Efficient Killing Research Articles

Magnesium Oxide Nanoparticles Synthesized from Solanum elaeagnifolium and their Effect Against Mealybugs Phenacoccus solenopsis Tinsley

The excessive use of chemical insecticides and the emergence of accompanying environmental problems has led to a return to environmentally friendly plant extracts through nanotechnology and the production of particles loaded on many metals to control insects. Accordingly, the study was conducted in 2023 to investigate the efficiency of the nano- extract of the seeds and leaves of the nightshade Solanum elaeagnifolium Cav loaded on magnesium, producing MgO nanoparticles and the regular alcoholic seed extract with a concentration of 5% on the relative efficiency of killing the first and third nymph instars as well as the adults of the mealybug Phenacoccus solenopsis Tinsley, grown on moss rose seedlings. The results showed that the highest relative efficiency in killing the insects at the first instar was 100% three days after treatment with nano seed extract, 84% at the third instar, and 75% for mealybug adults after seven days of treatment. In contrast, nano extract of nightshade leaves recorded a relative mortality efficiency of 91.6, 47.4 and 54.3% for the first and third nymph instars and the adults, respectively, and 97.1, 55.0 and 58.7% for the regular alcoholic seed extract after seven days of treatment. Keywords: Alcoholic extract, nightshade, nanotechnology, nanoextract, mealybug.

Unveiling the Cytotoxic Potential of Quercetin-Loaded Magnetic Bacterial Bots against Cervical Cancer.

Bacterial bots are potent vehicles in cancer theranostics where bacteria are used typically as cargos for drug delivery. However, living bacteria themselves may aid in their efficiency in killing the tissues. For example, living bacteria may be functionalized with magnetic and luminescent nanoparticles along with drugs in order to achieve the targeted delivery and release of payloads that would include the bacteria. In this study, we elucidate the synergistic impact of human-friendly living Lactobacillus rhamnosus bacteria decorated with gold nanoclusters and quercetin-loaded magnetic nanoparticles against the HeLa-cervical cancer cells. The cytotoxicity of the fabricated magnetic bacterial bots within the low dose (5 × 105 CFU/mL) against the HeLa monolayer and 3D spheroid model was found around 84%, which had remarkably enhanced up to more than 91% in the presence of an external magnet. The antiproliferative action of magnetic bacterial bots was demonstrated by the escalation of generated reactive oxygen species from 1.8- to 2.3-fold. Cells treated with magnetic bacterial bots showed a decrease in lipid droplet content, along with altered cell cycle patterns, which led to 74% (average population) of cells being exposed to necroptosis. The study highlights the cytotoxic potential of magnetic bacterial bots against cervical cancer cells outweighing that of the quercetin or the bacteria only, ensuring an external-magnetic-field-responsive targeted drug delivery system.

Profiles of Killer Systems and Volatile Organic Compounds of Rowanberry and Rosehip-Inhabiting Yeasts Substantiate Implications for Biocontrol.

Yeasts produce numerous antimicrobial agents such as killer toxins, volatile organic compounds (VOCs), and other secondary metabolites, establishing themselves in developing natural and sustainable biocontrol strategies for agriculture and food preservation. This study addressed the biocontrol potential of yeasts, isolated from spontaneous fermentations of rosehips (Rosa canina L.) and rowanberries (Sorbus aucuparia L.), focusing on their killer phenotypes and VOCs production. Yeasts were isolated using spontaneous fermentations with Hanseniaspora uvarum and Metschnikowia pulcherrima identified as the dominant species, comprising approximately 70% of the yeast population. Among 163 isolated strains, 20% demonstrated killing activity, with Saccharomyces cerevisiae exhibiting the strongest killing efficiency, as well as Pichia anomala and M. pulcherrima showing broad-spectrum antagonistic activity. This study identified dsRNA-encoded killer phenotypes in S. cerevisiae, S. paradoxus, and Torulaspora delbrueckii, revealing multiple distinct killer toxin types. The biocontrol potential of wild berry-inhabiting yeasts was demonstrated in a real food system, grape juice, where the S. cerevisiae K2-type killer strain significantly reduced fungal contaminants. The selected H. uvarum, M. pulcherrima, S. cerevisiae, and S. paradoxus yeast strains representing both berries were applied for VOC analysis and identification by gas chromatography-linked mass spectrometry. It was revealed that the patterns of emitted volatiles are yeast species-specific. Statistically significant differences between the individual VOCs were observed among killing phenotype-possessing vs. non-killer S. paradoxus yeasts, thus revealing the involvement of killer systems in multi-level biocontrol enablement. The performed studies deepen our understanding of potential yeast biocontrol mechanisms, highlight the importance of produced antimicrobials and volatiles in ensuring antagonistic efficacy, and prove the relevance of isolated biocontrol yeasts for improving food safety.

