected CSCs. In an in vitro assay of cancer cell infection by Bacillus Calmette Guerin (BCG) or mutant Mycobacterium tuberculosis (Mtb) strain 18b (Mtb-m18b), the pathogens preferentially replicated intracellular to SPm+/ABCG2+ CSCs of seven cell lines of diverse cancer types including SCC-25 oral squamous cancer cell line. The conditioned media (CM) of infected CSCs exhibited direct anti-microbial activity against Mtb and BCG, suggesting niche defense against pathogen. Importantly, the CM of infected CSCs exhibited marked in vitro bystander apoptosis toward non-infected CSCs. Moreover, the CM-treated xenograft bearing mice showed 10- to 15-fold reduction (p < 0.001; n = 7) in the number of CSCs residing in the hypoxic niches. Our in vitro studies indicated that BCG-infected SPm+/ABCG2+ equivalent EPCAM+/ABCG2+ CSCs of SCC-25 cells underwent pyroptosis and released a high mobility group box protein 1 (HMGB1)/p53 death signal into the tumor microenvironment (TME). The death signal can induce a Toll-like receptor 2/4–mediated bystander apoptosis in non-infected CSCs by activating p53/MDM2 oscillation and subsequent activation of capase-3–dependent intrinsic apoptosis. Notably, SPm+/ABCG2+ but not SP cells undergoing bystander apoptosis amplified the death signal by further release of HMGB1/p53 complex into the TME. These results suggest that post-hypoxia SPm+/ABCG2+ CSCs serve a functional role as a tumor stemness defense (TSD) phenotype to protect TME against bacterial invasion. Importantly, the CM of TSD phenotype undergoing bystander apoptosis may have therapeutic uses against CSCs residing in the hypoxic niche.

Prey defence phenotype mediates multiple-predator effects in tri-trophic food webs.

The emphasis on mechanisms governing the interaction among predators (e.g. cooperation, competition or intraguild predation) has driven the understanding of multiple-predator effects on prey survival and dynamics. However, overwhelming evidence shows that prey can adaptively respond to predators, exhibiting multiple defensive phenotypes to cope with predation. Nevertheless, there is still a relatively scarce theory connecting the emergence of prey defences in complex multi-predator scenarios and their ecological consequences. Using a mathematical approach, we evaluated the prevalence of defended prey phenotypes as a function of predator-induced mortality in a two-predator system, and how prey and phenotype dynamics affect trophic cascades. We also evaluated such responses when prey manifests a general defence against both predators (i.e. risk reducing) or a specialized defence against one predator at the expense of defence against the other predator (i.e. risk trade-off), and when such phenotypes induce fitness and foraging costs. We showed that the emergence of defended phenotypes under multiple predators depends on predator-induced mortality rates, the magnitude of phenotype costs and the effect of the defensive phenotype on the performance of all predators. Risk-reducing phenotypes enhance prioritized responses to predators with high killing rates, but prioritized responses are diminished when prey manifest risk trade-off phenotypes. Finally, we showed that resource abundance across the predation gradient directly depends on the prevalence of certain prey phenotypes and their effect on foraging costs. Ultimately, our results depict the implications of prey defences on prey and basal resources abundance in a multiple predators' environment, highlighting the role of the identity of defensive strategies in mediating the strength and nature of trophic cascades, via consumptive or non-consumptive effects.

