Riverine Discharge Fuels the Production of Methylmercury in a Large Temperate Estuary.

Estuaries are an important food source for the world's growing population, yet human health is at risk from elevated exposure to methylmercury (MeHg) via the consumption of estuarine fish. Moreover, the sources and cycling of MeHg in temperate estuarine ecosystems are poorly understood. Here, we investigated the seasonal and tidal patterns of mercury (Hg) forms in Long Island Sound (LIS), in a location where North Atlantic Ocean waters mix with the Connecticut River. We found that seasonal variations in Hg and MeHg in LIS followed the extent of riverine Hg delivery, while tides further exacerbated the remobilization of earlier deposited riverine Hg. The net production of MeHg near the river plume was significant compared to that in other locations and enhanced during high tide, possibly resulting from the enhanced microbial activity and organic carbon remineralization in the river plume. Statistical models, driven by our novel data, further support the hypothesis that the river-delivered organic matter and inorganic Hg drive net MeHg production in the estuarine water column. Our study sheds light on the significance of water column biogeochemical processes in temperate tidal estuaries in regulating MeHg levels and inspires new questions in our quest to understand MeHg sources and dynamics in coastal oceans.

Reducing Hypoxia in an Urban Estuary Despite Climate Warming.

Seasonal hypoxia is a serious threat to coastal ecosystems. This study on hypoxia in Long Island Sound (LIS), a large urbanized estuary, focuses on responses to managed nitrogen load reductions and climate change. At the analyzed station in western LIS, warming in bottom waters (0.8 °C per decade) favors hypoxia. Total nitrogen concentrations have decreased (0.06 mg L-1 per decade) with load reductions, but no linear temporal trend in chlorophyll is discernible. Bottom dissolved oxygen has increased (0.48 mg L-1 per decade), despite warming-induced solubility decreases (0.13 mg L-1 per decade). Decreasing trends in hypoxic area and volume (100 km2 and 1 km3 per decade) reflect improved conditions and are coincident with reducing loads. Regressions link hypoxic extent to nitrogen loads, chlorophyll, salinity, and winds. Though mitigation has reduced hypoxia, these improvements will not be sustained in the warming climate without continued intervention. The warming-induced oxygen solubility decrease forecasted for 2099 (0.4 mg L-1) would erode 35% of the observed oxygen gains. Implementing a nitrogen load reduction of 1.2 × 106 kg year-1 before the century's end would offset the oxygen solubility decline. This overall approach is applicable to areas experiencing warming and continued development that complicate efforts to reign in hypoxia.

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization.

Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41's binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue-NP41-bound nerves, followed by biotin hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10-7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve "ghosts" months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.

Rapid continuous 3D printing of customizable peripheral nerve guidance conduits.

Engineered nerve guidance conduits (NGCs) have been demonstrated for repairing peripheral nerve injuries. However, there remains a need for an advanced biofabrication system to build NGCs with complex architectures, tunable material properties, and customizable geometrical control. Here, a rapid continuous 3D-printing platform was developed to print customizable NGCs with unprecedented resolution, speed, flexibility, and scalability. A variety of NGC designs varying in complexity and size were created including a life-size biomimetic branched human facial NGC. In vivo implantation of NGCs with microchannels into complete sciatic nerve transections of mouse models demonstrated the effective directional guidance of regenerating sciatic nerves via branching into the microchannels and extending toward the distal end of the injury site. Histological staining and immunostaining further confirmed the progressive directional nerve regeneration and branching behavior across the entire NGC length. Observational and functional tests, including the von Frey threshold test and thermal test, showed promising recovery of motor function and sensation in the ipsilateral limbs grafted with the 3D-printed NGCs.

Early detection of thrombin activity in neuroinflammatory disease.

Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression.

Detection and monitoring of localized matrix metalloproteinase upregulation in a murine model of asthma.