In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma.

Chimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immunosuppression in the tumor microenvironment. CAR-T cell exhaustion is a major cause of relapse. Epigenetic modifications can regulate T cell activation, maturation and depletion; they can be applied to reduce T cell depletion, improve infiltration, and promote memory phenotype formation to reduce relapse after CAR-T cell therapy. We propose to develop and validate in vitro the function of novel CAR-T cells for the treatment of DLBCL, which simultaneously express an anti-CD19 CAR with lysine-specific demethylase 1 (LSD1) short hairpin (sh)RNA to prevent depletion and prolong the survival of CAR-T cells. We designed an shRNA sequence targeting LSD1 mRNA, and created a vector with the following elements: the U6 promoter driving expression of the LSD1 shRNA sequence, the EF1a promoter driving a second-generation anti-CD19 CAR sequence encoding an anti-CD19 single-chain variable fragment (FMC63), the CD8 hinge and transmembrane structural domains, the CD28 co-stimulatory structural domain, and the CD3ζ-activating structural domain. The MFG-LSD1 shRNA anti-CD19 CAR plasmid was first constructed, then packaged in retroviral vectors and transduced into human primary peripheral blood mononuclear cell-derived T cells to generate the corresponding CAR-T cells. We examined by flow cytometry the efficiency of two CAR-T cells in killing U-2932 cells (a human DLBCL line) upon co-culture with RNAU6 anti-CD19 CAR-T cells or LSD1 shRNA anti-CD19 CAR-T cells. We analyzed Ki-67 staining of the CAR-T cells by flow cytometry on days 0, 5, and 10, and counted the cells to assess expansion. We also used flow cytometry to detect the central memory T cell (TCM) proportion. We detected the expression of the CAR in the CAR-T cells by flow cytometry, and observed transduction rates of 31.5% for RNAU6 anti-CD19 CAR-T cells and 60.7% for LSD1 shRNA anti-CD19 CAR-T cells. The killing efficiency of LSD1 shRNA anti-CD19 CAR-T cells was significantly higher than that of RNAU6 anti-CD19 CAR-T cells at the low effector target ratio. We further found that LSD1 shRNA anti-CD19 CAR-T cells secreted more IFN-γ and granzyme B than RNAU6 anti-CD19 CAR-T cells. CAR-T cells proliferated after U-2932 cell stimulation and were able to sustain proliferation. After stimulation via U-2932 cell co-culture, both RNAU6 anti-CD19 CAR-T and LSD1 shRNA anti-CD19 CAR-T populations had increased proportions of cells with the TCM phenotype, with a higher percentage among LSD1 shRNA anti-CD19 CAR-T cells. We developed a novel, feasible CD19-LSD1 shRNA CAR-T cell strategy for the treatment of DLBCL. Our in vitro assay results showed that LSD1 shRNA anti-CD19 CAR-T cells more effectively killed target cells than RNAU6 anti-CD19 CAR-T cells, and developed a higher proportion of TCM phenotype cells. LSD1 shRNA anti-CD19 CAR-T cells may represent a potential treatment for DLBCL.

Construction of efficient ethylene removal and antibacterial cellulose paper-based packaging materials for avocado preservation.