A. thaliana Hybrids Develop Growth Abnormalities through Integration of Stress, Hormone and Growth Signaling.

Hybrids between Arabidopsis thaliana accessions are important in revealing the consequences of epistatic interactions in plants. F1 hybrids between the A. thaliana accessions displaying either defense or developmental phenotypes have been revealing the roles of the underlying epistatic genes. The interaction of two naturally occurring alleles of the OUTGROWTH-ASSOCIATED KINASE (OAK) gene in Sha and Lag2-2, previously shown to cause a similar phenotype in a different allelic combination in A. thaliana, was required for the hybrid phenotype. Outgrowth formation in the hybrids was associated with reduced levels of salicylic acid, jasmonic acid and abscisic acid in petioles and the application of these hormones mitigated the formation of the outgrowths. Moreover, different abiotic stresses were found to mitigate the outgrowth phenotype. The involvement of stress and hormone signaling in outgrowth formation was supported by a global transcriptome analysis, which additionally revealed that TCP1, a transcription factor known to regulate leaf growth and symmetry, was downregulated in the outgrowth tissue. These results demonstrate that a combination of natural alleles of OAK regulates growth and development through the integration of hormone and stress signals and highlight the importance of natural variation as a resource to discover the function of gene variants that are not present in the most studied accessions of A. thaliana.

Is PTSD an Evolutionary Survival Adaptation Initiated by Unrestrained Cytokine Signaling and Maintained by Epigenetic Change?

Treatment outcomes for PTSD with current psychological therapies are poor, with very few patients achieving sustained symptom remission. A number of authors have identified physiological and immune disturbances in Post Traumatic Stress Disorder (PTSD) patients, but there is no unifying hypothesis that explains the myriad features of the disorder. The medical literature was reviewed over a 6-year period primarily using the medical database PUBMED. The literature contains numerous papers that have identified a range of physiological and immune dysfunction in association with PTSD. This paper proposes that unrestrained cytokine signaling induces epigenetic changes that promote an evolutionary survival adaptation, which maintains a defensive PTSD phenotype. The brain can associate immune signaling with past threat and initiate a defensive behavioral response. The sympathetic nervous system is pro-inflammatory, while the parasympathetic nervous system is anti-inflammatory. Prolonged cholinergic withdrawal will promote a chronic inflammatory state. The innate immune cytokine IL-1β has pleiotropic properties and can regulate autonomic, glucocorticoid, and glutamate receptor functions, sleep, memory, and epigenetic enzymes. Changes in epigenetic enzyme activity can potentially alter phenotype and induce an adaptation. Levels of IL-1β correlate with severity and duration of PTSD and PTSD can be prevented by bolus administration of hydrocortisone in acute sepsis, consistent with unrestrained inflammation being a risk factor for PTSD. The nervous and immune systems engage in crosstalk, governed by common receptors. The benefits of currently used psychiatric medication may arise from immune, as well as synaptic, modulation. The psychedelic drugs (3,4-Methylenedioxymethamphetamine (MDMA), psilocybin, and ketamine) have potent immunosuppressive and anti-inflammatory effects on the adaptive immune system, which may contribute to their reported benefit in PTSD. There may be distinct PTSD phenotypes induced by innate and adaptive cytokine signaling. In order for an organism to survive, it must adapt to its environment. Cytokines signal danger to the brain and can induce epigenetic changes that result in a persistent defensive phenotype. PTSD may be the price individuals pay for the genomic flexibility that promotes adaptation and survival.

Development and experience-dependent modulation of the defensive behaviors of mice to visual threats

Rodents demonstrate defensive behaviors such as fleeing or freezing upon recognizing a looming shadow above them. Although individuals’ experiences in their habitat can modulate the defensive behavior phenotype, the effects of systematically manipulating the individual’s visual experience on vision-guided defensive behaviors have not been studied. We aimed to describe the developmental process of defensive behaviors in response to visual threats and the effects of visual deprivation. We found that the probability of escape response occurrence increased 3 weeks postnatally, and then stabilized. When visual experience was perturbed by dark rearing from postnatal day (P) 21 for a week, the developmental increase in escape probability was clearly suppressed, while the freezing probability increased. Intriguingly, exposure to the looming stimuli at P28 reversed the suppression of escape response development at P35. These results clearly indicate that the development of defensive behaviors in response to looming stimuli is affected by an individual’s sensory experience.