Extracellular proteases including matrix metalloproteinases (MMPs) are speculated to play a significant role in chronic lung diseases, such as asthma. Although increased protease expression has been correlated with lung pathogenesis, the relationship between localized enzyme activity and disease progression remains poorly understood. We report the application of MMP-2/9 activatable cell-penetrating peptides (ACPPs) and their ratiometric analogs (RACPPs) for in vivo measurement of protease activity and distribution in the lungs of mice that were challenged with the allergen ovalbumin. MMP-2/9 activity was increased greater than twofold in whole, dissected lungs from acutely challenged mice compared with control mice (P=1.8×10(-4)). This upregulation of MMP-2/9 activity was localized around inflamed airways with 1.6-fold higher protease-dependent ACPP uptake surrounding diseased airways compared with adjacent, pathologically normal lung parenchyma (P=0.03). MMP-2/9 activity detected by ACPP cleavage colocalized with gelatinase activity measured with in situ dye-quenched gelatin. For comparison, neutrophil elastase activity and thrombin activity, detected with elastase- and thrombin-cleavable RACPPs, respectively, were not significantly elevated in acutely allergen-challenged mouse lungs. The results demonstrate that ACPPs, like the MMP-2/9-activated and related ACPPs, allow for real-time detection of protease activity in a murine asthma model, which should improve our understanding of protease activation in asthma disease progression and help elucidate new therapy targets or act as a mechanism for therapeutic drug delivery.

Thrombin activity associated with neuronal damage during acute focal ischemia.

Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core. Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.

Ratiometric activatable cell-penetrating peptides provide rapid in vivo readout of thrombin activation.

In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.

Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment.

In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. Here we present INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity vary across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identifies a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing reveals a connection with cDC1s, and immunofluorescence confirms nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assist binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties.

Translating state-of-the-art brain magnetic resonance imaging (MRI) techniques into clinical practice: multimodal MRI differentiates dementia subtypes in a traditional clinical setting.

BACKGROUND: This study sought to validate the clinical utility of multimodal magnetic resonance imaging (MRI) techniques in the assessment of neurodegenerative disorders. We intended to demonstrate that advanced neuroimaging techniques commonly used in research can effectively be employed in clinical practice to accurately differentiate heathy aging and dementia subtypes. METHODS: Twenty patients with dementia of the Alzheimer's type (DAT) and 18 patients with Parkinson's disease dementia (PDD) were identified using gold-standard techniques. Twenty-three healthy, age and sex matched control participants were also recruited. All participants underwent multimodal MRI including T1 structural, diffusion tensor imaging (DTI), arterial spin labeling (ASL), and magnetic resonance spectroscopy (MRS). MRI modalities were evaluated by trained neuroimaging readers and were separately assessed using cross-validated, iterative discriminant function analyses with subsequent feature reduction techniques. In this way, each modality was evaluated for its ability to differentiate patients with dementia from healthy controls as well as to differentiate dementia subtypes. RESULTS: Following individual and group feature reduction, each of the multimodal MRI metrics except MRS successfully differentiated healthy aging from dementia and also demonstrated distinct dementia subtypes. Using the following ten metrics, excellent separation (95.5% accuracy, 92.3% sensitivity; 100.0% specificity) was achieved between healthy aging and neurodegenerative conditions: volume of the left frontal pole, left occipital pole, right posterior superior temporal gyrus, left posterior cingulate gyrus, right planum temporale; perfusion of the left hippocampus and left occipital lobe; fractional anisotropy (FA) of the forceps major and bilateral anterior thalamic radiation. Using volume of the left frontal pole, right posterior superior temporal gyrus, left posterior cingulate gyrus, perfusion of the left hippocampus and left occipital lobe; FA of the forceps major and bilateral anterior thalamic radiation, neurodegenerative subtypes were accurately differentiated as well (87.8% accuracy, 95.2% sensitivity; 85.0% specificity). CONCLUSIONS: Regional volumetrics, DTI metrics, and ASL successfully differentiated dementia patients from controls with sufficient sensitivity to differentiate dementia subtypes. Similarly, feature reduction results suggest that advanced analyses can meaningfully identify brain regions with the most positive predictive value and discriminant validity. Together, these advanced neuroimaging techniques can contribute significantly to diagnosis and treatment planning for individual patients.

Rapid and reversible nuclear accumulation of cytoplasmic tRNA in response to nutrient availability.