Fruits are susceptible to ethylene ripening and microbial infestation, which can lead to spoilage and further significant economic losses. Thus, using functional preservation materials is an effective controlling technology to improve the post-harvest quality and extend the shelf life of fruits. Hence, a dual-function cellulose-based paper with exceptional antibacterial efficiency, favourable ethylene removal performance, improved mechanical and hydrophobic properties was prepared by covalently grafted antibacterial guanidine salt and surface-modified ethylene scavenger. When the amount of guanidine salt grafted and ethylene scavenger is 1.85 wt% and 1.5 g·m-2, respectively, the prepared cellulose paper exhibited about 99 % ethylene conversion and killing efficiency with no leaching, which extending the post-harvest avocado preservation time to 20 days at room temperature. Packaging with the preservation paper effectively reduced hardness and consumption of soluble solids in avocado during storage, maintaining their appearance and nutritional quality. These findings highlight the excellent preservation capabilities of the dual-function paper, making it a promising material for fruit and vegetable preservation in small and medium planting areas and households.

Modulation of Donor in Purely Organic Triplet Harvesting AIE-TADF Photosensitizer for Image-guided Photodynamic Therapy.

Image-guided photodynamic therapy is acknowledged as one of the most demonstrative therapeutic modalities for cancer treatment because of its high precision, non-invasiveness, and improved imaging ability. A series of purely organic photosensitizers denoted as BTMCz, BTMPTZ, and BTMPXZ, have been designed and synthesized and are found to exhibit both thermally activated delayed fluorescence and aggregation-induced emission simultaneously. Experimental and theoretical studies are combined to reveal that modulation of the donor of the photosensitizer enables distinct thermally activated delayed fluorescence via a second-order spin-orbit perturbation mechanism involving lowest singlet charge-transfer and higher-lying triplet locally excited states, respectively. Further, different donor strengths and unique aggregations (H-, J- and X-type packings) greatly influence their color-tunable up-converted luminescence and endow them with superb dispersibility in water. The confocal microscopy-based cellular uptake study confirms the successful internalization of the nano-probes, while BTMCz enables the generation of reactive oxygen species (singlet oxygen) under white-light irradiation, enabling the efficient killing of cancer cells.

Bioinspired Synthesis of Silver Nanoparticles Using L. Reuteri: Antibacterial Efficacy, Molecular Docking Insights, and Cytotoxicity Assessment

AbstractThis study reports the green synthesis of silver nanoparticles (AgNps) by using Lactobacillus reuteri. Characterized AgNps exhibited near‐spherical morphology with an average size of 86.54 nm, as revealed by SEM (scanning electron microscopy), TEM (transmission electron microscopy), and AFM (atomic force microscopy) analyses. The nanoparticles displayed a slightly rough surface, with some particles exhibiting surface protrusions and sharp edges. The zeta potential measurement of −30.16 mV and electrophoretic mobility of −2.36 cm2/Vs that indicated the presence of negatively charged functional groups on the AgNps surface, suggesting stabilizing biomolecules that prevent nanoparticle aggregation in solution. The hydrodynamic size of the AgNps was determined to be 140.3 nm, and XRD (X‐ray diffraction) analysis revealed a crystallinity of 76.89% with an average crystal size of 8.35 nm and an interplanar distance of 0.233 nm. AgNps demonstrated crystallinity and potent antibacterial activity against Pseudomonas aeruginosa (92.9% killing efficiency) and an ATCC reference strain (76.8%). Molecular docking revealed moderate interactions between an optimized Ag3 cluster and key amino acid residues (Arg277, Gln279, Trp280, Asp273, and Val276) in the P. aeruginosa protein. These interactions, with binding energies ranging from −1.89 to −3.68 kcal/mol, suggested a potential mechanism for the observed antibacterial activity. A charge transfer interaction involving Asp273, contributing a stabilization energy of 3.66 kcal/mol, was identified as a key factor in the Ag‐protein complex formation.

