Low Cell Toxicity Research Articles

Novel active Trp- and Arg-rich antimicrobial peptides with high solubility and low red blood cell toxicity designed using machine learning tools.

Given the rising number of multidrug-resistant (MDR) bacteria, there is a need to design synthetic antimicrobial peptides (AMPs) that are highly active, non-hemolytic, and highly soluble to act as alternatives to antibiotics that are either no longer effective or used as drugs of last resort. Machine learning tools allow the straightforward in silico identification of non-hemolytic antimicrobial peptides. Here, we utilized a number of these tools to rank the best peptides from two libraries: 1) 8192 peptides with sequence bhxxbhbGAL, where b is the basic amino acid R or K, h is a hydrophobic amino acid, i.e. G, A, L, F, I, V, Y, or W and x is Q, S, A, or V; and 2) 512 peptides with sequence RWhxbhRGWL, where b and h are as for the first library and x is Q, S, A, or G. The top 100 sequences from each library, as well as 10 peptides predicted to be active but hemolytic (for a total of 220 peptides), were SPOT synthesized and their IC50 values were determined against S. aureus USA 300 (MRSA). Of these, 6 AMPs with low IC50's were characterized further in terms of: MICs against MRSA, E. faecalis, K. pneumoniae, E.coli and P. aeruginosa; RBC lysis; secondary structure in mammalian and bacterial model membranes; and activity against cancer cell lines HepG2, CHO, and PC-3. Overall, the approach yielded a large family of active antimicrobial peptides with high solubility and low red blood cell toxicity. It also provides a framework for future designs and improved machine learning tools.

Ag induced plasmonic TiO2 for photocatalytic degradation of pharmaceutical under visible light: Insights into mechanism, antimicrobial and cytotoxicity studies

Global concerns include water scarcity and shortage, which are escalated by organic pollutants such as naproxen (NPX) that deteriorate drinking water and taint access to safe drinking water by humans. Moreover, NPX may cause gastrointestinal difficulties, cardiovascular risks, kidney damage, allergic reactions, liver toxicity, bleeding issues and pregnancy risks, hence it is essential to remove it in water. Ag-TiO2 was synthesized by hydrothermal method for NPX degradation. Ag on TiO2 reduced the band gap and surface area of TiO2 and resulted in a plasmonic Ag-TiO2 composite of 0.2 % Ag. The photocatalytic degradation of 0.2 % Ag-TiO2 was 80 % in 180 minutes using a solar simulator during NPX degradation with a first order reaction rate that was 3.6 times faster than that of pure TiO2 and the catalyst showed good stability for four cycles. The dual activity of Ag0 surface plasmon resonance improved light absorption capability and enhanced charge transfer for increased photodegradation rate and stability. The antibacterial studies demonstrated that 0.2 % Ag-TiO2 posted strong antibacterial properties under light irradiation and less in the dark, with a greater effect on gram-negative than gram-positive bacteria. The minimum inhibitory concentration (MIC) values of 620, 1250, 2500, 2500 µg/mL were attained against B. subtilis, S. aureus, E. coli and S. typhimurium bacteria, respectively. The low cell toxicity of 0.2 % Ag-TiO2 was determined using human embryonic kidney (HEK293) cells with an inhibitory concentration (IC50) of 61.09 ± 0.24 µg/mL under light irradiation. Radical trapping experiments demonstrated that hydroxyl radicals (OH•) played a vital role during the degradation and bactericidal activity of NPX under light irradiation. This work advances new insights on the synthesis of less toxic nanoparticles with high photocatalytic and bactericidal activity for possible applications at industrial scale.

Bifunctional Copper Chelators Capable of Reducing Aβ Aggregation and Aβ-Induced Oxidative Stress.

Five bifunctional copper chelating agents, Alz-(1-5), designed to prevent beta-amyloid (Aβ) aggregation, were synthesized, and the leader compound (Alz-5) was chosen. Alz-5 acts as a bifunctional chelator that can interact with various Aβ aggregates and reduce their neurotoxicity. Reactive oxygen species measurements provided by the Pt-nanoelectrode technique in single Aβ42-affected human neuroblastoma SH-SY5Y cells revealed significant antioxidant activity of Alz-5. AFM data obtained on Aβ42 fibrils clearly indicate the antiaggregating property of Alz-5. To gain insights into the changes in the physiomechanical properties of Aβ42-affected cells, as well as in order to evaluate the antiaggregating ability of Alz-5, Young's modulus mapping on living SH-SY5Y cells affected consequently by Aβ42 and Alz-5 was conducted, and the ability of Alz-5 to decrease cell rigidity induced by Aβ42 was indisputably proven. Low cell toxicity and antioxidating properties, in conjunction with AFM and SICM-based biophysical provided on Aβ42-affected SH-SY5Y cells, support Alz-5 as a potential inhibitor of Aβ aggregation.

