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Glial activation with the production of pro-inflammatory mediators is a major driver of disease progression in neurological processes ranging from acute traumatic injury to chronic neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. Posttranscriptional regulation is a major gateway for glial activation as many mRNAs encoding pro-inflammatory mediators contain adenine- and uridine-rich elements (ARE) in the 3' untranslated region which govern their expression. We have previously shown that HuR, an RNA regulator that binds to AREs, plays a major positive role in regulating inflammatory cytokine production in glia. HuR is predominantly nuclear in localization but translocates to the cytoplasm to exert a positive regulatory effect on RNA stability and translational efficiency. Homodimerization of HuR is necessary for translocation and we have developed a small molecule inhibitor, SRI-42127, that blocks this process. Here we show that SRI-42127 suppressed HuR translocation in LPS-activated glia in vitro and in vivo and significantly attenuated the production of pro-inflammatory mediators including IL1β, IL-6, TNF-α, iNOS, CXCL1, and CCL2. Cytokines typically associated with anti-inflammatory effects including TGF-β1, IL-10, YM1, and Arg1 were either unaffected or minimally affected. SRI-42127 suppressed microglial activation in vivo and attenuated the recruitment/chemotaxis of neutrophils and monocytes. RNA kinetic studies and luciferase studies indicated that SRI-42127 has inhibitory effects both on mRNA stability and gene promoter activation. In summary, our findings underscore HuR's critical role in promoting glial activation and the potential for SRI-42127 and other HuR inhibitors for treating neurological diseases driven by this activation.

CRISPR–Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells., There have been reports of immune responses against Cas9 which may impair clinical use. Here the authors scan a cohort comparable to the North American population vis-à-vis distribution of MHC-II variants to identify Cas9 peptides presented by MHC-II proteins and can stimulate CD4 + T-cells.

Pear psylla, Cacopsylla pyricola (Förster), has remained the most challenging pest of commercial pears in Washington and Oregon, the top producers of pears in the United States. The lack of effective integrated pest management tactics for this pest has been a major barrier to effective management. In this study, we examined the potential for reflective plastic mulch affixed beneath pear trees to suppress pear psylla. In 2017 and 2018, single pear tree (cv. Bartlett) plots of reflective plastic mulch, black plastic mulch, and no mulch (check) were established in a research orchard to compare their effects on pear psylla. Arthropods were sampled every 7–14 d from March through late summer. In both years, reflective mulch plots had significantly fewer pear psylla adults, eggs, and nymphs of the first generation compared with black plastic and check plots. However, later generations of psylla were not suppressed by reflective mulch. Semi-field tests were conducted in 2019 and 2020 using uniformly pruned potted pear trees on either reflective plastic mulch or grass to determine whether summerform adults were tolerant to reflected light from mulch, or if shading from vegetative growth negated the effect of the mulch. In both years, significantly fewer summerform adults and eggs were found in reflective mulch plots, suggesting that shading, rather than summerform tolerance, reduced effects from reflective mulch in the summer. This study demonstrates the potential for reflective mulch to suppress pear psylla and justifies future examination as part of a season-long management program.

Purpose: Rindopepimut is a vaccine targeting the tumor-specific EGF driver mutation, EGFRvIII. The ReACT study investigated whether the addition of rindopepimut to standard bevacizumab improved outcome for patients with relapsed, EGFRvIII-positive glioblastoma. Patients and Methods: In this double-blind, randomized, phase II study (NCT01498328) conducted at 26 hospitals in the United States, bevacizumab-naïve patients with recurrent EGFRvIII-positive glioblastoma were randomized to receive rindopepimut or a control injection of keyhole limpet hemocyanin, each concurrent with bevacizumab. The primary endpoint was 6-month progression-free survival (PFS6) by central review with a one-sided significance of 0.2. Results: Between May 2012 and 2014, 73 patients were randomized (36 rindopepimut, 37 control). Rindopepimut toxicity included transient, low-grade local reactions. As primary endpoint, PFS6 was 28% (10/36) for rindopepimut compared with 16% (6/37) for control (P = 0.12, one-sided). Secondary and exploratory endpoints also favored the rindopepimut group including a statistically significant survival advantage [HR, 0.53; 95% confidence interval (CI), 0.32–0.88; two-sided log-rank P = 0.01], a higher ORR [30% (9/30) vs. 18% (6/34; P = 0.38)], median duration of response [7.8 months (95% CI, 3.5–22.2) vs. 5.6 (95% CI, 3.7–7.4)], and ability to discontinue steroids for ≥6 months [33% (6/18) vs. 0% (0/19)]. Eighty percent of rindopepimut-treated patients achieved robust anti-EGFRvIII titers (≥1:12,800), which were associated with prolonged survival (HR = 0.17; 95% CI, 0.07–0.45; P < 0.0001). Conclusions: Our randomized trial supports the potential for targeted immunotherapy among patients with GBM, but the therapeutic benefit requires validation due to the small sample size and potential heterogeneity of bevacizumab response among recurrent patients with GBM. See related commentary by Wick and Wagener, p. 1535

Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) who actively smoke during treatment have worse survival compared with never-smokers and former-smokers. We hypothesize the poor prognosis in tobacco smokers with HNSCC is, at least in part, due to ongoing suppression of immune response. We characterized the tumor immune microenvironment (TIM) of HNSCC in a retrospective cohort of 177 current, former, and never smokers. Experimental Design: Tumor specimens were subjected to analysis of CD3, CD8, FOXP3, PD-1, PD-L1, and pancytokeratin by multiplex immunofluorescence, whole-exome sequencing, and RNA sequencing. Immune markers were measured in tumor core, tumor margin, and stroma. Results: Our data indicate that current smokers have significantly lower numbers of CD8+ cytotoxic T cells and PD-L1+ cells in the TIM compared with never- and former-smokers. While tumor mutation burden and mutant allele tumor heterogeneity score do not associate with smoking status, gene-set enrichment analyses reveal significant suppression of IFNα and IFNγ response pathways in current smokers. Gene expression of canonical IFN response chemokines, CXCL9, CXCL10, and CXCL11, are lower in current smokers than in former smokers, suggesting a mechanism for the decreased immune cell migration to tumor sites. Conclusions: These results suggest active tobacco use in HNSCC has an immunosuppressive effect through inhibition of tumor infiltration of cytotoxic T cells, likely as a result of suppression of IFN response pathways. Our study highlights the importance of understanding the interaction between smoking and TIM in light of emerging immune modulators for cancer management.