Nanosecond Laser Pulses Facilitating Efficient and Specific Cell Killing with Doxorubicin‐Loaded Gold Nanoparticles Targeted to the Folate Receptor

Nanoparticle‐based drug carrier systems with active targeting and a controlled release capacity are of considerable interest to bypass side effects of conventional chemotherapy. One appealing approach involves chemotherapeutic‐loaded photothermal nanoparticles, where laser irradiation can release the loaded anticancer drug while also inducing local cytotoxicity through photothermal effects. This study investigates the potential of using nanosecond‐pulsed laser light for efficient and specific killing of folate receptor (FR)‐overexpressing cancer cells in combination with FR‐targeted doxorubicin‐loaded gold nanoparticles (AuNPs). Nanosecond pulsed laser irradiation allows the induction of mechanical forces alongside thermal effects. The effect of nanoparticle concentrations and laser fluences on cytotoxicity is systematically tested, achieving near‐complete tumor cell killing under the most stringent conditions. FR targeting is confirmed using FR‐positive and ‐negative cell lines, showing that folic acid functionalization of AuNPs results in more favorable nanoparticle–cell interactions and more efficient photothermal effects. Additionally, doxorubicin could be efficiently released from the AuNPs and endosomal compartments upon laser irradiation, adding to the observed cytotoxicity. Cell killing was precisely confined to irradiated cells, leaving surrounding cells unharmed. Overall, the significant reduction of tumor cell viability following the proposed combination demonstrates this approach to be a promising step toward safer, more effective anticancer therapies.

Required minimal protein domain of flower for synaptobrevin2 endocytosis in cytotoxic T cells

Flower, a highly conserved protein, crucial for endocytosis and cellular fitness, has been implicated in cytotoxic T lymphocyte (CTL) killing efficiency through its role in cytotoxic granule (CG) endocytosis at the immune synapse (IS). This study explores the molecular cues that govern Flower-mediated CG endocytosis by analyzing uptake of Synaptobrevin2, a protein specific to CG in mouse CTL. Using immunogold electron microscopy and total internal fluorescence microscopy, we found that Flower translocates in a stimulus-dependent manner from small vesicles to the IS, thereby ensuring specificity in CG membrane protein recycling. Using confocal live-cell imaging, we assessed the ability of a range of naturally occurring mouse, human and Drosophila isoforms to rescue defective endocytosis in Flower KO CTLs. This analysis demonstrated that the N-terminal portion of the protein, encompassing amino acids 1–106 in mice, is the minimal domain necessary for Synaptobrevin2 endocytosis. Additionally, we identified two pivotal sites through site-specific mutation: a putative AP2-binding site, and a tyrosine at position 104 in mouse Flower. These findings provide insights into Flower's specific functional domain essential for CG endocytosis, which is a key process in mediating T cell serial killing required for the effective fight against cancer.

Discovery of a pan anti-SARS-CoV-2 monoclonal antibody with highly efficient infected cell killing capacity for novel immunotherapeutic approaches

ABSTRACT Unlocking the potential of broadly reactive coronavirus monoclonal antibodies (mAbs) and their derivatives offers a transformative therapeutic avenue against severe COVID-19, especially crucial for safeguarding high-risk populations. Novel mAb-based immunotherapies may help address the reduced efficacy of current vaccines and neutralizing mAbs caused by the emergence of variants of concern (VOCs). Using phage display technology, we discovered a pan-SARS-CoV-2 mAb (C10) that targets a conserved region within the receptor-binding domain (RBD) of the virus. Noteworthy, C10 demonstrates exceptional efficacy in recognizing all assessed VOCs, including recent Omicron variants. While C10 lacks direct neutralization capacity, it efficiently binds to infected lung epithelial cells and induces their lysis via natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Building upon this pan-SARS-CoV-2 mAb, we engineered C10-based, Chimeric Antigen Receptor (CAR)-T cells endowed with efficient killing capacity against SARS-CoV-2-infected lung epithelial cells. Notably, NK and CAR-T-cell mediated killing of lung infected cells effectively reduces viral titers. These findings highlight the potential of non-neutralizing mAbs in providing immune protection against emerging infectious diseases. Our work reveals a pan-SARS-CoV-2 mAb effective in targeting infected cells and demonstrates the proof-of-concept for the potential application of CAR-T cell therapy in combating SARS-CoV-2 infections. Furthermore, it holds promise for the development of innovative antibody-based and cell-based therapeutic strategies against severe COVID-19 by expanding the array of therapeutic options available for high-risk populations.

Loss of AXIN1 regulates response to lenvatinib through a WNT/KDM5B/p15 signalling axis in hepatocellular carcinoma.

