Killing Agent Research Articles

Development of bait station to complement attract-and-kill agents of Zeugodacus tau (Diptera: Tephritidae).

Zeugodacus tau (Walker) (Diptera: Tephritidae) is an important agricultural pest currently managed primarily through the application of insecticides due to limited control strategies. Bait station devices are target specific and have emerged as a behaviorally based alternative to traditional insecticide sprays for managing Z. tau. In this study, we designed a bait station by integrating female-biased olfactory, visual, and gustatory elements, and a killing agent in a wax-matrix. Our results showed that the wax-matrix integrated with spinetoram showed the highest toxicity to immature and mature Z. tau females. Furthermore, the color and shape of the spinetoram bait station significantly influenced its attractiveness and toxicity to female Z. tau. Green sausage-shaped exteriors were the most effective color and shape examined. Subsequent experiments showed a length-dependent effect on mortality and visiting frequencies of Z. tau females when the bait stations were 9-13 cm long. The addition of the olfactory stimulus of 5% ammonium acetate to the bait station attracted a higher number of mature Z. tau females than the control. The killing efficacy of the weathered bait station was similar to that of the fresh station within an 8-week period (over 794 mm of rainfall). The bait station developed herein would provide new insight into the attract-and-kill strategy for Z. tau and alleviate the pressure of the actual management program for this pest.

Attractiveness, longevity, and release rates of multilure wafers for trapping males of the oriental fruit fly and melon fly (Diptera: Tephritidae).

Invasive fruit flies (Diptera: Tephritidae) pose a serious threat to the production and export of many commercially important fruits and vegetables. Detection of the agricultural pests Bactrocera dorsalis (Hendel) and Zeugodacus cucurbitae (Coquillett) relies heavily on traps baited with male-specific attractants. For B. dorsalis, traps are typically baited with liquid methyl eugenol (ME), and for Z. cucurbitae, traps are baited with liquid cue-lure (CL). Operating large-scale trapping networks is costly, consequently, there is much interest in identifying ways to maintain network sensitivity while reducing costs. One cost-cutting approach is the possibility of combining different male lures in the same dispenser, thus reducing the number of traps requiring servicing. The chief objective of this study was to compare captures of B. dorsalis and Z. cucurbitae males in Jackson traps baited with polymeric wafers impregnated with both ME and raspberry ketone (RK, a hydrolyzed form of CL) versus traps baited with liquid ME or CL freshly applied to cotton wicks. Captures were measured when the ME/RK wafers had been weathered for 12, 18, or 24wk. Captures of B. dorsalis and Z. cucurbitae males were similar between fresh lure and weathered wafers over all trapping periods, with a single exception apparently due to the lessened potency of the associated killing agent. The residual amount and release rate of ME and RK from the wafers were also measured to examine possible relationships between wafer chemistry and trap catch. The possible implications of the present results to area-wide trapping programs are discussed.

Degradable Microneedle Patch with Photothermal-Promoted Bacteria-Infected Wound Healing and Microenvironment Remodeling.

Bacteria-infected wound healing is one of the most challenging issues in health management that is attracting worldwide concerns. Despite great achievements with antibiotics, emergence of antibiotic-resistance retarded the wound healing process and also led to severe outcomes. Exploration of novel antibiotics together with amelioration of wound healing efficacy is desirable. Herein, a degradable microneedle patch (AAZH@MNs) was fabricated through incorporating near-infrared light responsive photothermal agents for sustained bacteria killing and prevention of biofilm formation. In addition, the antibacterial microneedle patch could even remold the microenvironment of bacteria-infected wounds through an antibacterial effect, significantly facilitating the wound healing process.

Comparison of the Trapping Efficacy of Locally Modified Gravid Aedes Trap and Autocidal Gravid Ovitrap for the Monitoring and Surveillance of Aedes aegypti Mosquitoes in Tanzania.