Surficial application of waste glycerol (WG) for enhanced bioremediation was tested in situ at an old military range site to address hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) contaminated groundwater. This treatment was effective in inducing strong reducing conditions (range: −4 to −205 mV) and increasing the concentrations of organic carbon (from 10 to 729 mg/L) and fatty acids (from 0 to 940 mg/L) concomitantly with a decrease in RDX concentrations (range: 17 to 143 μg/L) to below detection limits (0.1 μg/L) in 2 of the 3 monitoring wells (MWs) evaluated. None of these changes were observed in the control MW. RDX disappeared without the detection of any common anaerobic nitroso degradation intermediates, with the exception of one MW where the concentration of organics did not significantly increase (range: 10 to 20 mg/L), suggesting the conditions were not favourable for biodegradation. Ecotoxicological analysis suggested that the use of WG may have some dose-related deleterious effects on different soil and aquatic receptors. Analysis of the microbial community composition, using 16S rRNA gene amplicon sequences, which provided insight into whether the process design had selected for and stimulated the optimal microbial populations, indicated co-existence of numerous Operational Taxonomic Units (OTUs) belonging to groups known to be capable of RDX degradation under anaerobic conditions, with a positive link between Geobacter spp. enrichment and the presence of RDX nitroso metabolites. Overall, the results from this field test show that this treatment process can provide an effective long-term, semi-passive remediation option for RDX contaminated groundwater.

Psyllids (Hemiptera: Psylloidea) are phloem-feeding insects that tend to be highly specific in their host plants. Some species are well-known agricultural pests, often as vectors of plant pathogens. Many pest psyllids colonize agricultural fields from non-crop reproductive hosts or from non-host transitory and winter shelter plants. Uncertainty about which non-crop species serve as sources of psyllids hinders efforts to predict which fields or orchards are at greater risk of being colonized by psyllids. High-throughput sequencing of trnL, trnF, and ITS was used to examine the dietary histories of three pest and two non-pest psyllid species encompassing a diversity of lifecycles: Cacopsylla pyricola (Förster) (Psyllidae), Bactericera cockerelli (Šulc) (Triozidae), Diaphorina citri Kuwayama (Liviidae), Aphalara loca Caldwell (Aphalaridae), and a Cacopsylla species complex associated with Salix (Malphighiales: Salicaceae). Results revealed an unexpectedly high level of feeding on non-host species by all five psyllid species. The identification of the dietary history of the psyllids allowed us to infer their landscape-scale movements prior to capture. Our study demonstrates a novel use for gut content analysis-to provide insight into landscape-scale movements of psyllids-thus providing a means to pinpoint the non-crop sources of pest psyllids colonizing agricultural crops. We observed previously unknown psyllid behaviors during our efforts to develop this method and discuss new research directions for the study of psyllid ecology.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

El gen hyl Efm (que codifica una supuesta hialuronidasa) se ha encontrado casi exclusivamente en aislados clínicos de Enterococcus faecium y, recientemente, se ha demostrado que se encuentra en un plásmido que aumenta la capacidad de las cepas de E. faecium para colonizar el tracto gastrointestinal. En este trabajo, los resultados de los experimentos de apareamiento entre cepas de E. faecium que contienen hylz Efm pertenecientes al grupo clonal 17 y aisladas en los Estados Unidos y Colombia indicaron que el hyl EfmEl gen de estas cepas también se transporta en plásmidos grandes (> 145 kb) que mostramos que se transfieren fácilmente de cepas clínicas a huéspedes de E. faecium. También se observó cotransferencia de resistencia a vancomicina y resistencia de alto nivel (HLR) a aminoglucósidos (gentamicina y estreptomicina) y eritromicina. El grupo de genes vanA y los determinantes de resistencia a la gentamicina estaban genéticamente vinculados a hyl Efm , mientras que erm (B) y ant (6) -I, que confieren resistencia a macrólido-lincosamida-estreptogramina B y HLR a estreptomicina, respectivamente, no lo estaban. Un hyl EfmEl transconjugante positivo resultante de un apareamiento entre una cepa de endocarditis bien caracterizada [TX0016 (DO)] y un derivado de una cepa fecal de E. faecium de un voluntario humano sano (TX1330RF) exhibió una mayor virulencia en un modelo de peritonitis de ratón. Estos resultados indican que las cepas de E. faecium utilizan una estrategia que implica el reclutamiento en la misma unidad genética de genes de resistencia a antibióticos y determinantes que aumentan la capacidad de producir enfermedades. Nuestros hallazgos indican que la adquisición de los plásmidos hyl Efm puede explicar, al menos en parte, la reciente aparición exitosa de algunas cepas de E. faecium como patógenos nosocomiales. The hylEfm gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hylzEfm-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hylEfm gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hylEfm, whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hylEfm-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hylEfm plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

Enterococci are leading causes of antibiotic-resistant infection transmitted in hospitals. The intrinsic hardiness of these organisms allows them to survive disinfection practices and then proliferate in the gastrointestinal tracts of antibiotic-treated patients. The objective of this study was to identify the underlying genetic basis for its unusual hardiness. Using a functional genomic approach, we identified traits and pathways of general importance for enterococcal survival and growth that distinguish them from closely related pathogens as well as ancestrally related species. We further identified unique traits that enable them to survive antibiotic challenge, revealing a large set of genes that contribute to intrinsic antibiotic resistance and a smaller set of uniquely important genes that are rare outside enterococci., The enterococci, which are among the leading causes of multidrug-resistant (MDR) hospital infection, are notable for their environmental ruggedness, which extends to intrinsic antibiotic resistance. To identify genes that confer this unique property, we used Tn-seq to comprehensively explore the genome of MDR Enterococcus faecalis strain MMH594 for genes important for growth in nutrient-containing medium and with low-level antibiotic challenge. As expected, a large core of genes for DNA replication, expression, and central metabolism, shared with other bacteria, are intolerant to transposon disruption. However, genes were identified that are important to E. faecalis that are either absent from or unimportant for Staphylococcus aureus and Streptococcus pneumoniae fitness when similarly tested. Further, 217 genes were identified that when challenged by sub-MIC antibiotic levels exhibited reduced tolerance to transposon disruption, including those previously shown to contribute to intrinsic resistance, and others not previously ascribed this role. E. faecalis is one of the few Gram-positive bacteria experimentally shown to possess a functional Entner-Doudoroff pathway for carbon metabolism, a pathway that contributes to stress tolerance in other microbes. Through functional genomics and network analysis we defined the unusual structure of this pathway in E. faecalis and assessed its importance. These approaches also identified toxin-antitoxin and related systems that are unique and active in E. faecalis. Finally, we identified genes that are absent in the closest nonenterococcal relatives, the vagococci, and that contribute importantly to fitness with and without antibiotic selection, advancing an understanding of the unique biology of enterococci.