As a highly heterogeneous cancer, hepatocellular carcinoma (HCC) shows different response rates to the multi-kinase inhibitor lenvatinib. Thus, it is important to explore genetic biomarkers for precision lenvatinib therapy in HCC. The effect and mechanism of AXIN1 mutation on HCC were revealed by cell proliferation assay, long-term clone formation assay, sphere formation assay and small molecule inhibitor library screening. A new therapeutic strategy targeting HCC with AXIN1 mutation was evaluated in humanized models (patient-derived xenograft [PDX] and patient-derived organoid [PDO]). Based on The Cancer Genome Atlas (TCGA) data, we screened 6 most frequently lost tumour suppressor genes in HCC (TP53, ARID1A, AXIN1, CDKN2A, ARID2 and PTEN) and identified AXIN1 as the most crucial gene for lenvatinib sensitivity. Further study showed that AXIN1-knockout HCC cells had a more malignant phenotype and lower sensitivity to lenvatinib in vitro and in vivo. Mechanistically, the WNT pathway and its target gene c-Myc were activated when AXIN1 was missing, and the expression of tumour suppressor p15 was inhibited by transcription co-repressors c-Myc and Miz-1, resulting in the exacerbation of the resistant phenotype. Screening of a library of epigenetic-related enzyme inhibitors showed that a KDM5B inhibitor up-regulated p15 expression, leading to increased sensitivity to lenvatinib in vitro and in vivo. AXIN1-deficient patients have a lower response to lenvatinib, which may be associated with suppression of p15 mediated by WNT pathway activation. KDM5B inhibitors can restore p15 levels, resulting in efficient killing of resistant cells in HCC.

Pulsed Electric Field Treatment in Extracting Proteins from Legumes: A Review

A healthy diet rich in plant proteins can help in preventing chronic degenerative diseases. Plant-based protein consists of derivatives from algae, fungi (like mushrooms) and other plant products including stems, leaves, fruits, vegetables, grains, seeds, legumes and nuts. These sources are not only rich in protein, but also contain a high percentage of iron, calcium, folates, fiber, carbohydrates, fats etc. Hence, it is essential to explore plant-based protein sources and their other nutritional components to address existing food insecurity issues. Nowadays, the impact of food processing has produced promising results in extracting valuable bio-compounds including proteins from the plant matrix. In this view, PEF technology has secured an exceptional place in solving food quality issues through minimized thermal effects in the samples, improved extraction capabilities at a shorter time, higher extraction levels, high nutritional content of extracted samples, greater shelf-life extension and increased microbial killing efficiency. It is an energy efficient process which is used as a pre-treatment to increase selective extraction of intracellular compounds through electroporation technique. Here, the processing parameters play a significant role in obtaining enhanced extraction levels. These parameters have also considerably influenced the protein digestibility and amino acid modification. So far, PEF has been producing remarkable results in plant protein extraction research. Among various plant sources mentioned above, there is a limited literature available on the use of PEF-assisted protein extraction from legumes. In this review, the authors have discussed essential legumes and their nutritional components and have highlighted how PEF can be beneficial in extracting the protein levels from these sources. Further research should focus on PEF-assisted protein extraction from legumes, specifically analyzing the properties of protein quality and quantity.

Chlorine-resistant and dual anti-biofouling reverse osmosis membranes with zwitterionic and quaternary ammonium copolymers via mussel-inspired one-step codeposition

Reverse osmosis (RO) membrane technology encounters challenges such as membrane biofouling and oxidation during practical use, which significantly hinders its further development and application. To improve the chlorine and biofouling resistance of RO membrane, we suggest a method that modifies the mass ratios of zwitterionic and quaternary ammonium copolymers in the mussel-inspired coating layer. This method successfully controls the hydrophilicity and positive charge of the membrane surface, enhancing its overall performance. The plate counting results indicate that the bacterial killing efficiency of the blended modified TFC membrane (50:50) against E. coli and S. aureus is ≥98 %. Thanks to the combined effects of the anti-adhesion zwitterionic copolymer and the antibacterial quaternary ammonium copolymer, the blended modified TFC membrane (50:50) demonstrates superior anti-biofouling performance in dynamic biofouling tests. Furthermore, the desalination performance of the blending modified TFC membrane (50:50) remains stable after long-term exposure to 60,000 ppm·h of active chlorine, while the desalination performance of the pristine TFC membrane significantly declines. In conclusion, our advancements in chlorine-tolerant and anti-biofouling RO membranes could enhance the reliability of RO technology.