The study assessed the trapping efficacy of locally modified (1) Gravid Aedes Trap (GAT) lined with insecticide-treated net (ITN) as a killing agent and (2) Autocidal Gravid Ovitrap (AGO) with sticky board in the semi-field system (SFS) and field setting. Fully balanced Latin square experiments were conducted to compare GAT lined with ITN vs. AGO, both with either yeast or grass infusion. Biogent-Sentinel (BGS) with BG-Lure and no CO2 was used as a standard trap for Aedes mosquitoes. In the SFS, GAT outperformed AGO in collecting both nulliparous (65% vs. 49%, OR = 2.22, [95% CI: 1.89-2.60], p < 0.001) and gravid mosquitoes (73% vs. 64%, OR = 1.67, [95% CI: 1.41-1.97], p < 0.001). Similar differences were observed in the field. Yeast and grass infusion did not significantly differ in trapping gravid mosquitoes (OR = 0.91, [95% CI: 0.77-1.07], p = 0.250). The use of ITN improved mosquito recapture from 11% to 70% in the SFS. The same trend was observed in the field. Yeast was chosen for further evaluation in the optimized GAT due to its convenience and bifenthrin net for its resistance management properties. Mosquito density was collected when using 4× GATs relative to BGS-captured gravid mosquitoes 64 vs. 58 (IRR = 0.82, [95% CI: 0.35-1.95], p = 0.658) and showed no density dependence. Deployment of multiple yeast-baited GAT lined with bifenthrin net is cost-effective (single GAT < $8) compared to other traps such as BGS ($160).

Helical BODIPY Dyes as Heavy-Atom-Free Triplet Photosensitizers for Photodynamic Therapy of Cancer.

Despite of having many advantages over the popular cancer therapies, photodynamic therapy still is not widely acceptable for clinical applications. Scarcity of efficient triplet photosensitizers (PSs) is one of the main bottlenecks for this. Although numerous heavy atom attached PSs are developed as PDT agents, but these are not suitable for clinical applications due to several reasons. Thus, development of heavy-atom-free organic PSs is urgently required. In this context, development of new type of helical BODIPYs as PSs for PDT is discussed. BODIPYs have rich photophysical properties and structural helicity further enhances their triplet conversion rates. This new concept of structural helicity to enhance the triplet conversion of BODIPYs is discussed with reported helical BODIPYs. Helical geometries of these dyes are checked by X-ray crystallography studies and their high triplet conversions as compared to planar BODIPYs are also confirmed. Importantly, these dyes have high triplet lifetimes and are capable of generating high singlet oxygens even in hypoxia condition as compared to conventional heavy atom attached BODIPYs. All these make the helical BODIPYs excellent candidates as PDT agents. Finally, their successful applications as PDT agents in killing of various types of cancer cells are also discussed. The results are encouraging which indicate that helical BODIPYs could be next generation heavy-atom-free PSs for PDT applications.

Attract-and-kill for managing Popillia japonica (Coleoptera: Scarabaeidae) abundance and leaf injury in commercial vineyards.

Popillia japonica Newman (Coleoptera: Scarabaeidae) feeding negatively impacts many plant species, including grapes, potentially reducing fruit quality and yield. Chemical control, representing the current grower standard, relies on frequent broadcast applications of broad-spectrum insecticides, with alternative management strategies mostly lacking. Attract-and-kill (A&K) is a behavioral management strategy that combines semiochemical attractants and a killing agent on a substrate. This study assessed the impact of A&K on (i) the number of P. japonica adults and (ii) the percent of P. japonica feeding injury on grape foliage compared to the grower standard in commercial vineyards. This 2-year study was conducted at 3 commercial vineyards with 4 paired plots consisting of a grower standard control and an A&K treatment. The A&K treatment consisted of commercial lures, each placed on outside-edge grapevines, and weekly applications of carbaryl on the plants holding lures. The grower standard received broadcast insecticide applications at the grower's discretion. The A&K treatment experienced similar numbers of P. japonica adults and similar proportions of leaf injury compared to the grower standard. The use of A&K reduced by 96% the crop area treated with insecticides compared to the grower standard. The area treated by A&K was at the edge of the vineyards, where more leaf injury occurred regardless of treatment. A&K is a targeted approach that was effective at managing P. japonica and reducing chemical inputs on a small scale. It has the potential to be scaled up and refined to provide growers with a new management strategy.

Quantifying cathode biofilm resistance in a cdrAB modified Shewanella oneidensis MR-1 using electrochemical impedance spectroscopy