High-yield, plant-based production of an antimicrobial peptide with potent activity in a mouse model.

Plants offer a promising chassis for the large-scale, cost-effective production of diverse therapeutics, including antimicrobial peptides (AMPs). However, key advances will reduce production costs, including simplifying the downstream processing and purification steps. Here, using Nicotiana benthamiana plants, we present an improved modular design that enables AMPs to be secreted via the endomembrane system and sequestered in an extracellular compartment, the apoplast. Additionally, we translationally fused an AMP to a mutated small ubiquitin-like modifier sequence, thereby enhancing peptide yield and solubilizing the peptide with minimal aggregation and reduced occurrence of necrotic lesions in the plant. This strategy resulted in substantial peptide accumulation, reaching around 2.9 mg AMP per 20 g fresh weight of leaf tissue. Furthermore, the purified AMP demonstrated low collateral toxicity in primary human skin cells, killed pathogenic bacteria by permeabilizing the membrane and exhibited anti-infective efficacy in a preclinical mouse (Mus musculus) model system, reducing bacterial loads by up to three orders of magnitude. A base-case techno-economic analysis demonstrated the economic advantages and scalability of our plant-based platform. We envision that our work can establish plants as efficient bioreactors for producing preclinical-grade AMPs at a commercial scale, with the potential for clinical applications.

Copper-bridged fluorescent turn-on molecular system for high-performance identification and bioimaging of carbon monoxide

Novel high-performance fluorescent strategies have always been in great demand for carbon monoxide (CO) detection and cell-imaging at room temperature, which have two sides of a coin: its high toxicity even at low concentrations and crucial role in physiological and pathophysiological processes. Herein, we constructed a copper-bridged fluorescent turn-on molecular system, Nap-BC-Cu2+, in monitoring and cell-imaging of vaporous CO at room temperature. The absence of expensive palladium salts and the capability to detect both CORM-3 and CO are two major advantages of this strategy compared to previous reports. The sensing performance of the system is exceptional, demonstrated by less than 1 s response time and high sensitivity with remarkable fluorescence enhancement. The detection limit is calculated to be as low as approximately 82 nM and ∼ 14 ppb for CORM-3 and CO vapor, respectively. Besides, the presence of potential interferences had minimal impact on the detection. Moreover, the quantitative detection of CO vapor was also realized by naked-eye observation via paper tests, which provide a low-cost portable strategy for the real-time CO detection. Furthermore, the fluorescent turn-on molecular system exhibited low cell toxicity and high cellular uptake, and was effectively utilized for imaging CO in living Hela cells.

Peptide-mimetic treatment of Pseudomonas aeruginosa in a mouse model of respiratory infection

The rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year. Pseudomonas aeruginosa, in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2000 deaths in the U.S. annually. While the emergence of resistant bacteria has become ominously common, identification of useful new drug classes has been limited over the past over 40 years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killing P. aeruginosa and displays sufficiently low toxicity in mammalian cells to allow for use in treatment of infections. Interestingly, TM5 kills P. aeruginosa more rapidly than traditional antibiotics, within 30–60 min in vitro, and is effective against a range of clinical isolates, including extensively drug resistant strains. In vivo, TM5 significantly reduced bacterial load in the lungs within 24 h compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDR P. aeruginosa respiratory infections.

Membrane-localized magnetic hyperthermia promotes intracellular delivery of cell-impermeant probes.

In this work, we report the disruptive use of membrane-localized magnetic hyperthermia to promote the internalization of cell-impermeant probes. Under an alternating magnetic field, magnetic nanoparticles (MNPs) immobilized on the cell membrane via bioorthogonal click chemistry act as nanoheaters and lead to the thermal disruption of the plasma membrane, which can be used for internalization of different types of molecules, such as small fluorescent probes and nucleic acids. Noteworthily, no cell death, oxidative stress and alterations of the cell cycle are detected after the thermal stimulus, although cells are able to sense and respond to the thermal stimulus through the expression of different types of heat shock proteins (HSPs). Finally, we demonstrate the utility of this approach for the transfection of cells with a small interference RNA (siRNA), revealing a similar efficacy to a standard transfection method based on the use of cationic lipid-based reagents (such as Lipofectamine), but with lower cell toxicity. These results open the possibility of developing new procedures for "opening and closing" cellular membranes with minimal disturbance of cellular integrity. This on-demand modification of cell membrane permeability could allow the direct intracellular delivery of biologically relevant (bio)molecules, drugs and nanomaterials, thus overcoming traditional endocytosis pathways and avoiding endosomal entrapment.