IMMU-69. THERAPEUTIC WINDOW ENABLING ERADICATION OF RESIDUAL GLIOMA STEM CELLS BY INTRACRANIAL NK CELL THERAPY FOLLOWING TEMOZOLOMIDE-INDUCED TUMOUR DEPOPULATION – A POSSIBLE ROLE OF CYTOTOXIC EXTRACELLULAR VESICLES

Abstract Glioblastoma (GBM) is an incurable brain cancer, where dismal outcomes result from disease recurrence driven by tumour-initiating glioma stem cells (GSCs). GSCs survive and expand in the brain after surgery, radiation and temozolomide (TMZ) amidst weak immune and natural killer (NK) cell surveillance. Efficient NK cell-mediated killing of GBM cells occurs at high effector to target ratios precluding effective eradication of large tumour remnants, as enforced by clinical trials with autologous NK cells in an adjuvant setting. Here we explore in a human GSC xenograft model whether tumor depopulation using high dose Temozolomide (TMZ) could create a window of curative opportunity for endogenous or exogenous NK cells. We observed that while subcutaneous (sc) xenografts of patient derived GSCs are infiltrated by endogenous functional (NCR1+) NK cells in SCID mice, the corresponding intracranial (ic) tumours remained NK cell depleted. Notably, while TMZ caused near complete regression of intracranial GSC xenografts this was followed by inevitable recurrence of drug-resistant lesions. To explore whether direct delivery of NK cells into depopulated intracranial tumours would change the lethal course of the disease, mice were injected with GSCs intracranially, and upon tumour formation were treated with a sequence of TMZ (systemically), at 2, 7, 14, and 21 days later with irradiated NK92MI cells. Remarkably, this combined therapy completely obliterated recurrent disease at 2 and 7 days but not beyond 14 days. To assess whether NK92MI cells could be replaced by their derived extracellular vesicles (NK-EVs), the latter were injected i.c. post TMZ in GSC xenograft bearing mice. A single injection of NK-EVs resulted in tumour eradication in some but not all mice. Thus, chemotherapy-dependent tumour depopulation may create a unique window of opportunity for curative NK-mediated immunotherapy in GBM.

Evaluating the potential of off-the-shelf engineered mesenchymal stem cells for targeted Hepatocellular Carcinoma treatment: A multisite proof-of-concept study

Although combining 5-fluorouracil (5-FU) and Interferon-beta (IFNb) improves response rates in Hepatocellular Carcinoma (HCC), the outcomes remain suboptimal. This study investigates the feasibility of using highly transfected Mesenchymal Stem Cells (MSCs) to deliver a chemotherapeutic (5-FU) and an immunomodulator (IFNb) for localized HCC treatment. Considering the crucial role of cold-chain transportation in off-the-shelf allogeneic therapy, the study also assesses the quality and efficacy of frozen-thawed engineered MSCs, simulating a multisite study process. The engineered MSCs maintained their phenotypes and tumour tropism. With just 10 % engineered MSCs, a killing efficiency of over 70 % was achieved in Huh-7 and HepG2 cell lines in vitro. Coculture studies, soft agar assays, and in vivo experiments confirmed that MSCs are neither tumorigenic nor tumour-promoting. Tumour mass growth was inhibited by >80 % in the treated mice group. TUNEL, Annexin-V, and Ki67 staining confirmed DNA damage, cell death, and proliferation inhibition post-treatment. Blood chemistry and the weight of the mice were comparable to the control group, indicating a good safety profile. This proof-of-concept study demonstrates the efficacy and safety of off-the-shelf CDUPRT-IFNβ_MSCs in targeting hepatocellular carcinoma (HCC) growth. Evaluating the complete value chain of MSC therapy in early-stage preclinical studies is essential for justifying further investigation and clinical translation of this cell product.