A range of biotechnological applications for converting electricity to fixed metabolic substrates is fuelling the study of cathodic bioelectrochemical systems. Shewanella oneidensis MR-1 has emerged as an important model system for extracellular electron uptake on cathodes, as many of the proteins involved in this process overlap with the organism's previously characterized extracellular electron transport machinery. However, there are still many questions surrounding the mechanics of electron uptake in Shewanella stemming largely from the challenge of quantifying biomass on electrode surfaces. This limits our understanding of the physiologic and kinetic constraints of electron uptake, as well as our ability to make meaningful comparisons across systems. To investigate the relationship between cathodic activity and biomass, we used a Shewanella oneidensis strain genetically modified with cell aggregation protein CdrAB behind a blue light-controlled promotor. Using blue light exposure to control cell deposition, we then investigated the relationship between cathodic activity and biomass. Electrochemical impedance spectroscopy (EIS) confirmed a decrease in biofilm impedance over a range of blue light exposures (i.e., 2 to 8 h). Consistent with previous results, after this timepoint, a drop-in electrochemical activity was observed, and impedances increased. For biofilms within the 2–8 h light exposure range, we observed a trend towards increased biological current consumption by quantifying the difference between pre and post kill currents. Comparing EIS data between pre and post kill experiments supported an increase in impedance post addition of killing agents and a trend towards the lowest biofilm impedances observed in the longest blue light exposed systems. Using an equivalent circuit model to extrapolate specific biofilm parameters we quantified the charger transfer resistance within the biofilm that corresponds to varying biofilm thicknesses and matches previously observed activities. For example, electrochemical activity was highest for the 8 h blue light exposed biofilm condition, with a maximum cathodic biologic current of -5.49 ± 0.85 µA, and a biofilm charge transfer resistance measured at 6909.5 ± 2136.5 Ω. On an individual reactor basis, we correlate this biofilm charge transfer resistance with biologic cathodic current. We observed a linear trend with a correlation score of 0.87 (r2 = 0.773). To the best of our knowledge, this is the first investigation of biofilm physiology on Shewanella cathodes using EIS. Continued efforts in this direction will further our understanding of biofilm-electrode interface during extracellular electron uptake with the goal of enhancing applications to bioelectrochemical systems.

Evaluation of HOOK SWD attract-and-kill on captures, emergence, and survival of Drosophila suzukii in Florida

Abstract Attract-and-kill can be a useful tactic in managing insect pests as part of an integrated pest management (IPM) program. An attractant, usually a pheromone or food-based volatile blend, is mixed with a killing agent, usually an insecticide, in some form of applicator or dispenser. HOOK® products utilize a SPLAT® gel matrix (ISCA, Riverside, California). HOOK® SWD has been developed for the management of Drosophila suzukii Matsumura (Diptera: Drosophilidae), a major pest of thin-skinned fruit and berry crops. The attractant is a blend of food-based volatiles, and the killing agent is spinosad. This study looked at the use of HOOK® SWD in combination with insecticide applications to manage D. suzukii in conventional and organic blackberries and blueberries. The experiment was conducted on four farms: a conventional commercial blueberry farm, a commercial organic blueberry farm, a conventional u-pick blackberry farm, and a conventional organic blackberry farm all in north-central Florida. HOOK® SWD applied at 7- and 14-day intervals demonstrated efficacy in reducing D. suzukii trap catch and emergence at the organic blackberry and conventional blueberry farms. Drosophila suzukii trap catch and emergence counts at the conventional u-pick blackberry and the organic blueberry farms were not significantly different among treatments. Several factors may have contributed to this lack of efficacy including understory weeds, proximity of unmanaged areas, etc. Therefore, HOOK® SWD shows promise as a D. suzukii management tactic, but further research is needed to optimize its efficacy.

Green Synthesized Polymeric Iodophors with Thyme as Antimicrobial Agents.

Antimicrobial resistance (AMR) is a growing concern for the future of mankind. Common antibiotics fail in the treatment of microbial infections at an alarming rate. Morbidity and mortality rates increase, especially among immune-compromised populations. Medicinal plants and their essential oils, as well as iodine could be potential solutions against resistant pathogens. These natural antimicrobials abate microbial proliferation, especially in synergistic combinations. We performed a simple, one-pot synthesis to prepare our formulation with polyvinylpyrrolidone (PVP)-complexed iodine (I2), Thymus Vulgaris L. (Thyme), and Aloe Barbadensis Miller (AV). SEM/EDS, UV-vis, Raman, FTIR, and XRD analyses verified the purity, composition, and morphology of AV-PVP-Thyme-I2. We investigated the inhibitory action of the bio-formulation AV-PVP-Thyme-I2 against 10 selected reference pathogens on impregnated sterile discs, surgical sutures, cotton gauze bandages, surgical face masks, and KN95 masks. The antimicrobial properties of AV-PVP-Thyme-I2 were studied by disc diffusion methods and compared with those of the antibiotics gentamycin and nystatin. The results confirm AV-PVP-Thyme-I2 as a strong antifungal and antibacterial agent against the majority of the tested microorganisms with excellent results on cotton bandages and face masks. After storing AV-PVP-Thyme-I2 for 18 months, the inhibitory action was augmented compared to the fresh formulation. Consequently, we suggest AV-PVP-Thyme-I2 as an antimicrobial agent against wound infections and a spray-on contact killing agent.