Background Echinococcus multilocularis is one of the most severe and lethal parasitic diseases of humans, most often reported in Europe and Asia. Only 1 previous case has been documented in the contiguous United States from Minnesota in 1977. European haplotypes have been identified in carnivores and domestic dogs as well as recently in patients in western and central Canada. Methods We used immunohistochemical testing with the monoclonal antibody Em2G11 and a species-specific enzyme-linked immunosorbent assay affinity-purified antigen Em2, as well as COX1 gene sequencing. Results Using pathology, immunohistochemical staining, specific immunodiagnostic testing, and COX1 gene sequencing, we were able to definitively identify E. multilocularis as the causative agent of our patient’s liver and lung lesions, which clustered most closely with the European haplotype. Conclusions We have identified the first case of a European haplotype E. multilocularis in the United States and the first case of this parasitic infection east of the Mississippi River. Given the identification of this haplotype in Canada, this appears to be an emerging infectious disease in North America.

Although immunotherapy works well in glioblastoma (GBM) pre-clinical mouse models, the therapy has not demonstrated efficacy in GBM patients. Since recent studies have linked the gut microbial composition to the success with immunotherapy for other cancers, we utilized a novel humanized microbiome (HuM) model in order to study the response to immunotherapy in a pre-clinical mouse model of GBM. We used five healthy human donors for fecal transplantation of gnotobiotic mice since it is now recognized that microbe strain level differences render individual humans with a unique microbial community composition. After the transplanted microbiomes stabilized, the mice were bred to generate 5 independent humanized mouse lines (humanized microbiome HuM1-HuM5). Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome composition with significant differences in diversity and microbial composition among HuM1-HuM5 lines. We next analyzed the growth of intracranial glioma cells in the HuM lines. All HuM mouse lines were susceptible to GBM transplantation, and exhibited similar median survival ranging from 19-26 days. Interestingly, we found that HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM1, HuM4, and HuM5 mice are non-responders to anti-PD-1 resulting in the death of the mice from the intracranial tumors, while HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls. Bray-Curtis cluster analysis of the 5 HuM gut microbial communities revealed that HuM2 and HuM3 were closely related. Detailed taxonomic comparison analysis at the top 5 across all HuM mouse lines revealed that Bacteroides cellulosilyticus was commonly found between HuM2 and HuM3 with high abundances. The results of our study establish the utility of humanized microbiome mice as avatars to delineate features of the host interaction with gut microbe communities needed for effective immunotherapy against GBM.

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis in various solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. In particular, small molecules do not exhibit binding specificity for PRL-3 over highly homologous family members PRL-1 and PRL-2, and antibodies directed against PRL-3 are limited by assay type. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards PRL-1 and PRL-2. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and co-immunoprecipitation with a known PRL-3 substrate showed the nanobodies bind PRL-3 outside of the active site, meaning they can be used to study PRL-3 interaction with binding partners. The nanobodies were also specific to PRL-3 over other PRLs in immunoprecipitation and immunofluorescence experiments in human cancer cells that overexpressed the PRL family. We found that N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression.

Intratumor spatial heterogeneity facilitates therapeutic resistance in glioblastoma (GBM). Nonetheless, understanding of GBM heterogeneity is largely limited to the surgically resectable tumor core lesion while the seeds for recurrence reside in the unresectable tumor edge. In this study, stratification of GBM to core and edge demonstrates clinically relevant surgical sequelae. We establish regionally derived models of GBM edge and core that retain their spatial identity in a cell autonomous manner. Upon xenotransplantation, edge-derived cells show a higher capacity for infiltrative growth, while core cells demonstrate core lesions with greater therapy resistance. Investigation of intercellular signaling between these two tumor populations uncovers the paracrine crosstalk from tumor core that promotes malignancy and therapy resistance of edge cells. These phenotypic alterations are initiated by HDAC1 in GBM core cells which subsequently affect edge cells by secreting the soluble form of CD109 protein. Our data reveal the role of intracellular communication between regionally different populations of GBM cells in tumor recurrence., Intratumoural spatial heterogeneity is crucial to enhance therapeutic resistance in glioblastoma. Here, the authors show a paracrine signaling mechanism where glioblastoma-initiating cells located in the tumour edge elevate their malignancy by interaction with core-located tumour cells.

Mexican bean beetle, Epilachna varivestis Mulsant (Coleoptera: Coccinellidae), is a serious pest of snap bean, Phaseolus vulgaris L. (Fabales: Fabaceae), lima bean, Phaseolus lunatus L. (Fabales: Fabaceae), and an occasional pest of soybean, Glycine max (L.) Merr. (Fabales: Fabaceae), in the United States. Past research indicates that some bean cultivars endure greater injury from this pest than others, suggesting that such crops are either more preferred, better hosts, and/or have poorer defenses. This study characterizes injury potential among three snap bean and three lima bean cultivars by measuring ovipositional preference, survival, and feeding injury of E. varivestis in field and greenhouse experiments. Snap beans, as a group, were more preferred and susceptible to injury than lima beans. The purple wax snap bean, Dragon's Tongue, was the most susceptible overall; while the dwarf bush lima bean, Henderson, was the least susceptible. The results of this study will aid in the development of integrated pest management strategies for E. varivestis, while also providing broader biological information for future research on this pest and others.