Macropinocytosis-targeted peptide-docetaxel conjugate for bystander pancreatic cancer treatment

Oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are highly prevalent in pancreatic ductal adenocarcinoma (PDAC) and have garnered attention as potential targets for targeted therapies, such as KRAS inhibitors. However, the limited therapeutic efficacy of KRAS allele-specific inhibitors necessitate an efficient pan-KRAS cancer cell killing strategy. Here, we have examined enhanced macropinocytosis pathway in KRAS mutant cancer cells and report improved intracellular delivery of albumin-based therapeutics. We further established an albumin-binding peptide-docetaxel conjugate platform (MPD3), which has a caspase-3 cleavable feature, for macropinocytosis-targeted bystander payload delivery and realization of bystander killing of pan-KRAS cancer cells, complemented with caspase-3 mediated activation of MPD3 to bolster tumoral accumulation of cytotoxic payloads. Utilization of in vitro co-culture system of pan-KRAS cancer cells and pharmacodynamic marker staining revealed potent bystander killing effects of MPD3, highlighting MPD3 as an efficient delivery platform against pan-KRAS cancer. Moreover, MPD3 elicited robust anti-tumor activities in both local and liver metastatic PDAC tumor models in mice. Overall, this work establishes a paradigm for developing translational pan-KRAS cancer treatment and broadens the applicability of albumin binding peptide-drug conjugate against albumin-metabolism enriched cancers.

Race for the surface between THP-1 macrophages and Staphylococcus aureus on various titanium implants with well-defined topography and wettability.

Gristina et al. (1987) suggested the fate of a biomaterial is decided in a “race for the surface” between pathogens and the host. To gain deeper insight into the mechanisms behind this concept, we investigated the “race for the surface” across three co-culture scenarios with THP-1 macrophages and Staphylococcus aureus (1:1 ratio), varying the order of addition: (i) simultaneous, (ii) macrophages first, and (iii) S. aureus first, on six Ti6Al4V-ELI surfaces modified with specific topographies and wettability. The outcome of the race for the surface was not influenced by these biomaterials but by the chronological introduction of macrophages and S. aureus. When macrophages and S. aureus arrived simultaneously, macrophages won the race, leading to the lowest number of viable S. aureus through rapid phagocytosis and killing. When macrophages arrived and established first, macrophages still prevailed but under greater challenge resulting from the lower bacterial killing efficiency of adherent macrophages and numerous viable intracellular bacteria, supporting the concept of the so-called immunocompromised zone around implants (upregulation of TLR-2 receptor and pro-inflammatory IL-1β). When S. aureus arrived first establishing a biofilm, bacteria won the race, leading to macrophage dysfunction and cell death (upregulation of FcγR and TLR-2 receptors, NF-κB signaling, NOX2 mediated reactive oxygen species), contributing to a persistent biofilm phenotype (upregulation of clfA, icaA, sarA, downregulation of agrA, hld, lukAB) and intracellular survival of S. aureus (lipA upregulation). The clinical implications are bacterial colonization of the implant and persistence of intracellular bacteria in periprosthetic tissues, which can lead to infection chronicity. Statement of SignificanceGristina et al. (1987) suggested the fate of a biomaterial is decided in a “race for the surface” between bacterial pathogens and host cells. There is a lack of in vitro co-culture models and knowledge on macrophage-S. aureus interactions on biomaterial surfaces, and none has evaluated the expression of virulence factors in S. aureus biofilms.We have successfully developed co-culture models and molecular panels and elucidated important cellular and molecular interactions between macrophages and S. aureus on a broad range of titanium biomaterials with well-defined surface topography and wettability. Our findings highlight the critical role of biofilm formation and the chronological order of bacteria or macrophage arrival in determining the fate of the surface.