Targeting the Cgas-Sting Pathway Inhibits the Progression and Enhances the Chemotherapy Efficacy of Peripheral T-Cell Lymphoma

Objective Peripheral T-cell lymphoma (PTCL) is a highly heterogeneous group of mature T-cell malignancies. Owing to its complex clinicopathological characteristics and diagnostic difficulties, the underlying molecular mechanism leading to the high invasiveness and rapid progression remains significantly unidentified. The efficacy of current first-line treatment is dismal, novel agents and therapeutic strategies are urgently needed to improve the prognosis of PTCL patients, especially for relapsed/refractory (RR) ones. Recent studies have indicated a close association between the cGAS-STING pathway and tumor promotion, which may provide new therapeutic targets. Herein, we investigated the role of the cGAS-STING pathway and its underlying mechanism in PTCL progression. Methods Firstly, we conducted single-cell RNA sequencing (scRNA-seq) to examine cellular identity and investigate the expression heterogeneity of cGAS-STING pathway. The cGAS-STING pathway score (CSPS) was calculated based on the expression of seven marker genes of this pathway. Immunofluorescence was performed to detect cGAS and STING expression in PTCL tissue microarray containing 35 PTCL samples and 8 normal lymph node biopsies. To assess the tumor-killing activity of cGAS-STING pathway inhibitors, here we tested five drugs including cGAS inhibitors G150 and G140, STING inhibitor H151, TANK binding kinase-1 (TBK1) inhibitor TBK1/Ikkε-IN-5 and signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic. Protein mass spectrometry was used to evaluate the protein changes in PTCL cell lines after G150 treatment. Next, we established a xenograft model to test the anti-tumor effect of G150 and TG003 (an inhibitor of CLK1) single-agent or combination therapy in vivo. Furthermore, we applied single-cell dynamic RNA sequencing to investigate the mechanism on the synergistic effect of TG003 and G150 in inhibition of cell proliferation. CCK8 and flow cytometric analysis were performed to detected the cellular viability and the apoptotic rate with the drug combination compared with single-drug treatment. Results scRNA-seq and showed the cGAS-STING pathway was overexpressed in PTCL with high heterogeneity and could be used as a prognostic marke (Fig. A-D). cGAS expression was positively correlated with DNA repair. cGAS inhibition effectively suppressed tumor growth via cGAS-STING-RELB-STAT3 signaling pathway and impairing DNA damage repair in PTCL. G150 treatment led to a concentration-dependent DNA damage (Fig. E-G). Moreover, single-cell dynamic RNA sequencing revealed that TG003 functions through inducing the reduction of proliferation-related nascent RNAs on the basis of G150 treatment (Fig. H,I). CLK1 played a critical role in the survival of residual tumor cells after treatment with cGAS inhibitors, and CLK1 suppression could enhance the sensitivity to cGAS inhibitors (Fig. J). In first-line therapy, chemotherapy-induced DNA damage activated the cGAS-STING pathway, which may ultimately lead to relapse or progression. cGAS inhibitors could synergize with chemotherapeutic agents in tumor killing (Fig. K-N). Fig. O summarized the oncogenic role of cGAS-STING pathway and the interaction between Tfh cells and B cells in AITL. Conclusion Herein, we demonstrated for the first time that the activated cGAS-STING pathway plays an oncogenic role in PTCL progression, in part by enhancing DNA repair and proliferation, which may also trigger chemotherapy resistance and ultimate relapse. CLK1 may serve as an indicator for the sensitivity to cGAS inhibitors. This may partly unveil the deficiency of first-line therapy and lay foundation for clinical application of cGAS-STING pathway-associated inhibitors.