A roadmap for future research on insularity effects on plant–herbivore interactions

AbstractState of the artTheory predicts that herbivore pressure should be weaker on islands than on the mainland, owing to lower herbivore abundance and diversity because of dispersal constraints and environmental filtering. As a result, plants on islands should invest less in defences against herbivory. Although early empirical studies supported these predictions, recent systematic island–mainland comparisons have questioned this paradigm, with some studies reporting either no difference between islands and mainland or higher herbivory and plant defences on islands. Current data therefore appear to be unsupportive of predictions on insularity effects on plant–herbivore interactions, calling for more research to reassess predictions and to test underlying mechanisms for observed patterns.Research opportunitiesTo meet this challenge, a renewed research programme based on the accrual of studies with specific features is needed. These should include more robust experimental designs with replication within and across systems, integrative and more nuanced assessments of plant defensive phenotypes and herbivory, a food web approach that considers the multi‐trophic context in which plant–herbivore interactions are embedded, and a consideration of historical factors (e.g., island origin and biogeographical factors, defensive anachronisms).OutlookThis new research programme will require integration of evolutionary ecology research on plant–herbivore interactions with island biogeography, palaeoecology and community ecology to understand the influence of factors acting at different scales, from local factors driving herbivory and plant defences to historical processes and regional drivers of species composition determining species traits and their interactions.

Plant invasion alters latitudinal pattern of plant-defense syndromes.

The relationship between herbivory and latitude may differ between native and introduced populations of invasive plants, which can generate latitudinal heterogeneity in the strength of enemy release. However, still little is known about how latitudinal heterogeneity in herbivore pressure influences latitudinal variation in defense phenotypes of invasive plants. We tested how latitudinal patterns in multi-variate defense syndromes differed between native (Argentinian) and introduced (Chinese) populations of the invasive herb Alternanthera philoxeroides. In addition, to better understand the drivers underlying latitudinal patterns, we also tested whether associations of defense syndromes with climate and herbivory differed between native and introduced ranges. We found that native plant populations clustered into three main defense syndromes associated with latitude. In contrast, we only found two defense syndromes in the introduced range. One matched the high-latitude syndrome from the native range, but was distributed at both the northern and southern range limits in the introduced range. The other was unique to the introduced range and occurred at mid-latitudes. Climatic conditions were associated with variation in syndromes in the native range, and climatic conditions and herbivory were associated with variation in syndromes in the introduced range. Together, our results demonstrate that plants may under the new environmental conditions in the introduced range show latitudinal patterns of defense syndromes that are different from those in their native range. This emphasizes that geographical dependence of population differentiation should be explicitly considered in studies on the evolution of defense in invasive plants.

Genome-Wide Association Study Reveals Genomic Region Associated with Mite-Recruitment Phenotypes in the Domesticated Grapevine (Vitis vinifera).

Indirect defenses are plant phenotypes that reduce damage by attracting natural enemies of plant pests and pathogens to leaves. Despite their economic and ecological importance, few studies have investigated the genetic underpinnings of indirect defense phenotypes. Here, we present a genome-wide association study of five phenotypes previously determined to increase populations of beneficial (fungivorous and predacious) mites on grape leaves (genus Vitis): leaf bristles, leaf hairs, and the size, density, and depth of leaf domatia. Using a common garden genetic panel of 399 V. vinifera cultivars, we tested for genetic associations of these phenotypes using previously obtained genotyping data from the Vitis9kSNP array. We found one single nucleotide polymorphism (SNP) significantly associated with domatia density. This SNP (Chr5:1160194) is near two genes of interest: Importin Alpha Isoform 1 (VIT_205s0077g01440), involved in downy mildew resistance, and GATA Transcription Factor 8 (VIT_205s0077g01450), involved in leaf shape development. Our findings are among the first to examine the genomic regions associated with ecologically important plant traits that facilitate interactions with beneficial mites, and suggest promising candidate genes for breeding and genetic editing to increase naturally occurring predator-based defenses in grapevines.