Rapid synthesis of ferulic acid-derived lignin coated silver nanoparticles with low cytotoxicity and high antibacterial activity

Antibiotic resistance and the rise of untreatable bacterial infections pose severe threats to human health. Silver nanoparticles (AgNPs) have emerged as a promising antibacterial solution due to their broad-spectrum effectiveness. However, their relatively high cytotoxicity has limited their widespread application. In this study, ferulic acid (FA) was used as a reducing agent, while silver oxide served as a silver precursor to rapidly prepare FA-derived lignin (FAL) coated AgNPs (AgNPs@FAL) with a size ranging from 34.8 to 77.1 nm. Density functional theory (DFT) calculations indicated that the coating of FAL endowed AgNPs@FAL with high stability, preventing the oxidation of AgNPs prior to antibacterial applications. Cell experiments further indicated that AgNPs@FAL exhibited lower cell toxicity (∼80 % viability of normal kidney cells cultured at 25 μg/mL AgNPs@FAL) compared to fully exposed commercially available citrate-modified AgNPs (AgNPs@CA). Antibacterial experiments revealed that the minimum inhibitory concentrations (MIC) of AgNPs@FAL against E. coli and S. aureus were 12.5 μg/mL and 25 μg/mL, respectively, surpassing the antibacterial effect of AgNPs@CA, as well as ampicillin and penicillin. Additionally, AgNPs@FAL was capable of disrupting E. coli and S. aureus biofilm formation. This novel AgNPs@FAL formulation presents a promising antibacterial solution, addressing limitations observed in conventional drugs.

Evaluation of the efficacy of Chitosan nanoparticles based on Rosuvastatin in the treatment of acute toxoplasmosis: An In vitro and In vivo study

Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan that infects warm-blooded animals and has a global distribution. Acute toxoplasmosis is commonly reported in patients with acquired/congenital toxoplasmosis and immune deficiency. New methods are needed to prevent the sideffects of classical treatment. In this study, Rosuvastatin loaded chitosan nanoparticle (CH-NP-ROS) were synthesized and zeta potential and size were determined, and an MTT assay was performed to evaluate the cell toxicity on Macrophage cells (MQ) and anti-Toxoplasma activity using Trypan-blue staining by different concentrations of Rosuvastatin (ROS), and Rosuvastatin loaded chitosan nanoparticle (CH-NP-ROS). The cell viability assay demonstrated that CH-NP-ROS had lower cell toxicity (<15 %) compared to ROS (<30 %). Statistical analysis showed that CH-NP-ROS significantly killed 98.950 ± 1.344; P < 0.05) of Toxoplasma gondii tachyzoites. In vivo results of perituneal fluid showed that CH-NP significantly reduced the parasite load in the CH-NP-ROS group, compared to that in negative control group (P < 0.001). Growth inhibition rates of tachyzoites in mice receiving free ROS and CH-NP-ROS (injection and oral form) were found to be 166.125 + 4.066, 118.750 + 4.596 and 124.875 + 2.652, respectively, compared to mice in Sulfadiazine/Pyrimethamine treated group (positive control). In the infected untreated mice (control +), the mean tachyzoite counts per oil immersion field in the spleen was 8.25 respectively. The mean survival time in all the groups treated with ROS and CH-NP-ROS was longer than that in the negative control group Therefore, nanoformulation is a promising approach for the delivery and is safe for using therapeutic effects in acute toxoplasmosis.