A comparison of live versus kill pitfall traps to assess the diet of carabids through a metabarcoding approach

AbstractMetabarcoding approaches are powerful tools to unravel trophic relationships between predators and prey. To apply metabarcoding analyses on invertebrate gut contents, specimens must be well preserved from DNA degradation, thus the trapping method should be selected accordingly. Dry pitfall traps are commonly assumed to provide a better DNA preservation than traps that use a killing agent. However, this assumption has never been specifically tested for gut content analyses. In our study, we compared how two types of pitfall trapping, dry vs. with brine, affect the conservation of prey DNA contained in the digestive tract of predators and subsequent metabarcoding analyses. We placed dry and ‘classic’ pitfall traps in oilseed rape fields within an intensive agricultural area in the French Nouvelle‐Aquitaine region. Traps were set up in autumn and compared for carabid trapping efficiency as well as our capacity to retrieve dietary information from the digestive tract of the main carabid species, Nebria salina (Fairmaire &amp; Laboulbène) and Calathus fuscipes (Goeze) (both Coleoptera: Carabidae). The PCR success rate was higher in dry pitfall traps compared to classic ones for N. salina. We hypothesise that this was due to the presence of PCR inhibitors in the gut of this species. The ability to sequence prey DNA did not differ between specimens caught in both trap types. The list of preyed species was similar between both trap types. However, sequencing yielded more prey operational taxonomic units (OTUs) from specimens caught in dry pitfall traps, leading to differences in prey community composition and a greater ability to reconstruct prey community. Our analyses also shed light on the prey spectrum of C. fuscipes and N. salina in oilseed rape in autumn.

The neutrophil oxidant hypothiocyanous acid causes a thiol-specific stress response and an oxidative shift of the bacillithiol redox potential in Staphylococcus aureus.

During infections, Staphylococcus aureus is exposed to hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), which are produced by the neutrophil myeloperoxidase as potent antimicrobial killing agents. In this work, we applied RNAseq transcriptomics, Brx-roGFP2 biosensor measurements, and phenotype analyses to investigate the stress responses and defense mechanisms of S. aureus COL toward HOSCN stress. Based on the RNAseq transcriptome profile, HOSCN exerts strong thiol-specific oxidative, electrophile, and metal stress responses as well as protein damage in S. aureus, which is indicated by the strong induction of the HypR, TetR1, PerR, QsrR, MhqR, CstR, CsoR, CzrA, AgrA, HrcA, and CtsR regulons. Phenotype analyses of various mutants in HOSCN-responsive genes revealed that the HOSCN reductase MerA conferred the highest resistance toward HOSCN stress in S. aureus COL, whereas the QsrR and MhqR electrophile stress regulons do not contribute to protection. Brx-roGFP2 biosensor measurements and bacillithiol (BSH)-specific Western blot analyses revealed a strong oxidative shift of the bacillithiol redox potential (EBSH) and increased S-bacillithiolations in S. aureus, indicating that BSH is oxidized to bacillithiol disulfide (BSSB) under HOSCN stress. While the ΔmerA mutant was delayed in recovery of the reduced EBSH, overproduction of MerA in the ΔhypR mutant enabled faster recovery of EBSH due to efficient HOSCN detoxification. Moreover, both MerA and BSH were shown to contribute to HOSCN resistance in growth assays. In summary, HOSCN provokes a thiol-specific oxidative, electrophile, and metal stress response, an oxidative shift in EBSH and increased S-bacillithiolation in S. aureus.IMPORTANCEStaphylococcus aureus colonizes the skin and the airways but can also lead to life-threatening systemic and chronic infections. During colonization and phagocytosis by immune cells, S. aureus encounters the thiol-reactive oxidant HOSCN. The understanding of the adaptation mechanisms of S. aureus toward HOSCN stress is important to identify novel drug targets to combat multi-resistant S. aureus isolates. As a defense mechanism, S. aureus uses the flavin disulfide reductase MerA, which functions as HOSCN reductase and protects against HOSCN stress. Moreover, MerA homologs have conserved functions in HOSCN detoxification in other bacteria, including intestinal and respiratory pathogens. In this work, we studied the comprehensive thiol-reactive mode of action of HOSCN and its effect on the reversible shift of the EBSH to discover new defense mechanisms against the neutrophil oxidant. These findings provide new leads for future drug design to fight the pathogen at the sites of colonization and infections.

A Family of Spore Lipoproteins Stabilizes the Germination Apparatus by Altering Inner Spore Membrane Fluidity in Bacillus subtilis Spores.