Background Patients with glioblastoma (GBM) have a dismal prognosis. Nearly all will relapse with no clear standard of care for recurrent disease (rGBM). Approximately 50% of patients have tumors harboring epidermal growth factor receptor (EGFR) amplification. The antibody–drug conjugate depatuxizumab mafodotin (depatux-m) binds cells with EGFR amplification, is internalized, and releases a microtubule toxin, killing the cell. Here we report efficacy, safety and pharmacokinetics (PK) of depatux-m + temozolomide (TMZ) in patients with EGFR-amplified rGBM. Methods M12-356 (NCT01800695) was an open-label study encompassing patients with newly diagnosed or rGBM across 3 treatment arms. Results are reported for adults with EGFR-amplified, measurable rGBM who received depatux-m (0.5–1.5 mg/kg) on days 1 and 15, and TMZ (150–200 mg/m2) on days 1–5 in a 28-day cycle. Patients were bevacizumab and nitrosourea naïve. Results There were 60 patients, median age 56 years (range, 20–79). Fifty-nine patients previously received TMZ. Common adverse events (AEs) were blurred vision (63%), fatigue (38%), and photophobia (35%). Grades 3/4 AEs were split between ocular and non-ocular AEs, occurring in 22% of patients each. Systemic PK exposure of depatux-m was dose proportional. The objective response rate was 14.3%, the 6-month progression-free survival rate was 25.2%, and the 6-month overall survival rate was 69.1%. Conclusions Depatux-m + TMZ displayed an AE profile similar to what was described previously. Antitumor activity in this TMZ-refractory population was encouraging. Continued study of depatux-m in patients with EGFR-amplified, newly diagnosed, or recurrent GBM is ongoing in 2 global, randomized trials (NCT02573324, NCT02343406).

Erythritol is a dietary sweetener that is used for low-calorie or diabetic diets. Although safe for human consumption, erythritol is lethal to certain Dipteran pests, but insecticidal effects of erythritol on phloem-feeding insects have yet to be examined. Our goal was to determine whether erythritol has insecticidal activity against pear psylla, Cacopsylla pyricola (Foerster) (Hemiptera: Psyllidae). We first demonstrated that ingestion of erythritol solutions compared with water by pear psylla caused reduced feeding, impaired motor functions, and reduced survival time of adults. We then tested whether foliar treatment of pear leaves with erythritol was also lethal to pear psylla. Foliar treatment of erythritol led to reduced 3-d survival of pear psylla nymphs and adults, and reduced rates of oviposition by pear psylla adults. Psylla adults also preferred to settle on untreated leaves than on erythritol-treated leaves in preference assays. Finally, we conducted field experiments to test whether applications of erythritol provided pear trees with protection against pear psylla under natural field conditions. Those experiments showed a reduction in pear psylla nymphs on erythritol-treated trees compared with untreated trees, but only if the erythritol was completely dissolved into solution by heating. Laboratory trials confirmed the importance of heating. Results of our experiments demonstrate that erythritol is insecticidal to pear psylla nymphs and adults and provide the first report that erythritol is lethal to a phloem-feeding insect. These findings suggest that erythritol may provide a new safe and effective tool for the management of pear psylla.

Enterococci, one of the most common causes of hospital-associated infections, are responsible for substantial morbidity and mortality. Enterococcus faecalis, the more common and virulent species, causes serious high-inoculum infections, namely infective endocarditis, that are associated with cardiac surgery and mortality rates that remained unchanged for the last 30 years. The best cures for these infections are observed with combination antibiotic therapy; however, optimal treatment has not been fully elucidated. It is the purpose of this review to highlight treatment options and their limitations, and provide direction for future investigative efforts to aid in the treatment of these severe infections. While ampicillin plus ceftriaxone has emerged as a preferred treatment option, mortality rates continue to be high, and from a safety standpoint, ceftriaxone, unlike other cephalosporins, promotes colonization with vancomycin resistant-enterococci due to high biliary concentrations. More research is needed to improve patient outcomes from this high-mortality disease.

Objective— Pulmonary artery smooth muscle cells (PASMCs) from neprilysin (NEP) null mice exhibit a synthetic phenotype and increased activation of Rho GTPases compared with their wild-type counterparts. Although Rho GTPases are known to promote a contractile SMC phenotype, we hypothesize that their sustained activity decreases SM-protein expression in these cells. Approach and Results— PASMCs isolated from wild-type and NEP −/− mice were used to assess levels of SM-proteins (SM-actin, SM-myosin, SM22, and calponin) by Western blotting, and were lower in NEP −/− PASMCs compared with wild-type. Rac and Rho (ras homology family member) levels and activity were higher in NEP −/− PASMCs, and ShRNA to Rac and Rho restored SM-protein, and attenuated the enhanced migration and proliferation of NEP −/− PASMCs. SM-gene repressors, p-Elk-1, and Klf4 (Kruppel lung factor 4), were higher in NEP −/− PASMCs and decreased by shRNA to Rac and Rho. Costimulation of wild-type PASMCs with PDGF (platelet-derived growth factor) and the NEP substrate, ET-1 (endothelin-1), increased Rac and Rho activity, and decreased SM-protein levels mimicking the NEP knock-out phenotype. Activation of Rac and Rho and downstream effectors was observed in lung tissue from NEP −/− mice and humans with chronic obstructive pulmonary disease. Conclusions— Sustained Rho activation in NEP −/− PASMCs is associated with a decrease in SM-protein levels and increased migration and proliferation. Inactivation of RhoGDI (Rho guanine dissociation inhibitor) and RhoGAP (Rho GTPase activating protein) by phosphorylation may contribute to prolonged activation of Rho in NEP −/− PASMCs. Rho GTPases may thus have a role in integration of signals between vasopeptides and growth factor receptors and could influence pathways that suppress SM-proteins to promote a synthetic phenotype.

Recapitulation of the tumor microenvironment is critical for probing mechanisms involved in cancer, and for evaluating the tumor-killing potential of chemotherapeutic agents, targeted therapies and immunotherapies. Microfluidic devices have emerged as valuable tools for both mechanistic studies and for preclinical evaluation of therapeutic agents, due to their ability to precisely control drug concentrations and gradients of oxygen and other species in a scalable and potentially high throughput manner. Most existing in vitro microfluidic cancer models are comprised of cultured cancer cells embedded in a physiologically relevant matrix, collocated with vascular-like structures. However, the recent emergence of immune checkpoint inhibitors (ICI) as a powerful therapeutic modality against many cancers has created a need for preclinical in vitro models that accommodate interactions between tumors and immune cells, particularly for assessment of unprocessed tumor fragments harvested directly from patient biopsies. Here we report on a microfluidic model, termed EVIDENT (ex vivo immuno-oncology dynamic environment for tumor biopsies), that accommodates up to 12 separate tumor biopsy fragments interacting with flowing tumor-infiltrating lymphocytes (TILs) in a dynamic microenvironment. Flow control is achieved with a single pump in a simple and scalable configuration, and the entire system is constructed using low-sorption materials, addressing two principal concerns with existing microfluidic cancer models. The system sustains tumor fragments for multiple days, and permits real-time, high-resolution imaging of the interaction between autologous TILs and tumor fragments, enabling mapping of TIL-mediated tumor killing and testing of various ICI treatments versus tumor response. Custom image analytic algorithms based on machine learning reported here provide automated and quantitative assessment of experimental results. Initial studies indicate that the system is capable of quantifying temporal levels of TIL infiltration and tumor death, and that the EVIDENT model mimics the known in vivo tumor response to anti-PD-1 ICI treatment of flowing TILs relative to isotype control treatments for syngeneic mouse MC38 tumors.

The carbamate insecticide methomyl is sometimes used to control slugs in field corn and soybean by foliar applications, but control outcomes in research trials and commercial operations have been mixed. In this study, laboratory bioassays were conducted on dusky slug, Arion subfuscus Muller, a common pest of corn and soybean in the Mid-Atlantic United States, to evaluate residual toxicity of Lannate LV (methomyl) at low and high concentrations corresponding to label recommended field rates, and if toxicity may be affected by ambient moisture or repellency to treated plants. Without wetting events, methomyl residues on corn plants caused 90–100% mortality of A. subfuscus for two days and 70–90% mortality for six days. When corn plants were briefly misted with ca. 0.3 cm of water 6 h after methomyl application, mortality was 36% 12 h after treatment, and 0 to 5% 24 h after treatment for both low and high rates. Repellency of A. subfuscus to corn plants treated with the high rate of methomyl was narrowly significant (P = 0.04) and low rate was not significant. These results suggest that high ambient moisture needed to elicit slug activity in the field also abates toxicity of methomyl residues, explaining why field control is usually poor despite high mortality in the lab.

Background: The aim of this study was to evaluate the outcomes of priming salvage radioimmunotherapy (RIT) with a low dose of external beam radiotherapy (EBRT) in patients with relapsed low grade non-Hodgkin lymphoma (LG-NHL). Methods: Patients who received salvage RIT with or without 2 × 2 Gy EBRT between March 2009 and February 2013 were retrospectively reviewed at a single institution. Planning target volume (PTV) for EBRT was created by adding a 1–2 cm expansion to the gross tumor volume depending on the anatomical location. Kaplan−Meier method via log-rank was employed to analyze the endpoints freedom from progression (FFP) and overall survival (OS). Results: We identified 22 patients who received salvage RIT without chemotherapy with a median follow up of 34 months. Of these, 9 (41%) patients were treated with EBRT immediately prior to RIT, and 13 (59%) received salvage RIT alone. Median FFP was not reached in patients who underwent combination treatment, while it was 9 months for patients treated with RIT alone ( p = 0.02). OS for all patients at 36 months was 80.3% with no significant difference between the two groups ( p = 0.88). On univariate analysis, the addition of EBRT was associated with improved FFP [hazard ratio (HR) = 4.17; 95% confidence interval (CI), 1.24–19.1; p = 0.02)]. No long term toxicities were reported in both groups. Conclusions: RIT outcomes and effects were improved with addition of low-dose EBRT immediately prior to it, in the treatment of relapsed LG-NHL with no additional toxicity. This study is hypothesis-generating and the findings should be validated in prospective studies.

Feeding behaviour, feeding intensity and staying behaviour of neonate western corn rootworm (Diabrotica virgifera virgifera LeConte) larvae were evaluated in response to synthetic feeding stimulant blends. All of the treatments contained a 3-sugar blend (glucose : fructose : sucrose, 30 : 4 : 4 mg/ml) and one of twelve free fatty acids. Each free fatty acid was tested in this blend at three different concentrations. The addition of the 12 : 0, 16 : 0, 16 : 1, 18 : 0, 18 : 1, 18 : 2 and 18 : 3 free fatty acids to the sugar blend significantly (P

Methyl anthranilate was identified as the active compound in extracts of maize (Zea mays L.) roots that were shown to be repellent to neonate western corn rootworm (Diabrotica virgifera virgifera LeConte) larvae. A bioassay-driven approach was used to isolate the active material from diethyl ether extracts of roots from germinating maize seeds. Separation of the extract on a Florisil column yielded an active fraction of 90:10 hexane:diethyl ether. Analysis with gas chromatography-mass spectrometry identified two compounds in the active fraction: indole (2,3-benzopyrrole) and methyl anthranilate (methyl 2-aminobenzoate). When tested in behavioral bioassays, methyl anthranilate elicited a significant (P

Pear psylla and honeydew marking to fruit cause significant economic damage to pears in Washington, a key pear growing region of the United States. The goal of this project is to compare an integrated pest management (IPM) program using materials which selectively target pests and relies on large natural enemy populations to grower standard conventional and organic pear pest management. In six locations throughout the growing region three management techniques organic, conventional and IPM were designated to 2 ha plots in grower managed orchards. Natural enemy and pest populations were monitored using beat tray, leaf brush and trapping methods. Pest excreted honeydew levels on leaves as well as fruit marking were measured to compare the relative success of pest management programs. IPM programs, using materials which selectively target pests, successfully increased natural enemy populations and kept psylla and honeydew levels low at the end of the season. However, early season pest populations resulted in fruit marking in some years. Fruit quality in IPM programs was similar to organic orchards in 2018, and organic and conventional in 2019, but fruit quality varied by orchard. In order to maintain consistent season-long control, further revision of IPM programs for Pacific Northwest pears is necessary including management tools such as targeted insecticide applications, honeydew washing systems using designated overhead sprinklers, and natural enemy thresholds.

Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 μg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays.

The purpose of this study is to determine the role of the complement immune system in glioma and/or glioma therapy. Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is accompanied by a devastating prognosis; the median survival is 12-14 months, with less than 10 percent of patients living for more than two years after diagnosis. Unfortunately, current treatment options are limited, and many initially promising drugs have failed phase three clinical trials. One potential and relatively unexplored target for co-therapy in GBM is the complement immune system. Historically, the focus of work with complement has been on its role in innate immunity, where it can aid in the recognition and elimination of pathogens or undesired host material. More recent work, however, has revealed a key function of complement as a “double-edged sword” in the CNS. While the cascade is necessary for CNS development and homeostasis, overactive complement can lead to the hallmark neuroinflammation and neurodegeneration seen in conditions like Alzheimer's, multiple sclerosis, and traumatic brain injuries. Yet despite the presence of complement receptors on nearly all CNS cells and the direct role that complement plays in multiple neuroinflammatory diseases, very few studies have examined complement expression in brain tumors. The current project seeks to bridge this gap in knowledge by assessing the impact of complement on glioma. This effort began by selecting candidate genes, as the complement family contains over 50 members. To do so, data from the publicly available Cancer Genome Atlas (TCGA) was mined, providing 14 targets of interest for further analysis. Cell-based experiments were performed in three GBM patient-derived xenolines (PDx): XD456, JX6, and JX10. mRNA expression was determined via TaqMan real-time PCR. Protein levels were assessed via Western blot. Overall, seven of the 14 initial targets demonstrated clear over-expression in all three human GBM PDx cell lines. This expression was not changed upon treatment with glioma growth factors such as epidermal or fibroblast growth factor (EGF or FGF). Intriguingly, however, the degree of over-expression varied by cell line, even when these lines were derived from patients assigned to the same molecular GBM subtype. For example, at the RNA level and in comparison to other cell lines, complement factor H (a C3 inhibitor) was up to six times higher in XD456, and clusterin (a MAC inhibitor) was nearly 18-fold higher in JX10. These data suggest that the current system for classifying tumor subtype may be missing important factors. Finally, when compared back to TCGA data, overexpression of these targets was associated with significantly worse tumor phenotype and patient survival. This project has critically identified components of the complement system that are upregulated in GBM and strongly correlated to survival. Ultimately, these data may provide the first potential targets for complement-based co-therapeutics to be explored in future studies. Citation Format: Emily Nicole Hayward, Xiuhua Yang, Natalia Filippova, Jennifer A. Calano, David Namkoong, Stefan Kovac, Louis B. Nabors. Characterization and analysis of the complement immune system in glioblastoma (GBM) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3335.

Mexican bean beetle, Epilachna varivestis Mulsant, is a pest of snap bean and lima bean in the eastern United States. This pest is susceptible to many insecticides available to conventional growers; however, organic management using parasitoid releases and organic insecticides have inconsistent results. In the interest of developing cultural management techniques such as trap crops or push-pull systems, five bean cultivars were evaluated for preferential host selection by E. varivestis using marked beetles in field cages and open plots. Beetles were marked with a water-based paint pen and their locations on plants monitored over time. In field cages, the purple wax bean, Dragon's Tongue (DT), was preferred over yellow wax, green bean and lima bean; soybean was the least preferred overall. Recaptures of E. varivestis adults in open field plots progressively decreased following beetle release, suggesting the affinity of adults to disperse despite being on or near acceptable hosts. The two wax beans were equally preferred in open field experiments, partially more than the green and lima bean and consistently more than the soybean. These experiments suggest that DT may be a suitable crop for trap cropping or attract and kill strategies for E. varivestis. © The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. We thank Jon Wooge, Hallie Harriman, Katlin Mooneyham, Liz Fread, Alex Hessler, and other employees of Virginia Tech and Kentland Farm for their assistance with sampling, preparation, and maintenance of plots. We also thank Drs. Ames Herbert, Peter Schultz, Ramon Arancibia, Timothy Kring, Tobin Northfield and W. Rodney Cooper for experimental development and statistical guidance. Funding for this research was provided by Southern SARE (Sustainable Agricultural Research and Education), project number GS13-120.

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

Continued loss of natural habitats with native prairies and wildflower patches is eliminating diverse sources of pollen, nectar and phytochemicals therein for foraging bees. The longstanding plant-pollinator mutualism reiterates the role of phytochemicals in sustaining plant-pollinator relationship and promoting honey bee health. We studied the effects of four phytochemicals&mdash, caffeine, gallic acid, kaempferol and p-coumaric acid, on survival and pathogen tolerance in the European honey bee, Apis mellifera (L.). We recorded longevity of worker bees that were provided ad libitum access to sugar solution supplemented with different concentrations of phytochemicals. We artificially infected worker bees with the protozoan parasite, Nosema ceranae. Infected bees were provided access to the same concentrations of the phytochemicals in the sugar solution, and their longevity and spore load at mortality were determined. Bees supplemented with dietary phytochemicals survived longer and lower concentrations were generally more beneficial. Dietary phytochemicals enabled bees to combat infection as seen by reduced spore-load at mortality. Many of the phytochemicals are plant defense compounds that pollinators have evolved to tolerate and derive benefits from. Our findings support the chemical bases of co-evolutionary interactions and reiterate the importance of diversity in floral nutrition sources to sustain healthy honey bee populations by strengthening the natural mutualistic relationships.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an important economic pest of maize (Zea mays L.) in North America and Europe. Previous efforts to formulate an artificial diet for western corn rootworm larvae highlighted an important role of corn root powder, which had a significant positive impact on several larval developmental traits. Unfortunately, this ingredient is not available for purchase. Toward the goal of developing an artificial diet for western corn rootworm larvae with all ingredients readily accessible, we conducted research to isolate essential growth factors for larval development from corn root powder to improve the performance of diet without corn root powder. For all experiments, multiple life history parameters (survival, weight, and molting) were recorded from 15-d diet bioassays. Corn roots may contain factors that assist in larval growth, but some of these factors were not fully extracted by methanol and remained in the extracted root. Methanolic extracts significantly increased molting to second instar, but did not significantly increase survival, dry weight, or molting to third instar, suggesting the primary corn root substituents affecting these factors cannot be extracted or other extraction methods may be required to extract the essential factors from corn roots. We showed that whole corn root powder was best when used in combination with all the other nutrient sources in the published western corn rootworm formulation. Corn root powder made from proprietary seed and Viking seed has similar value.