Cytoplasmic tRNAs have recently been found to accumulate in the nucleus during amino acid starvation in yeast. The mechanism and regulation by which tRNAs return to the nucleus are unclear. Here, we show accumulation of cytoplasmic tRNA in the nucleus also occurs during glucose starvation. Nuclear accumulation of tRNA in response to acute glucose or amino acid starvation is rapid, reversible, requires no new transcription, and is independent of the aminoacylation status of tRNA. Gradual depletion of nutrients also results in the accrual of tRNA in the nucleus. Distinct signal transduction pathways seem to be involved in the accumulation of cytoplasmic tRNA in the nucleus in response to amino acid versus glucose starvation. These findings suggest tRNA nucleocytoplasmic distribution may play a role in gene expression in response to nutritional stress.

ATF4 is necessary and sufficient for ER stress-induced upregulation of REDD1 expression.

In response to a variety of cell stresses, e.g. endoplasmic reticulum (ER) stress, expression of REDD1 (regulated in development and DNA damage responses) is transcriptionally upregulated. However, the mechanism through which ER stress acts to upregulate REDD1 expression is unknown. In the present study, REDD1 expression was found to be upregulated by ER stress in several cell lines. However, in MEF cells lacking the eIF2alpha kinase PERK, ER stress failed to upregulate REDD1 expression, demonstrating that phosphorylation of eIF2alpha was necessary for the effect. Moreover, ER stress led to upregulated expression of the transcription factor ATF4, but in MEF cells lacking ATF4, REDD1 mRNA expression was not increased by ER stress. In contrast, exogenous expression of ATF4 was sufficient to induce REDD1 expression. Overall, the results suggest that REDD1 expression is upregulated during ER stress through a mechanism involving activation of PERK, phosphorylation of eIF2alpha, and increased ATF4 expression.

Citizen science observations reveal rapid, multi-decadal ecosystem changes in eastern Long Island Sound.

Long-term environmental records are among the most valuable assets for understanding the trajectory and consequences of climate change. Here we report on a newly recovered time-series from Project Oceanology, a non-profit ocean science organization serving New England schools (USA) since 1972. As part of its educational mission, Project Oceanology has routinely and consistently recorded water temperature, pH, and oxygen as well as invertebrate and fish abundance in nearshore waters of the Thames River estuary in eastern Long Island Sound (LIS). We digitized these long-term records to test for decadal trends in abiotic and biotic variables including shifts in species abundance, richness, and diversity. Consistent with previous studies, the data revealed an above-average warming rate of eastern LIS waters over the past four decades (+0.45 °C decade-1), a non-linear acidification trend twice the global average (-0.04 pH units decade-1), and a notable decline in whole water-column dissolved oxygen concentrations (-0.29 mg L-1 decade-1). Trawl catches between 1997 and 2016 suggested a significant decrease in overall species diversity and richness, declines in cold-water adapted species such as American lobster (Homarus americanus), rock crab (Cancer irroratus), and winter flounder (Pseudopleuronectes americanus), but concurrent increases in the warm-water decapod Libinia emarginata (spider crab). Our study confirmed that Long Island Sound is a rapidly changing urban estuary, while demonstrating the value of long-term observations made by citizen-scientists, educators, and other stakeholders.

Occurrence and partitioning behavior of perfluoroalkyl acids in wastewater effluent discharging into the Long Island Sound.

Perfluoroalkyl acids (PFAAs) were measured in aqueous and suspended particulate matter (SPM) fractions in the final effluents from 12 wastewater treatment facilities located around the Connecticut shoreline. Aqueous phase concentrations ranged from 53 to 198 ng/L for ∑PFAAs with ≤7 perfluorinated carbons (CF2) and 2-73 ng/L for >7 CF2 PFAAs. Predominant PFAAs associated with effluent derived SPM were perfluorodecanoic acid and perflurorooctane sulfonic acid, detected in 48% and 52% of samples in concentrations ranging from 7 CF2 PFAAs. Partitioning coefficients (log KOC) derived for effluent water and SPM phases (4.2 ±â€¯0.3, 4.4 ±â€¯0.4, 5.1 ±â€¯0.2 and 5.3 ±â€¯0.2 for PFOA, PFNA PFDA and PFUnA; 4.5 ±â€¯0.2 and 5.2 ±â€¯0.2 for PFOS and PFHsX respectively) were found to be of similar magnitude to aeration tank particles, though 0.5 to 2 log units greater than sludge solids and to natural system particulates including riverine SPM, estuarine SPM and sediments. Results from this study suggest that effluent derived suspended particulate matter could be an effective vector in the transport of long-chained PFAAs through wastewater treatment into receiving waters, and a potential vector to the local food chain.