Dormant bacterial spores undergo the process of germination to return to a vegetative state. In most species, germination involves the sensing of nutrient germinants, the release of various cations and a calcium-dipicolinic acid (DPA) complex, spore cortex degradation, and full rehydration of the spore core. These steps are mediated by membrane-associated proteins, and all these proteins have exposure on the outer surface of the membrane, a hydrated environment where they are potentially subject to damage during dormancy. A family of lipoproteins, including YlaJ, which is expressed from the sleB operon in some species, are present in all sequenced Bacillus and Clostridium genomes that contain sleB. B. subtilis possesses four proteins in this family, and prior studies have demonstrated two of these are required for efficient spore germination and these proteins contain a multimerization domain. Genetic studies of strains lacking all combinations of these four genes now reveal all four play roles in ensuring efficient germination, and affect multiple steps in this process. Electron microscopy does not reveal significant changes in spore morphology in strains lacking lipoproteins. Generalized polarization measurements of a membrane dye probe indicate the lipoproteins decrease spore membrane fluidity. These data suggest a model in which the lipoproteins form a macromolecular structure on the outer surface of the inner spore membrane, where they act to stabilize the membrane and potentially interact with other germination proteins, and thus stabilize the function of multiple components of the germination machinery. IMPORTANCE Bacterial spores exhibit extreme longevity and resistance to many killing agents, and are thus problematic agents of several diseases and of food spoilage. However, to cause disease or spoilage, germination of the spore and return to the vegetative state is necessary. The proteins responsible for initiation and progression of germination are thus potential targets for spore-killing processes. A family of membrane-bound lipoproteins that are conserved across most spore-forming species was studied in the model organism Bacillus subtilis. The results indicate that these proteins reduce the membrane fluidity and increase the stability of other membrane associated proteins that are required for germination. Further understanding of such protein interactions on the spore membrane surface will enhance our understanding of the germination process and its potential as a decontamination method target.

Co2+ Doped Biotinylated Carbon Dot: A Theranostic Agent for Target Specific Killing of Cancer Cells via Hypoxia Induced Apoptosis.

Conventional cancer treatments have systematic side effects that stand against its desirable therapeutic efficacy. Alternative strategies using biochemical features of cancer cells to promote apoptosis are finding notable significance. One such important biochemical feature of malignant cells is hypoxia, alteration of which can lead to cell death. Hypoxia inducible factor 1α (HIF-1α) has the key role in hypoxia generation. Herein, we synthesized biotinylated Co2+ -integrated carbon dot (CoCDb ) that specifically diagnose and selectively killed cancer cells with 3-3.1-fold higher efficiency over non-cancer cells by hypoxia induced apoptosis in absence of traditional therapeutic intervention. Immunoblotting assay in CoCDb treated MDA-MB-231 cells confirmed the increased expression of HIF-1α that was responsible for efficient killing of cancer cells. In 2D cells and 3D tumor spheroid, CoCDb treated cancer cells showed significant apoptosis that make CoCDb a potential theranostic agent.

Abstract 698: Developing a self-targeted cancer immunotherapy using engineered whole tumor cell-based vaccine

Abstract The administration of inactivated tumor cell lysate is known to induce a potent antitumor immune response, however, their therapeutic efficacy as shown in Phase I-III clinical trials is limited. This could be attributed to the lack of direct cytotoxic effect on tumor cells and the inability to trigger a strong antitumor immune response. Unlike inactivated tumor cells, living tumor cells possess a unique potential to home to and self-target tumors. Therefore, repurposing the tumor cells’ self-homing property and natural source of neoantigens is advantageous for self-targeted cancer immunotherapy. In this study, we developed a genetically engineered living whole cancer cell-based therapeutic with direct tumor killing and immunostimulatory roles. We switched the tumor cells from death ligands sensitive to resistant using CRISPR/Cas9 and subsequently engineered them to release dual cell killing and immunomodulatory agents. These engineered therapeutic tumor cells (ThTC) eliminated established tumor and activating antitumor immune cell trafficking and antigen-specific T cell activation signaling. This mechanism-based efficacy of ThTC translated into a marked survival benefit and long-term immunity in primary, recurrent, and metastatic cancer models in immunocompromised, immunocompetent and humanized mice. Arming naturally neoantigen-rich tumor cells with multidisciplinary therapeutics represents promising cell-based immunotherapy for solid tumors and establishes a roadmap towards clinical translation. Citation Format: Kok-Siong Chen, Natalia Claire Mendonca, Paulo Borges, Khalid Shah. Developing a self-targeted cancer immunotherapy using engineered whole tumor cell-based vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 698.

Should ocular Demodex be checked and treated in refractory keratitis patients without blepharitis?