Establishment of a rapid, cost-effective, and accurate method for assessing insect sperm viability

High-quality sperm cells are crucial to reproductive success for both males and post-mating females in animals. Sperm viability, defined as the proportion of viable sperm cells, is used as a sperm quality index and this method has provided new insights into research on reproductive strategies. Sperm viability has been assessed by fluorescent staining of sperm cells. However, current staining protocols could potentially underestimate viability due to cell damage caused by cell treatments such as high dye concentration and long time for post-mounting. In this study, we established a method that enables rapid sperm viability assessment, has low sperm cell toxicity, and provides precise results regardless of operator expertise, and cost-effective using sperm cells from an ant, Crematogaster osakensis (Hymenoptera). First, to shorten the time for observation of a sufficient number of sperm cells, the volume per field of view was increased by height elevation between the glass slide and the coverslip, thereby we increased the number of sperm cells in a field of view. Second, to reduce sperm cell toxicity, we optimized the minimum dye concentration and incubation time using acridine orange (AO) and Hoechst in addition to SYBR 14 and propidium iodide (PI), which has been used in most previous studies. We determined the optimal protocol to be 1 µg/mL AO and 150 µM PI without incubation. Besides, we automated counting sperm cells with ImageJ software and combined with manual correction for more accurate results. We employed the improved method for sperm samples from mealworm beetles (Tenebrio molitor) and silkmoths (Bombyx mori). This method, established through our study, will advance research on reproductive strategies, including sperm competition and sperm quality maintenance in females.

Silymarin suppresses proliferation and PD-L1 expression in colorectal cancer cells and increases inflammatory CD8+ cells in tumor-bearing mice

IntroductionSilymarin as an herbal medicine has shown anticancer effects on tumor cells, while having low toxicity in normal cells. In this study, the effects of Silymarin on proliferation and apoptosis of colorectal cancer cells and its impact on immune response against cancer cells were evaluated in vitro and in vivo. Methods and materialsThe effect of Silymarin on CT-26 and Caco-2 cells proliferation and apoptosis were demonstrated by MTT assay and PI staining. A subcutaneous tumor of colorectal cancer was developed. Silymarin and Doxorubicin were administrated by intravenous injection. qRT-PCR analyses was performed on blood samples and tumor tissues. Spleen tissue was used to evaluate CD8+ T cell immune responses. Histological study was carried out on tumor tissues. ResultsSilymarin showed anti-proliferative effects on CT-26 and Caco-2 cells. The markers of immunogenic cell death (Calreticulin exposure, ATP secretion, and HMGB1 secretion) significantly increased in both cell lines in the presence of silymarin. The expression of genes related to cell proliferation particularly β-Catenin and Cycline D1, and also anti-apoptotic ones such as Bcl-2 significantly reduced in mice treated with Silymarin while the expression of pro-apoptotic Bax increased. The RNA level of PD-L1 decreased in tumor tissues exposed by Silymarin. Moreover, the number of CTLs increased in the spleen of mice treated with Silymarin in comparison with untreated mice. Decreased tumor size and also survival of colorectal cancer cells in Silymarin-treated mice were observed in histological analysis. ConclusionSilymarin treatment showed a suppressive role on colorectal cancer cells almost as much as Doxorubicin. Our study indicated that having a low toxicity profile, cost-effectiveness, and availability of raw materials, plant-derived Silymarin can be a good candidate for further investigation to treat CRC.

152 Three-Dimensional Printing Bespoke Polycaprolactone Tracheal Splint for Treatment of Tracheomalacia

Abstract Aim Tracheomalacia can be a fatal airway disease with limited treatment options. Three-dimensional (3D) printing has led to various patient-specific tracheal stents and splints. However, these are limited by mismatched stiffness and resulting granulation tissue formation and fibrosis. We aimed to develop a non-toxic and biodegradable polycaprolactone expandable tracheal splint by 3D printing. By fine-tuning mechanical properties, we hypothesised that the splint would match the native trachea and accommodate tracheal growth in tracheomalacia patients. Method We designed splints consisting of concave topological patterns with varying angles and aspect ratios. Fabrication occurred using polycaprolactone filaments in a 3D fuse deposition modelling printer. Uniaxial tensile studies were performed on splints to assess Young’s Modulus, Toughness, Ultimate strain, Strength, and Poisson ratio. Splints displaying optimal mechanical properties were tested for human lung fibroblast cell viability, proliferation, and morphology. Results On a mechanical testing rig, uniaxial tensile studies confirmed that splint designs with smaller angles and negative Poisson ratios of up to strains of 25%, better mimicked mechanical properties of native human trachea, accommodating tracheal growth. In vitro studies using these tracheal splints demonstrated successful human lung cell proliferation with low cell toxicity. Conclusions This study characterised 3D printed bespoke tracheal splints with various 2D structures, showing mechanical properties that match the native trachea. However, in vivo studies investigating cellular interactions on our splint must be performed to further evaluate the ability for endogenous tissue formation.

