Quantitative Analysis of Therapeutic Antibody Interactions with Fcγ Receptors Using High-Speed Atomic Force Microscopy.

This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.

Virtual Screening for Biomimetic Anti-Cancer Peptides from Cordyceps militaris Putative Pepsinized Peptidome and Validation on Colon Cancer Cell Line.

Colorectal cancer is one of the leading causes of cancer-related death in Thailand and many other countries. The standard practice for curing this cancer is surgery with an adjuvant chemotherapy treatment. However, the unfavorable side effects of chemotherapeutic drugs are undeniable. Recently, protein hydrolysates and anticancer peptides have become popular alternative options for colon cancer treatment. Therefore, we aimed to screen and select the anticancer peptide candidates from the in silico pepsin hydrolysate of a Cordyceps militaris (CM) proteome using machine-learning-based prediction servers for anticancer prediction, i.e., AntiCP, iACP, and MLACP. The selected CM-anticancer peptide candidates could be an alternative treatment or co-treatment agent for colorectal cancer, reducing the use of chemotherapeutic drugs. To ensure the anticancer properties, an in vitro assay was performed with "CM-biomimetic peptides" on the non-metastatic colon cancer cell line (HT-29). According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results from peptide candidate treatments at 0-400 µM, the IC50 doses of the CM-biomimetic peptide with no toxic and cancer-cell-penetrating ability, original C. militaris biomimetic peptide (C-ori), against the HT-29 cell line were 114.9 µM at 72 hours. The effects of C-ori compared to the doxorubicin, a conventional chemotherapeutic drug for colon cancer treatment, and the combination effects of both the CM-anticancer peptide and doxorubicin were observed. The results showed that C-ori increased the overall efficiency in the combination treatment with doxorubicin. According to the acridine orange/propidium iodine (AO/PI) staining assay, C-ori can induce apoptosis in HT-29 cells significantly, confirmed by chromatin condensation, membrane blebbing, apoptotic bodies, and late apoptosis which were observed under a fluorescence microscope.

Degradation products of crosslinked silk fibroin scaffolds modulate the immune response but not cell toxicity.

Silk fibroin (SF) scaffolds have widely been used as functional materials for tissue engineering and implantation. For long-term applications, many cross-linking strategies have been developed to enhance the stability and enzymatic degradation of scaffolds. Although the biocompatibility of SF scaffolds has been investigated, less is known about the extent to which the degradation products of these scaffolds affect the host response in the long term after implantation. In this work, we first studied the effect of two different crosslinkers, namely, 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride) (EDC) and glutaraldehyde (GA), on the topology, mechanical stability and enzymatic degradation of SF scaffolds. We found that the SF scaffolds treated with GA (GA-SF) appeared to show an increase in the sheet thickness and a higher elastic modulus when compared to that treated with EDC (EDC-SF) at a similar level of crosslinking degree. The uncrosslinked and both crosslinked SF scaffolds were completely digested by proteinase K but were not susceptible to degradation by collagenase type IV and trypsin. We next investigated the effect of the degradation of SF on the cytotoxicity, genotoxicity, and immunogenicity. The results demonstrated that the degradation products of the uncrosslinked and crosslinked SFs did not trigger cell proliferation, cell death, or genotoxicity in primary human cells, while they appeared to modulate the phenotypes of macrophages. The degradation products of GA-SF promoted pro-inflammatory phenotypes, while those from EDC-SF enhanced polarization towards anti-inflammatory macrophages. Our results demonstrated that the degradation products of SF scaffolds can mediate the immune modulation of macrophages, which can be implemented as a therapeutic strategy to control the long-term immune response during implantation.

Investigation of genotoxicity, mutagenicity, and cytotoxicity in erythrocytes of Nile tilapia (Oreochromis niloticus) after fluoxetine exposure.

Fluoxetine (FLX) is an antidepressant that is increasingly being detected in aquatic environments. However, this contaminated FLX can affect aquatic organisms. Therefore, the aim of this study was to evaluate the genotoxic, mutagenic, and cytotoxic potential of FLX on erythrocytes in Nile tilapia (Oreochromis niloticus) after acute exposure. Fish were exposed to different concentrations of FLX (10, 100 and 1000 µg/L) for 96 h. Then, the condition factor (K value) was used to assess the general fish condition. The genotoxicity was investigated using a comet assay, and the mutagenicity was examined using micronucleus (MN) and erythrocytic nuclear abnormalities (ENAs) assays. In addition, the cytotoxicity was analyzed by erythrocyte morphometry and erythrocyte maturity index (EMI). The results showed that FLX did not affect the fish's health. Nevertheless, 100 and 1000 µg/L FLX significantly increased DNA damage. Furthermore, a higher concentration of FLX presented a significantly increased frequency of MNs and ENAs, also leading to changes in some erythrocyte morphometric indices and significantly decreased mature erythrocytes. In conclusion, our results indicate that FLX induces genotoxic, mutagenic, and cytotoxic effects in erythrocytes of O. niloticus.