Effective drug design screening in bacterial glycolytic enzymes via targeting alternative allosteric sites

Three glycolytic enzymes phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GADPH) and pyruvate kinase (PK) that belong to Staphylococcus aureus were used as targets for screening a dataset composed of 7229 compounds of which 1416 were FDA-approved. Instead of catalytic sites, evolutionarily less conserved allosteric sites were targeted to identify compounds that would selectively bind the bacteria's glycolytic enzymes instead of the human host. Seven different allosteric sites provided by three enzymes were used in independent screening experiments via docking. For each of the seven sites, a total of 723 compounds were selected as the top 10 % which displayed the highest binding affinities. All compounds were then united to yield the top 54 drug candidates shared by all seven sites. Next, 17 out of 54 were selected and subjected to in vitro experiments for testing their inhibition capability for antibacterial growth and enzymatic activity. Accordingly, four compounds displaying antibacterial growth inhibition above 40 % were determined as Candesartan cilexetil, Montelukast (sodium), Dronedarone (hydrochloride) and Thonzonium (bromide). In a second round of experiment, Candesartan cilexetil and Thonzonium displayed exceptionally high killing efficiencies on two bacterial strains of S.aureus (methicillin-sensitive and methicillin-resistant) with concentrations as low as 4 μg/mL and 0.5 μg/mL. Yet, their enzymatic assays were not in accordance with their killing effectiveness. Different inhibitory effects was observed for each compound in each enzymatic assay. A more effective target strategy would be to screen for drug compounds that woud inhibit a combination of glycolytic enzymes observed in the glycolytic pathway.

Evaluation of Potency and Specificity of Cryptophycin-Loaded Antibody-Drug Conjugates.

An enhanced variant of the antimitotic toxin cryptophycin was conjugated to the anti-Her2 monoclonal antibody (mAb) Trastuzumab upon Michael addition. Either antibodies with freed hinge-region cysteines or THIOMAB formats with engineered cysteines in the mAbs light chain were added to a maleimide derivative of cryptophycin. These Antibody-Drug Conjugates (ADCs) showed retained binding to Her2 positive tumor cells and highly efficient cell killing in double-digit pM range on high Her2-expressing SK-BR-3 cells. Two ADCs (DAR 6, DAR 3) showed superior cell killing of the cell lines JIMT-1 and RT112 with medium receptor expression level in comparison with a DAR 6 MMAE ADC serving as reference. The observed cell cytotoxicity is target-dependent since no impact on cell viability was observed for low Her2-expressing MDA-MB468 cells. Particularly the DAR 3 ADC in THIOMAB format exhibiting desirable biophysical properties and high potency emerged as a promising candidate for further in vivo investigations.

Multifunctional hyaluronic acid ligand-assisted construction of CD44- and mitochondria-targeted self-assembled upconversion nanoparticles for enhanced photodynamic therapy.

Upconversion nanoparticles (UCNPs) have been used as a potential nanocarrier for photosensitizers (PSs), which have demonstrated a great deal of promise in achieving an effective photodynamic therapy (PDT) for deep-seated tumors. However, overcoming biological barriers to achieve mitochondria-targeted PDT remains a major challenge. Herein, CD44- and mitochondria-targeted photodynamic nanosystems were fabricated through the self-assembly of hyaluronic acid-conjugated-methoxy poly(ethylene glycol)-diethylenetriamine-grafted-(chlorin e6-dihydrolipoic acid-(3-carboxypropyl)triphenylphosphine bromide) polymeric ligands (HA-c-mPEG-Deta-g-(Ce6-DHLA-TPP)) and NaErF4:Tm@NaYF4 core-shell UCNPs (termed CMPNs). The CMPNs presented ideal physiological stability, a good drug loading capacity and an improved capacity for the generation of singlet oxygen (1O2) based on the FRET mechanism. Significantly, confocal images revealed that CMPNs not only facilitated cellular uptake through CD44-receptor-targeted endocytosis, subsequently enabling rapid evasion from endo-lysosomal sequestration, but also specifically targeted mitochondria, ultimately inducing a profound disruption of mitochondrial membrane potential, which triggered apoptosis upon laser irradiation, thereby significantly enhancing the therapeutic effect. Furthermore, in vitro antitumor experiments further confirmed the substantial enhancement in cancer cell killing efficiency achieved by treating with CMPNs upon near-infrared (NIR) laser irradiation. This innovative approach holds promise for the development of NIR-laser-activated photodynamic nanoagents specifically designed for mitochondria-targeted PDT, thus addressing the limitations of the current PDT treatments.