Nerve-targeted probes for fluorescence-guided intraoperative imaging.

A fundamental goal of many surgeries is nerve preservation, as inadvertent injury can lead to patient morbidity including numbness, pain, localized paralysis and incontinence. Nerve identification during surgery relies on multiple parameters including anatomy, texture, color and relationship to surrounding structures using white light illumination. We propose that fluorescent labeling of nerves can enhance the contrast between nerves and adjacent tissue during surgery which may lead to improved outcomes. Methods: Nerve binding peptide sequences including HNP401 were identified by phage display using selective binding to dissected nerve tissue. Peptide dye conjugates including FAM-HNP401 and structural variants were synthesized and screened for nerve binding after topical application on fresh rodent and human tissue and in-vivo after systemic IV administration into both mice and rats. Nerve to muscle contrast was quantified by measuring fluorescent intensity after topical or systemic administration of peptide dye conjugate. Results: Peptide dye conjugate FAM-HNP401 showed selective binding to human sural nerve with 10.9x fluorescence signal intensity (1374.44 ± 425.96) compared to a previously identified peptide FAM-NP41 (126.17 ± 61.03). FAM-HNP401 showed nerve-to-muscle contrast of 3.03 ± 0.57. FAM-HNP401 binds and highlight multiple human peripheral nerves including lower leg sural, upper arm medial antebrachial as well as autonomic nerves isolated from human prostate. Conclusion: Phage display has identified a novel peptide that selectively binds to ex-vivo human nerves and in-vivo using rodent models. FAM-HNP401 or an optimized variant could be translated for use in a clinical setting for intraoperative identification of human nerves to improve visualization and potentially decrease the incidence of intra-surgical nerve injury.

Impact of MMP-2 and MMP-9 enzyme activity on wound healing, tumor growth and RACPP cleavage.

Matrix metalloproteinases-2 and -9 (MMP-2/-9) are key tissue remodeling enzymes that have multiple overlapping activities critical for wound healing and tumor progression in vivo. To overcome issues of redundancy in studying their functions in vivo, we created MMP-2/-9 double knockout (DKO) mice in the C57BL/6 background to examine wound healing. We then bred the DKO mice into the polyomavirus middle T (PyVmT) model of breast cancer to analyze the role of these enzymes in tumorigenesis. Breeding analyses indicated that significantly fewer DKO mice were born than predicted by Mendelian genetics and weaned DKO mice were growth compromised compared with wild type (WT) cohorts. Epithelial wound healing was dramatically delayed in adult DKO mice and when the DKO was combined with the PyVmT oncogene, we found that the biologically related process of mammary tumorigenesis was inhibited in a site-specific manner. To further examine the role of MMP-2/-9 in tumor progression, tumor cells derived from WT or DKO PyVmT transgenic tumors were grown in WT or DKO mice. Ratiometric activatable cell penetrating peptides (RACPPs) previously used to image cancer based on MMP-2/-9 activity were used to understand differences in MMP activity in WT or knockout syngeneic tumors in WT and KO animals. Analysis of an MMP-2 selective RACPP in WT or DKO mice bearing WT and DKO PyVmT tumor cells indicated that the genotype of the tumor cells was more important than the host stromal genotype in promoting MMP-2/-9 activity in the tumors in this model system. Additional complexities were revealed as the recruitment of host macrophages by the tumor cells was found to be the source of the tumor MMP-2/-9 activity and it is evident that MMP-2/-9 from both host and tumor is required for maximum signal using RACPP imaging for detection. We conclude that in the PyVmT model, the majority of MMP-2/-9 activity in mammary tumors is associated with host macrophages recruited into the tumor rather than that produced by the tumor cells themselves. Thus therapies that target tumor-associated macrophage functions have the potential to slow tumor progression.