Liposomal Thiostrepton Formulation and Its Effect on Breast Cancer Growth Inhibition.

Forkhead box M1 (FOXM1) is known to play a role in breast cancer progression. FOXM1 inhibition becomes one of the strategies in developing the novel cancer therapy. Recently, thiostrepton has been recognized as a potent FOXM1 inhibitor. To improve its potential, we aimed to develop a nanodelivery system for thiostrepton. Here, liposome-encapsulated thiostrepton (TSLP) was developed. Physiochemical properties were characterized by TEM and dynamic light scattering technique. The biological activities were also evaluated, by cellular internalization, MTT assay, spheroid formation assay and RT-PCR. The result showed that the range sizes of TSLP were 152 ± 2 nm, polydispersity index (PdI) of 0.23 ± 0.02 and zeta potential of -20.2 ± 0.1 mV. As expected, TSLP showed a higher potential in reducing FOXM1 levels in MCF-7 cells than free thiostrepton. Additionally, TSLP significantly improved the efficiently and specificity of thiostrepton in reducing cell viability of MCF-7, but not of the fibroblast (HDFn) cells. Interestingly, TSLP had an ability to induce MCF-7 cell death in both 2D monolayer and 3D spheroid culture. In conclusions, TSLP could possibly be one of the potential developments using nano-delivery system to improve abilities and specificity of thiostrepton in breast cancer cell inhibition and death inducing, with decreasing non-specific toxicity.

Fabrication of calcium phosphate composite polymer/SLS-stabilized emulsion-based bioactive gels and their application for dentine tubule occlusion.

OBJECTIVES: Calcium phosphate/SLS/P123 composite bioactive gels were prepared to achieve dentine tubule occlusion. METHODS: Gels containing calcium phosphate particles were prepared in a water-in-oil microemulsion system with a mixture of triblock copolymer pluronic (P123) as a co-surfactant and sodium lauryl sulfate (SLS) as a surfactant in cyclohexane. Subsequently, calcium chloride dihydrate and sodium hydrogen phosphate aqueous solutions were added in a water phase. Finally, slow evaporation of the oil phase at room temperature was performed to produce a hybrid gel. The obtained gels were investigated for their toxicity by the sulforhodamine B (SRB) assay and applied on human dentine specimens to examine their ability to occlude dentine tubules. RESULTS: The size and morphology of the calcium phosphate particles embedded in the gel depended on the concentration of P123 and SLS, which were used as a template for mineral precipitation. The prepared calcium phosphate particles (200-500 nm in diameter) with the maximum polymer and surfactant content exhibited spherical shapes. Further, on reducing their content twice and tenfold yields micro-particles with flower-like shapes. These bioactive gels were able to occlude into dentine tubules after 3 days of application with a plugging rate of 79.22% when using the smallest particles. In addition, calcium phosphate nanorods were transformed into dentine tubules with a maximum depth of 6 µm on increasing the amount of gel. CONCLUSIONS: The bioactive gels were effectively used as bioactive fillers to occlude exposed human dentine tubules.

Biocompatibility and biodegradability of filler encapsulated chloroacetated natural rubber/polyvinyl alcohol nanofiber for wound dressing.

The novel biodegradable films of chloroacetated natural rubber/polyvinyl alcohol (CNR/PVA) (55/45 wt%) non-woven nanofiber films encapsulated with kaolin and starch (2.5 and 5 wt%) were produced successfully by green electrospinning technique. The effect of fillers with different content on the physical, chemical, mechanical, biocompatibility and biodegradation properties of CNR/PVA nanofiber films were investigated. The higher crystallinity obtained in CNR/PVA encapsulate with 2.5 wt% kaolin and nanofibers were formed with the maximum diameter distribution and mean value of 40-160 nm and 94.15 ±â€¯54.19 nm respectively. DSC and DMA revealed the kaolin can improve the interfacial adhesion between CNR and PVA and contribute to enhancing the chemical interactions. The mechanical properties improved upon encapsulation of starch and kaolin and more favourable nanofibers with smaller diameter obtained using kaolin rather than starch. The cytotoxicity results revealed the viability of the prepared nanofiber films with human dermal fibroblast cell. Furthermore, the incorporation of starch and kaolin accelerated the degradation rate and the highest enzymatic degradation obtained with 2.5 wt% of starch. The prepared nanofiber films have the potential to be applied for the skin tissue engineering scaffold applications.