To evaluate the correlation between Demodex infestation and keratitis, and to assess demodicosis using a simple approach. A modified slit lamp illumination (at 40× magnification) was used to observe Demodex tails in 40 patients with refractory keratitis and 80 healthy controls. Bacterial smear and culture of the conjunctival sac and corneal lesion were performed to identify the pathogen. Tea tree oil ointment (TTOO) was added as a Demodex killing agent for lid scrubs to the treatment when Demodex infestation was confirmed. Demodex tails were found in all patients compared to 42/80 of the controls (P<0.01). Seventeen patients presented blepharitis, while 23 were free of scales and inflammation at the lid margin. The demodicosis was mild, moderate, and severe in 8, 19, and 13 patients, respectively, compared to mild in 42 controls (P<0.01). The keratitis was mild, moderate, and severe in 13, 19, and 8 patients, respectively. The severity of Demodex infestation was not correlated to the severity of keratitis (P=0.126). The growth of Staphylococcus was revealed in nine patients who did not react to antibiotic eye drops prior to the TTOO treatment. Patients' signs and symptoms got resolved after the lid scrub with TTOO. Ocular Demodex needs to be checked and treated in refractory keratitis patients with or without blepharitis. A slit-lamp illumination under high magnification favors the judgment of the severity of Demodex infestation.

Potential Alternatives to Spinosad as the Killing Agent Mixed With Two Attractant Products in Attract-and-Kill Formulations Used to Manage the Spotted-Wing Drosophila, Drosophila suzukii (Diptera: Drosophilidae).

Spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is a key pest of many berry and fruit crops worldwide. The primary method of controlling this pest is the application of insecticides. Attract-and-kill is a management tactic that may reduce the number of insecticide applications needed to manage D. suzukii. ACTTRA SWD OR1 and ACTTRA SWD TD, developed by ISCA Technologies Inc., combine D. suzukii attractants with a gel matrix. Growers add an insecticide as a killing agent. The only USDA National Organic Program approved organic insecticide that has been shown to be effective as a killing agent is spinosad. This study aimed to determine the efficacy of other USDA National Organic Program approved organic insecticides, including Grandevo 30 WDG (Chromobacterium subtsugae strain PRAA4-1 30%), MBI-203 SC2 (C. subtsugae strain PRAA4-1 98%), Venerate XC (Burkholderia spp. Strain A396 94.45%), MBI-306 SC1 (B. rinojensis Strain A396 94.45%), Azera (azadirachtin 1.2% + pyrethrins 1.4%), and PyGanic (pyrethrins 1.4%), when used as the killing agent with the two ACTTRA SWD products. Lab and cage bioassays were conducted. Entrust (spinosad 22.5%) and PyGanic were the only compounds that showed some efficacy when used with ACTTRA SWD OR1 and ACTTRA SWD TD.

Winning Formulas to Fulfill Cancer Moonshot

The objective of this study was to develop winning formulas to fulfill cancer moonshot declared by President Biden on Sept. 12, 2022, the 60th anniversary of the moonshot speech of President Kennedy. The intention of President Biden was to use the spirit of successful moonshot project to salvage the failure of war on cancer project declared by President Nixon in 1971 [1]. Cancer therapies based on killing of cancer cells were the choice of cancer establishments in the past. The choice of killing cancer cells is understandable because the perpetual replication of cancer cells constitutes the most outstanding feature of cancer. Cancer is contributed by multiple factors. Factors other than replication of cancer cells also play essential roles on the development of cancer. Cancer arises as a consequence of wound not healing properly due to the collapse of chemo-surveillance, thus allowing Progenitor Stem Cells (PSCs) to evolve into Cancer Stem Cells (CSCs), and then to progress to faster growing Cancer Cells (CCs) through activation of oncogenes and/or inactivation suppressor genes. Cell killing creates more wounds to aggravate the already bad situation on the functionality of chemo-surveillance, which is an important mechanism to suppress the build-up of cells with abnormal Methylation Enzymes (MEs) that include PSCs, CSCs, and CCs. PSCs and their immediate derivatives CSCs are protected by drug resistant and anti-apoptosis mechanisms. Cytotoxic agents can wipe out CCs but cannot affect CSCs. The damage to chemo-surveillance contributed by cell killing agents allows CSCs to become dominant cells to cause he failure of cancer therapy, thus losing the war on cancer. Cell Differentiation Agent (CDA) formulations are the preparations consisting of Differentiation Inducers (DIs) and Differentiation Helper Inducers (DHIs), plus phenylacetylglutamine as an anti-cachexia agent. Such preparations are perfect cancer drugs to take out both CCs and CSCs by the induction of terminal differentiation through destabilization of abnormal MEs, and to restore the functionality of chemo-surveillance. Cancer therapies mediated by CDA formulations are the nature’s choice to combat cancer that can fulfill the goal of President Biden’s cancer moonshot. CDA formulations, however, cannot make the tumor to disappear, which can be easily accomplished by therapies based on cell killing. President Biden’s goal is very modest, requiring reduction of cancer mortality 50% in 25 years. That goal can be easily accomplished by winning formulas that include CDA formulations and therapies to eliminate or to kill CCs. We offer the following winning formulas: 1. Therapy with CDA-CSC, a preparation made up by an approved DI and an approved DHI, to eradicate CSCs and CCs by the induction of terminal differentiation and to restore the functionality of chemo-surveillance, followed by the surgical removal of the residual tumor. 2. Surgical removal of the primary tumor, followed by the administration of CDA-CSC to prevent recurrence and metastasis, and to restore the functionality of chemo-surveillance. 3. Radiotherapy, immunotherapy, or chemotherapy to kill CCs, followed by the administration of CDA-CSC to eradicate CSCs and to restore the functionality of chemo-surveillance.