The susceptibility of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae to nine insecticides from five different classes and to Bt proteins eCry3.1Ab and mCry3A in the presence or absence of feeding stimulants, was estimated in filter paper and diet toxicity assays, respectively. The use of a synthetic feeding stimulant blend of the sugars glucose, sucrose, and fructose plus linoleic acid at a ratio of 30:4:4:0.3 mg/ml of distilled water was evaluated to determine whether they increase the efficacy of insecticides and Bt proteins. The efficacy of thiamethoxam diluted in solutions with feeding stimulants was significantly increased when compared to thiamethoxam dilutions in water (>60-fold). Differences in the efficacy of the other insecticide classes when diluted in feeding stimulant solutions were no greater than fivefold when compared to the insecticides diluted in water. The presence of corn root juice as a natural feeding stimulant diminished toxicity of the insecticides, except for thiamethoxam, even though larval fresh weight was higher when fed on root juice compared to feeding stimulant or water. The use of feeding stimulants in diet toxicity assays did not enhance efficacy of eCry3.1Ab nor mCry3A proteins. Feeding stimulants can be recommended in combination with thiamethoxam to increase larval mortality. These results are discussed in terms of applicability of feeding stimulants to improve susceptibility of western corn rootworm larvae to pesticides in general.

Methyl anthranilate, a natural compound in maize roots, was previously shown to be repellent to larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte. In this study, MA was tested in a soil environment for behavioral effects on neonate larvae. In tests with a single maize seedling surrounded by MA-coated granules, MA prevented larvae from locating the roots and the number of larvae recovered from roots decreased with increasing rates of MA. When MA was employed in a simple push-pull strategy between an MA-treated seedling and an untreated seedling, concentrations of MA as low as 0.01% caused significantly more larvae to be “pushed” to and recovered from roots of the untreated seedling. The effect strengthened as the dosage increased, with up to 90% repellency occurring with the highest dose tested (10% MA). A commercial product containing MA gave similar results, with 96% repellency observed for the highest dose tested. Chemical analysis showed that amounts of MA remained high for 4 weeks in dry, sterilized or unsterilized soil, but MA levels declined rapidly in moist soil. After 7 days, MA levels in moist, sterilized soil were 50% lower than in dry soil and in unsterilized moist soil, only a trace of MA remained, suggesting that both moisture and microbial activity contributed to the loss of MA. A variety of carrier granules were coated with MA and tested in soil bioassays after aging in moist soil. After 1 day in the soil, all of the MA granules were repellent at the 1% rate and clay granules were also effective at 0.1%. After one week in the soil, only the molecular sieve granules elicited repellency, but this activity was gone after two weeks of aging. These results demonstrate that MA elicits a repellent effect by western corn rootworm larvae in the soil environment and may have future potential as a rootworm treatment if formulations can be developed to protect the material from decomposition in the soil.

Enterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 −35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems., IMPORTANCE Enterococcus faecalis is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an E. faecalis strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to “suppress” infections in prosthetic joints and offer novel insights into the interaction of β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.

Amyloid-beta (Aβ) is a group of aggregation-prone, 38- to 43-amino acid peptides generated in the eye and other organs. Numerous studies suggest that the excessive build-up of low-molecular-weight soluble oligomers of Aβ plays a role in the progression of Alzheimer's disease and other brain degenerative diseases. Recent studies raise the hypothesis that excessive Aβ levels may contribute also to certain retinal degenerative diseases. These findings, together with evidence that a major portion of Aβ is released as monomer into the extracellular space, raise the possibility that a technology enabling the enzymatic break-down of monomeric Aβ in the living eye under physiological conditions could prove useful for research on ocular Aβ physiology and, perhaps ultimately, for therapeutic applications. Neprilysin (NEP), an endopeptidase known to cleave Aβ monomer into inactive products, is a membrane-associated protein. However, sNEP, a recombinant form of the NEP catalytic domain, is soluble in aqueous medium. With the aim of determining the Aβ-cleaving activity of exogenous sNEP in the microenvironment of the intact eye, we analyzed the effect of intra-vitreally delivered sNEP on ocular Aβ levels in mice that exhibit readily measurable, aqueous buffer-extractable Aβ40 and Aβ42, two principal forms of Aβ. Anesthetized 10-month wild-type (C57BL/6J) and 2–3-month 5XFAD transgenic mice received intra-vitreal injections of sNEP (0.004–10 μg) in one eye and were sacrificed at defined post-treatment times (30 min - 12 weeks). Eye tissues (combined lens, vitreous, retina, RPE and choroid) were homogenized in phosphate-buffered saline, and analyzed for Aβ40 and Aβ42 (ELISA) and for total protein (Bradford assay). The fellow, untreated eye of each mouse served as control, and concentrations of Aβ (pmol/g protein) in the treated eye were normalized to that of the untreated control eye. In C57BL/6J mice, as measured at 2 h after sNEP treatment, increasing amounts of injected sNEP yielded progressively greater reductions of Aβ40, ranging from 12% ± 3% (mean ± SEM; n = 3) with 4 ng sNEP to 85% ± 13% (n = 5) with 10 μg sNEP. At 4 ng sNEP the average Aβ40 reduction reached >70% by 24 h following treatment and remained near this level for about 8 weeks. In 5XFAD mice, 10 μg sNEP produced an Aβ40 decrease of 99% ± 1% (n = 4) and a substantial although smaller decrease in Aβ42 (42% ± 36%; n = 4) within 24 h. Electroretinograms (ERGs) were recorded from eyes of C57BL/6J and 5XFAD mice at 9 days following treatment with 4 ng or 10 μg sNEP, conditions that on average led, respectively, to an 82% and 91% Aβ40 reduction in C57BL/6J eyes, an 87% and 92% Aβ40 reduction in 5XFAD eyes, and a 23% and 52% Aβ42 reduction in 5XFAD eyes. In all cases, sNEP-treated eyes exhibited robust ERG responses, consistent with a general tolerance of the posterior eye tissues to the investigated conditions of sNEP treatment. The sNEP-mediated decrease of ocular Aβ levels reported here represents a possible approach for determining effects of Aβ reduction in normally functioning eyes and in models of retinal degenerative disease.

Insulin-degrading enzyme (IDE) functions in the catabolism of bioactive peptides. Established roles include degrading insulin and the amyloid beta peptide (Aβ), linking it to diabetes and Alzheimer’s disease. IDE is primarily located in the cytosol, and a longstanding question is how it gains access to its peptide substrates. Reports suggest that IDE secreted by an unconventional pathway participates in extracellular hydrolysis of insulin and Aβ. We find that IDE release from cultured HEK-293 or BV-2 cells represents only ~1% of total cellular IDE, far less than has been reported previously. Importantly, lactate dehydrogenase (LDH) and other cytosolic enzymes are released at the same relative level, indicating that extracellular IDE results from a loss of cell integrity, not secretion. Lovastatin increases IDE release from BV-2 cells as reported, but this release is mirrored by LDH release. Cell viability assays indicate lovastatin causes a loss of cell integrity, explaining its effect on IDE release. IDE is present in an exosome-enriched fraction from BV-2 cell conditioned media, however it represents only ~0.01% of the total cellular enzyme and is unlikely to be a significant source of IDE. These results call into question the secretion of IDE and its importance in extracellular peptide degradation.

Levels of amyloid beta (Aβ) in the central nervous system are regulated by the balance between its synthesis and degradation. Neprilysin (NEP) is associated with Alzheimer's disease (AD) by its ability to degrade Aβ. Some studies have involved the exposure to mercury (Hg) in AD pathogenesis; therefore, our aim was to investigate the effects on the anabolism and catabolism of Aβ in differentiated SH-SY5Y cells incubated with 1-20 μM of Hg. Exposure to 20 µM of Hg induced an increase in Aβ-42 secretion, but did not increase the expression of the amyloid precursor protein (APP). Hg incubation (10 and 20 µM) increased NEP protein levels; however, it did not change NEP mRNA levels nor the levels of the amyloid intracellular domain peptide, a protein fragment with transcriptional activity. Interestingly, Hg reduced NEP activity at 10 and 20 µM, and circular dichroism analysis using human recombinant NEP showed conformational changes after incubation with molar equivalents of Hg. This suggests that the Hg-induced inhibition of NEP activity may be mediated by a conformational change resulting in reduced Aβ-42 degradation. Finally, the comparative effects of lead (Pb, 50 μM) were evaluated. We found a significant increase in Aβ-42 levels and a dramatic increase in APP protein levels; however, no alteration in NEP levels was observed nor in the enzymatic activity of this metalloprotease, despite the fact that Pb slightly modified the rhNEP conformation. Overall, our data suggest that Hg and Pb increase Aβ levels by different mechanisms.

Complex interactions occur between tumor and host immune system at each site in the metastatic setting, the outcome of which can determine behavior ranging from dormancy to rapid growth. An additional layer of complexity arises from the understanding that cytotoxic T cells can traffic through the host circulatory system. Coupling mathematical models of local tumor-immune dynamics and systemic T cell trafficking allows us to simulate the evolution of tumor and immune cell populations in anatomically distant sites following local therapy and thus computationally evaluate immune interconnectivity. Results suggest that the presence of a secondary site may either inhibit or promote growth of the primary, depending on the capacity for immune recruitment of each tumor and the resulting systemic redistribution of T cells. Treatment such as surgical resection and radiotherapy can be simulated to estimate both the decrease in tumor volume at the local treatment-targeted site, and the change in overall tumor burden and tumor growth trajectories across all sites. Qualitatively similar responses of distant tumors to local therapy (positive and negative abscopal effects) to those reported in the clinical setting were observed. Such findings may facilitate an improved understanding of general disease kinetics in the metastatic setting: if metastatic sites are interconnected through the immune system, truly local therapy does not exist.

Thiamethoxam is a neonicotinoid insecticide commonly applied directly to the seeds (seed-treatment) of commercial snap beans, Phaseolus vulgaris L. While previous studies have examined target and nontarget effects of thiamethoxam seed-treatments in snap beans and other crops, to our knowledge, none have been conducted in agroecosystems predominated by the pest Mexican bean beetle, Epilachna varivestis Mulsant (Coleoptera: Coccinellidae). This study examined the effects of thiamethoxam-treated snap beans on E. varivestis, other arthropods, and crop performance in southwestern Virginia. Greenhouse experiments were conducted to evaluate residual toxicity of treated snap beans to E. varivestis and a key predator, Podisus maculiventris (Say) (Hemiptera: Pentatomidae). Treated plants were highly toxic to E. varivestis at 13 d, moderately toxic from 16 to 20 d, and minimally toxic at 24 d. P. maculiventris was unaffected by exposure to treated plants or by feeding on E. varivestis that consumed treated plants. Small plot field experiments in 2014 and 2015 showed no significant effects of thiamethoxam seed-treatments on E. varivestis densities, other arthropods, crop injury, or yield. In 2016, planting was delayed by persistent rain, resulting in early E. varivestis colonization. In this year, thiamethoxam-treated plants had significantly lower densities and feeding injury from E. varivestis, followed by significantly higher yields. Natural enemies were unaffected by seed-treatments in all field experiments. These experiments demonstrated that thiamethoxam seed-treatments provide control of E. varivestis when beetles infest fields within 2 to 3 wk after planting; but otherwise provide negligible advantages. Negative effects from thiamethoxam seed-treatments on nontarget arthropods appear minimal for snap beans in this region.

Lowe syndrome is a rare X-linked congenital disease that presents with congenital cataracts and glaucoma, as well as renal and cerebral dysfunction. OCRL, an inositol polyphosphate 5-phosphatase, is mutated in Lowe syndrome. We previously showed that OCRL is involved in vesicular trafficking to the primary cilium. Primary cilia are sensory organelles on the surface of eukaryotic cells that mediate mechanotransduction in the kidney, brain, and bone. However, their potential role in the trabecular meshwork (TM) in the eye, which regulates intraocular pressure, is unknown. Here, we show that TM cells, which are defective in glaucoma, have primary cilia that are critical for response to pressure changes. Primary cilia in TM cells shorten in response to fluid flow and elevated hydrostatic pressure, and promote increased transcription of TNF-α, TGF-β, and GLI1 genes. Furthermore, OCRL is found to be required for primary cilia to respond to pressure stimulation. The interaction of OCRL with transient receptor potential vanilloid 4 (TRPV4), a ciliary mechanosensory channel, suggests that OCRL may act through regulation of this channel. A novel disease-causing OCRL allele prevents TRPV4-mediated calcium signaling. In addition, TRPV4 agonist GSK 1016790A treatment reduced intraocular pressure in mice; TRPV4 knockout animals exhibited elevated intraocular pressure and shortened cilia. Thus, mechanotransduction by primary cilia in TM cells is implicated in how the eye senses pressure changes and highlights OCRL and TRPV4 as attractive therapeutic targets for the treatment of glaucoma. Implications of OCRL and TRPV4 in primary cilia function may also shed light on mechanosensation in other organ systems.