Molecular mechanism of Forkhead box M1 inhibition by thiostrepton in breast cancer cells.

Breast cancer is the most common type of malignancies in women worldwide, and genotoxic chemotherapeutic drugs are effective by causing DNA damage in cancer cells. However, >90% of patients with metastatic cancer are resistant to chemotherapy. The Forkhead box M1 (FOXM1) transcription factor plays a pivotal role in the resistance of breast cancer cells to chemotherapy by promoting DNA damage repair following genotoxic drug treatment. The aim of the present study was to investigate the inhibition of the FOXM1 protein by thiostrepton, a natural antibiotic produced by the Streptomyces species. Experimental studies were designed to examine the effectiveness of thiostrepton in downregulating FOXM1 mRNA expression and activity, leading to senescence and apoptosis of breast cancer cells. The cytotoxicity of thiostrepton in breast cancer was determined using cell viability assay. Additionally, thiostrepton treatment decreased the mRNA expression of cyclin B1 (CCNB1), a downstream target of FOXM1. The present results indicated that thiostrepton inhibited FOXM1 mRNA expression and its effect on CCNB1. Molecular dynamic simulations were performed to study the interactions between FOXM1­DNA and thiostrepton after molecular docking. The results revealed that the possible mechanism underlying the inhibitory effect of thiostrepton on FOXM1 function was by forming a tight complex with the DNA and FOXM1 via its binding domain. Collectively, these results indicated that thiostrepton is a specific and direct inhibitor of the FOXM1 protein in breast cancer. The findings of the present study may lead to the development of novel therapeutic strategies for breast cancer and help overcome resistance to conventional chemotherapeutic drugs.

Detection of hTERT mRNA in gastrointestinal tract cancer specimens.

Human telomerase consisting of telomerase RNA template (hTR) and telomerase reverse transcriptase (hTERT) provides a mechanism for synthesis of telomere repeats that prolongs life span of cells. Telomerase activity is present in germ-line and malignant tumor cells but not in most normal human somatic cells. This study determined hTERT mRNA level in tissue samples from patients with gastrointestinal tract (GI) cancers. Tissue samples were obtained from 22 GI cancer patients, 3 gastrointestinal stomal tumors (GIST) and 25 corresponding non-cancerous tissues. hTERT expression was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using Taqman probe, hTERT mRNA was detected in 12 of 22 cancerous tissue samples. Six of 8 tissue samples obtained from patients with hepatocellular carcinoma and cholangiocarcinoma were positive for hTERT. However, hTERT mRNA was not detected in GIST and non-cancerous tissues. These results suggest that hTERT may be an effective target for cancer therapies to treat many type of GI cancers including cholangiocarcinoma and hepatocellular carcinoma.

In Vitro Methods for Studying the Mechanisms of Resistance to DNA-Damaging Therapeutic Drugs.

Most commonly used anticancer drugs exert their effects mainly by causing DNA damage. The enhancement in DNA damage response (DDR) is considered a key mechanism that enables cancer cells to survive through eliminating the damaged DNA lesions and thereby developing resistance to DNA-damaging agents. This chapter describes the four experimental approaches for studying DDR and genotoxic drug resistance, including the use of γ-H2AX and comet assays to monitor DNA damage and repair capacity as well as the use of clonogenic and ß-galactosidase staining assays to assess long-term cell fate after DNA-damaging treatment. Finally, we also present examples of these methods currently used in our laboratory for studying the role of FOXM1 in DNA damage-induced senescence and epirubicin resistance.

Detection of hTERT mRNA in gastrointestinal tract cancer specimens.

Human telomerase consisting of telomerase RNA template (hTR) and telomerase reverse transcriptase (hTERT) provides a mechanism for synthesis of telomere repeats that prolongs life span of cells. Telomerase activity is present in germ-line and malignant tumor cells but not in most normal human somatic cells. This study determined hTERT mRNA level in tissue samples from patients with gastrointestinal tract (GI) cancers. Tissue samples were obtained from 22 GI cancer patients, 3 gastrointestinal stomal tumors (GIST) and 25 corresponding non-cancerous tissues. hTERT expression was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using Taqman probe, hTERT mRNA was detected in 12 of 22 cancerous tissue samples. Six of 8 tissue samples obtained from patients with hepatocellular carcinoma and cholangiocarcinoma were positive for hTERT. However, hTERT mRNA was not detected in GIST and non-cancerous tissues. These results suggest that hTERT may be an effective target for cancer therapies to treat many type of GI cancers including cholangiocarcinoma and hepatocellular carcinoma.