Antibody drug conjugates, targeting cancer-expressed EGFR, exhibit potent and specific antitumor activity

The monoclonal antibody ‘40H3′ binds to EGFRvIII and to full-length EGFR when it is overexpressed on cancer cells. To generate candidate cytotoxic antibody-drug conjugates (ADCs), 40H3 was modified by the addition of small molecular weight payloads that included two tubulin-modifying agents, two topoisomerase inhibitors and a pyrrolobenzodiazepine (PBD) dimer. Conjugates retained antigen binding activity comparable to the unmodified 40H3 antibody. The cytotoxicity of five distinct ADCs was evaluated on a variety of EGFR-expressing cells including three triple negative breast cancer (TNBC) lines. Generally, the 40H3 conjugate with the PBD dimer (40H3-Tesirine) was the most active killing agent. The killing of EGFR-positive cells by 40H3-Tesirine correlated with the number of surface binding sites for 40H3. However, bystander killing was also evident in experiments with antigen-negative cells. In vivo tumor xenograft experiments were conducted on two TNBC tumor lines. Three treatments with the 40H3-Tesirine ADC at 1 mg/kg were sufficient to achieve complete remissions without evidence of mouse toxicity. Data support the development of ADCs derived from the 40H3 antibody for the treatment of cancers that express EGFRvIII or high levels of EGFR.

240 (PB020) - Antitumor activity of Antibody-Drug Conjugates targeting cancer-expressed EGFR in Preclinical Models

Background: The epidermal growth factor receptor (EGFR) plays a key role in the growth and survival of many human tumors of epithelial origin. The monoclonal antibody, 40H3, targeting overexpressed EGFR and the truncated form EGFR variant III (EGFRvIII) on tumor cells, was conjugated to small molecules with potent cytotoxic activity, generating five antibody-drug conjugates (ADCs). Their lethality for tumor cells was evaluated in vitro and in vivo. Materials and methods: ADC construction: Purified 40H3 antibody was conjugated to five different payloads: two tubulin inhibitors, monomethyl auristatin E (MMAE) and DM1, two topoisomerase inhibitors, SN38 and deruxtecan (DXd1) and a PBD dimer (SG-3199). Binding assay: The binding affinity constants of the various 40H3-based ADCs were determined against the His-tagged EGFR peptide loop (aa 287– 302) immobilized on Ni-NTA biosensors with the Octet Red96 analyzer. Bystander assay: Cocultured F98npEGFRvIII and F98 cells were treated with media, free payload (SG-3199), 40H3 antibody, 40H3-tesirine or IgG-tesirine at the indicated concentrations. After 48 hours, the cells were labeled with cetuximab-PE and SYTOXTM Red viability dye and then analyzed for bystander killing by flow cytometry. In vivo studies: MDA-MB-468 or BT-20 tumor xenografts were treated with unmodified 40H3 antibody or 40H3-tesirine. Tumor volumes and mouse weights were measured at least three times weekly. Results: The five ADCs retained antigen binding activity of the unmodified 40H3 antibody. They showed a potent cytotoxicity on a panel of EGFR-expressing cells including three triple negative breast cancer lines. Cell killing was correlated to the number of binding sites for the 40H3 antibody. The 40H3 conjugate with the PBD dimer (40H3-tesirine) was the most active killing agent and it also exhibited bystander killing of cells not expressing human EGFR. Moreover, in vivo, on two different models of tumor xenografts, treatment with 40H3-tesirine achieved complete remissions. Conclusions: The 40H3 antibody is a valid delivery agent for toxic payloads. Among the five ADCs, 40H3-tesirine showed the highest cytotoxic effect toward EGFR-expressing tumor cells in vitro and in vivo. No conflict of interest.