Detection of Subclinical Arthritis in Mice by a Thrombin Receptor-Derived Imaging Agent.

OBJECTIVE: Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. METHODS: K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPPNleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPPNleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. RESULTS: In chronic arthritis, RACPPNleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPPNleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPPNleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. CONCLUSION: RACPPNleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPPNleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression.

Early detection of squamous cell carcinoma in carcinogen induced oral cancer rodent model by ratiometric activatable cell penetrating peptides.

OBJECTIVES: Ratiometric cell-penetrating-peptides (RACPP) are hairpin-shaped molecules that undergo cleavage by tumor-associated proteases resulting in measurable Cy5:Cy7 fluorescence ratiometric change to label cancer in vivo. We evaluated an MMP cleavable RACPP for use in the early detection of malignant lesions in a carcinogen-induced rodent tumor model. METHODS: Wild-type immune-competent mice were given 4-nitroquinoline-oxide (4NQO) for 16weeks. Oral cavities from live mice that had been intravenously administered MMP cleavable PLGC(Me)AG-RACPP were serially imaged from week 11 through week 21 using white-light reflectance and Cy5:Cy7 ratiometric fluorescence. RESULTS: In an initial study we found that at week 21 nearly all mice (13/14) had oral cavity lesions, of which 90% were high-grade dysplasia or invasive carcinoma. These high-grade lesions were identifiable with white light reflectance and RACPP Cy5:Cy7 ratiometric fluorescence with similar detectability, Area Under Curve (AUC) for RACPP detection was 0.97 (95% Confidence interval (CI)=0.92-1.02, p<0.001), sensitivity=89%, specificity=100%. In a follow up study, oral cavity lesions generated by 4NQO were imaged and histologically analyzed at weeks 16, 18 and 21. In this study we showed that RACPP-fluorescence detection positively identified 15 squamous cell carcinomas (in 6 separate mice) that were poorly visible or undetectable by white light reflectance. CONCLUSIONS: RACPP ratiometric fluorescence can be used to accurately detect carcinogen-induced carcinoma in immunocompetent mice that are poorly visible or undetectable by white light reflectance.

Improved facial nerve identification during parotidectomy with fluorescently labeled peptide.

OBJECTIVES/HYPOTHESIS: Additional intraoperative guidance could reduce the risk of iatrogenic injury during parotid gland cancer surgery. We evaluated the intraoperative use of fluorescently labeled nerve binding peptide NP41 to aid facial nerve identification and preservation during parotidectomy in an orthotopic model of murine parotid gland cancer. We also quantified the accuracy of intraoperative nerve detection for surface and buried nerves in the head and neck with NP41 versus white light (WL) alone. STUDY DESIGN: Twenty-eight mice underwent parotid gland cancer surgeries with additional fluorescence (FL) guidance versus WL reflectance (WLR) alone. Eight mice were used for additional nerve-imaging experiments. METHODS: Twenty-eight parotid tumor-bearing mice underwent parotidectomy. Eight mice underwent imaging of both sides of the face after skin removal. Postoperative assessment of facial nerve function measured by automated whisker tracking were compared between FL guidance (n = 13) versus WL alone (n=15). In eight mice, nerve to surrounding tissue contrast was measured under FL versus WLR for all nerve branches detectable in the field of view. RESULTS: Postoperative facial nerve function after parotid gland cancer surgery tended to be better with additional FL guidance. Fluorescent labeling significantly improved nerve to surrounding tissue contrast for both large and smaller buried nerve branches compared to WLR visualization and improved detection sensitivity and specificity. CONCLUSIONS: NP41 FL imaging significantly aids the intraoperative identification of nerve braches otherwise nearly invisible to the naked eye. Its application in a murine model of parotid gland cancer surgery tended to improve functional preservation of the facial nerve. LEVEL OF EVIDENCE: NA Laryngoscope, 126:2711-2717, 2016.

Anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize.

Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery.