Potential evidence of peripheral learning and memory in the arms of dwarf cuttlefish, Sepia bandensis.

CREB (cAMP response element-binding) transcription factors are conserved markers of memory formation in the brain and peripheral circuits. We provide immunohistochemical evidence of CREB phosphorylation in the dwarf cuttlefish, Sepia bandensis, following the inaccessible prey (IP) memory experiment. During the IP experiment, cuttlefish are shown prey enclosed in a transparent tube, and tentacle strikes against the tube decrease over time as the cuttlefish learns the prey is inaccessible. The cues driving IP learning are unclear but may include sensory inputs from arms touching the tube. The neural activity marker, anti-phospho-CREB (anti-pCREB) was used to determine whether IP training stimulated cuttlefish arm sensory neurons. pCREB immunoreactivity occurred along the oral surface of the arms, including the suckers and epithelial folds surrounding the suckers. pCREB increased in the epithelial folds and suckers of trained cuttlefish. We found differential pCREB immunoreactivity along the distal-proximal axis of trained arms, with pCREB concentrated distally. Unequal CREB phosphorylation occurred among the 4 trained arm pairs, with arm pairs 1 and 2 containing more pCREB. The resulting patterns of pCREB in trained arms suggest that the arms obtain cues that may be salient for learning and memory of the IP experiment.

Evidence of learning and memory in the juvenile dwarf cuttlefish Sepia bandensis.

Measuring behavior in the form of numerical data is difficult, especially for studies involving complex actions. DanioVision is a closed-chamber system that utilizes subject tracking to comprehensively record behavior, while also mitigating the influence of environmental conditions. We used DanioVision to record activity of juvenile dwarf cuttlefish (Sepia bandensis) during the inaccessible prey (IP) procedure, a memory experiment in which cuttlefish learn to inhibit capture attempts towards inaccessible prey. By quantifying total movement and orientation of the body, we found that cuttlefish show memory by selectively inhibiting tentacle strikes without reducing total movement, or orientation towards the prey. We show that DanioVision can be used to assess multiple components of dynamic responses that are not measurable by direct observation alone and provide new evidence that strike inhibition is the product of learning, and not motor fatigue.

A pheromone receptor in cichlid fish mediates attraction to females but inhibits male parental care.

Reproductive behaviors differ across species, but the mechanisms that control variation in mating and parental care systems remain unclear. In many animal species, pheromones guide mating and parental care. However, it is not well understood how vertebrate pheromone signaling evolution can lead to new reproductive behavior strategies. In fishes, prostaglandin F2α (PGF2α) drives mating and reproductive pheromone signaling in fertile females, but this pheromonal activity appears restricted to specific lineages, and it remains unknown how a female fertility pheromone is sensed for most fish species. Here, we utilize single-cell transcriptomics and CRISPR gene editing in a cichlid fish model to identify and test the roles of key genes involved in olfactory sensing of reproductive cues. We find that a pheromone receptor, Or113a, detects fertile cichlid females and thereby promotes male attraction and mating behavior, sensing a ligand other than PGF2α. Furthermore, while cichlid fishes exhibit extensive parental care, for most species, care is provided solely by females. We find that males initiate mouthbrooding parental care if they have disrupted signaling in ciliated sensory neurons due to cnga2b mutation or if or113a is inactivated. Together, these results show that distinct mechanisms of pheromonal signaling drive reproductive behaviors across taxa. Additionally, these findings indicate that a single pheromone receptor has gained a novel role in behavior regulation, driving avoidance of paternal care among haplochromine cichlid fishes. Lastly, a sexually dimorphic, evolutionarily derived parental behavior is controlled by central circuits present in both sexes, while olfactory signals gate this behavior in a sex-specific manner.

Pheromone Perception in Fish: Mechanisms and Modulation by Internal Status.

Pheromones are chemical signals that facilitate communication between animals, and most animals use pheromones for reproduction and other forms of social behavior. The identification of key ligands and olfactory receptors used for pheromonal communication provides insight into the sensory processing of these important cues. An individual's responses to pheromones can be plastic, as physiological status modulates behavioral outputs. In this review, we outline the mechanisms for pheromone sensation and highlight physiological mechanisms that modify pheromone-guided behavior. We focus on hormones, which regulate pheromonal communication across vertebrates including fish, amphibians, and rodents. This regulation may occur in peripheral olfactory organs and the brain, but the mechanisms remain unclear. While this review centers on research in fish, we will discuss other systems to provide insight into how hormonal mechanisms function across taxa.

A Novel 3DNA® Nanocarrier effectively delivers payloads to pancreatic tumors.

INTRODUCTION: Standard-of-care systemic chemotherapies for pancreatic ductal adenocarcinoma (PDAC) currently have limited clinical benefits, in addition to causing adverse side effects in many patients. One factor known to contribute to the poor chemotherapy response is the poor drug diffusion into PDAC tumors. Novel treatment methods are therefore drastically needed to improve targeted delivery of treatments. Here, we evaluated the efficacy of the 3DNA® Nanocarrier (3DNA) platform to direct delivery of therapeutics to PDAC tumors in vivo. MATERIALS AND METHODS: A panel of PDAC cell lines and a patient tissue microarray were screened for established tumor-specific proteins to identify targeting moieties for active targeting of the 3DNA. NRG mice with or without orthotopic MIA PaCa-2-luciferase PDAC tumors were treated intraperitoneally with 100 µl of fluorescently labeled 3DNA. RESULTS: Folic acid and transferrin receptors were significantly elevated in PDAC compared to normal pancreas. Accordingly, both folic acid- and transferrin-conjugated 3DNA treatments significantly increased delivery of 3DNA specifically to tumors in comparison to unconjugated 3DNA treatment. In the absence of tumors, there was an increased clearance of both folic acid-conjugated 3DNA and unconjugated 3DNA, compared to the clearance rate in tumor-bearing mice. Lastly, delivery of siLuciferase by folic acid-conjugated 3DNA in an orthotopic model of luciferase-expressing PDAC showed significant and prolonged suppression of luciferase protein expression and activity. CONCLUSION: Our study progresses the 3DNA technology as a reliable and effective treatment delivery platform for targeted therapeutic approaches in PDAC.

MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium.

We have shown that smoking impacts bronchial airway gene expression and that heterogeneity in this response associates with smoking-related disease risk. In this study, we sought to determine whether microRNAs (miRNAs) play a role in regulating the airway gene expression response to smoking. We examined whole-genome miRNA and mRNA expression in bronchial airway epithelium from current and never smokers (n = 20) and found 28 miRNAs to be differentially expressed (P < 0.05) with the majority being down-regulated in smokers. We further identified a number of mRNAs whose expression level is highly inversely correlated with miRNA expression in vivo. Many of these mRNAs contain potential binding sites for the differentially expressed miRNAs in their 3'-untranslated region (UTR) and are themselves affected by smoking. We found that either increasing or decreasing the levels of mir-218 (a miRNA that is strongly affected by smoking) in both primary bronchial epithelial cells and H1299 cells was sufficient to cause a corresponding decrease or increase in the expression of predicted mir-218 mRNA targets, respectively. Further, mir-218 expression is reduced in primary bronchial epithelium exposed to cigarette smoke condensate (CSC), and alteration of mir-218 levels in these cells diminishes the induction of the predicted mir-218 target MAFG in response to CSC. These data indicate that mir-218 levels modulate the airway epithelial gene expression response to cigarette smoke and support a role for miRNAs in regulating host response to environmental toxins.

Tuning Design Parameters of ICAM-1-Targeted 3DNA Nanocarriers to Optimize Pulmonary Targeting Depending on Drug Type.

3DNA holds promise as a carrier for drugs that can be intercalated into its core or linked to surface arms. Coupling 3DNA to an antibody targeting intercellular adhesion molecule 1 (ICAM-1) results in high lung-specific biodistributions in vivo. While the role of individual parameters on ICAM-1 targeting has been studied for other nanocarriers, it has never been examined for 3DNA or in a manner capable of revealing the hierarchic interplay among said parameters. In this study, we used 2-layer vs. 4-layer anti-ICAM 3DNA and radiotracing to examine biodistribution in mice. We found that, below saturating conditions and within the ranges tested, the density of targeting antibodies on 3DNA is the most relevant parameter driving lung targeting over liver clearance, compared to the number of antibodies per carrier, total antibody dose, 3DNA dose, 3DNA size, or the administered concentration, which influenced the dose in organs but not the lung specific-over-liver clearance ratio. Data predicts that lung-specific delivery of intercalating (core loaded) drugs can be tuned using this biodistribution pattern, while that of arm-linked (surface loaded) drugs requires a careful parametric balance because increasing anti-ICAM density reduces the number of 3DNA arms available for drug loading.

Sustained Release of Antibody-Conjugated DNA Nanocarriers from a Novel Injectable Hydrogel for Targeted Cell Depletion to Treat Cataract Posterior Capsule Opacification.

Purpose: To compare a novel, sustained release formulation and a bolus injection of a targeted nanocarrier for the ability to specifically deplete cells responsible for the development of posterior capsule opacification (PCO) in week-long, dynamic cell cultures. Methods: A novel, injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymer hydrogel was engineered for the sustained release of targeted, nucleic acid nanocarriers loaded with cytotoxic doxorubicin (G8:3DNA:Dox). Human rhabdomyosarcoma (RD) cells were used due to their expression of brain-specific angiogenesis inhibitor 1 (BAI1), a specific marker for the myofibroblasts responsible for PCO. Under constant media flow, nanocarriers were injected into cell cultures as either a bolus or within the hydrogel. Cells were fixed and stained every other day for 7 days to compare targeted depletion of BAI1+ cells. Results: The formulation transitions to a gel at physiological temperatures, is optically clear, noncytotoxic, and can release G8:3DNA:Dox nanocarriers for up to 4 weeks. In RD cell cultures, G8:3DNA:Dox nanocarriers specifically eliminated BAI1+ cells. The bolus nanocarrier dose showed significantly reduced cell depletion overtime, while the sustained release of nanocarriers showed increased cell depletion over time. By day 7, <2% of BAI1+ cells were depleted by the bolus injection and 74.2% BAI1+ cells were targeted by the sustained release of nanocarriers. Conclusions: The sustained release of nanocarriers from the hydrogel allows for improved therapeutic delivery in a dynamic system. This method can offer a more effective and efficient method of prophylactically treating PCO after cataract surgery.

Limited nutrient availability in the tumor microenvironment renders pancreatic tumors sensitive to allosteric IDH1 inhibitors.

Nutrient-deprived conditions in the tumor microenvironment (TME) restrain cancer cell viability due to increased free radicals and reduced energy production. In pancreatic cancer cells a cytosolic metabolic enzyme, wild-type isocitrate dehydrogenase 1 (wtIDH1), enables adaptation to these conditions. Under nutrient starvation, wtIDH1 oxidizes isocitrate to generate α-ketoglutarate (αKG) for anaplerosis and NADPH to support antioxidant defense. In this study, we show that allosteric inhibitors of mutant IDH1 (mIDH1) are potent wtIDH1 inhibitors under conditions present in the TME. We demonstrate that low magnesium levels facilitate allosteric inhibition of wtIDH1, which is lethal to cancer cells when nutrients are limited. Furthermore, the Food & Drug Administration (FDA)-approved mIDH1 inhibitor ivosidenib (AG-120) dramatically inhibited tumor growth in preclinical models of pancreatic cancer, highlighting this approach as a potential therapeutic strategy against wild-type IDH1 cancers.

A method to improve quantitative radiotracing-based analysis of the in vivo biodistribution of drug carriers.

Biodistribution studies are essential in drug carrier design and translation, and radiotracing provides a sensitive quantitation for this purpose. Yet, for biodegradable formulations, small amounts of free-label signal may arise prior to or immediately after injection in animal models, causing potentially confounding biodistribution results. In this study, we refined a method to overcome this obstacle. First, we verified free signal generation in animal samples and then, mimicking it in a controllable setting, we injected mice intravenously with a radiolabeled drug carrier formulation (125I-antibody/3DNA) containing a known amount of free radiolabel (125I), or free 125I alone as a control. Corrected biodistribution data were obtained by separating the free radiolabel from blood and organs postmortem, using trichloroacetic acid precipitation, and subtracting the confounding signal from each tissue measurement. Control free 125I-radiolabel was detected at ≥85% accuracy in blood and tissues, validating the method. It biodistributed very heterogeneously among organs (0.6-39 %ID/g), indicating that any free 125I generated in the body or present in an injected formulation cannot be simply corrected to the free-label fraction in the original preparation, but the free label must be empirically measured in each organ. Application of this method to the biodistribution of 125I-antibody/3DNA, including formulations directed to endothelial target ICAM-1, showed accurate classification of free 125I species in blood and tissues. In addition, this technique rendered data on the in vivo degradation of the traced agents over time. Thus, this is a valuable technique to obtain accurate measurements of biodistribution using 125I and possibly other radiotracers.