Analyzing the Antibacterial, Anticancer, and Antioxidant Qualities of the Stink Bean (Parkia Speciosa) by Various Extraction Techniques

In various Southeast Asian countries, people have long incorporated the young green seeds of Parkia speciosa, commonly known as stink beans, into their culinary traditions. These seeds can be prepared using various cooking methods, including boiling, microwaving, stir-frying, or even consuming them raw. While several studies have explored the pharmaceutical properties of raw stink beans, this study seeks to expand our knowledge of how different extraction methods can influence the release of bioactive compounds from these young stink bean seeds. Three distinct extraction methods, namely (1) microwave-assisted water extraction, (2) boiling water extraction and (3) 50 % ethanol extraction were employed in this research. The study focused on total phenolic content and antioxidant activities, with the 50 % ethanol extraction method yielding the most promising results. Moreover, only the extract obtained from the 50 % ethanol method exhibited antimicrobial activity against 4 gram-positive bacteria, including Methicillin-Resistant Staphylococcus aureus, Staphylococcus aureus, Bacillus cereus, Micrococcus luteus and 1 gram-negative bacterium, Salmonella typhi. Notably, extracts obtained from the 3 extraction methods of young stink bean seeds exhibit varying degrees of inhibition of cancer cell growth with low cell toxicity. Taken together, our findings suggest that distinct extraction methods play a key role in obtaining different sets of bioactive compounds from the young seeds of stink beans. Therefore, various cooking methods, including stir-frying, microwaving and pickling in alcohol, may offer diverse sources of bioactive compounds for promoting consumer health. HIGHLIGHTS A 50 % ethanol extraction of young stink bean seeds yields the highest concentrations of phenolic compounds and exhibits the highest antioxidant capacity The sole extract derived from the 50 % ethanol extraction of young stink bean seeds demonstrated notable antimicrobial efficacy against both selected gram-positive and gram-negative bacteria Three distinct extraction methodologies—microwave-assisted water extraction, boiling water extraction and 50 % ethanol extraction—exhibited varying degrees of cytotoxicity against normal cell lines and cancer cells GRAPHICAL ABSTRACT

A highly selective Ru(II) complex-based phosphorescent probe for the sequential sensing Cu2+ and H2S and its applications

A highly selective Ru(II) complex-based phosphorescent probe Ru-Cyclen for the sequential sensing Cu2+ and H2S in pure aqueous medium was synthesized and characterized. Probe Ru-Cyclen exhibited a turn-off phosphorescence response toward Cu2+ by using a complex formation with a 1:1 binding mode, and the resulting complex enabled to specifically detect H2S based on the displacement approach with a turn-on phosphorescence response. Also, Ru-Cyclen possessed high selectivity and sensitivity with a low detection limit of 0.045 μM and 0.44 μM for Cu2+ and H2S respectively, and worked best in the range of pH 3–11. The kinetics response toward Cu2+ and H2S were very fast, completing within 4 min and 10 s, respectively. Additionally, the sensing mechanism was confirmed by fluorescence Job’s plot analysis, 1H NMR titration, EPR, TOF-mass spectrometry and phosphorescence lifetime analysis. Moreover, Ru-Cyclen showed low cell toxicity and successfully detected Cu2+ and exogenously or endogenously generated H2S in living zebrafish. Finally, combining the unique merits of the probe, including excellent water-solubility, a long emission wavelength of 600 nm, large Stoke shift of 150 nm and rapid response, probe Ru-Cyclen was applied in real water samples, human serum and test strips successfully.

A reanalysis and integration of transcriptomics and proteomics datasets unveil novel drug targets for Mekong schistosomiasis

Schistosomiasis, caused by Schistosoma trematodes, is a significant global health concern, particularly affecting millions in Africa and Southeast Asia. Despite efforts to combat it, the rise of praziquantel (PZQ) resistance underscores the need for new treatment options. Protein kinases (PKs) are vital in cellular signaling and offer potential as drug targets. This study focused on focal adhesion kinase (FAK) as a candidate for anti-schistosomal therapy. Transcriptomic and proteomic analyses of adult S. mekongi worms identified FAK as a promising target due to its upregulation and essential role in cellular processes. Molecular docking simulations assessed the binding energy of FAK inhibitors to Schistosoma FAK versus human FAK. FAK inhibitor 14 and PF-03814735 exhibited strong binding to Schistosoma FAK with minimal binding for human FAK. In vitro assays confirmed significant anti-parasitic activity against S. mekongi, S. mansoni, and S. japonicum, comparable to PZQ, with low toxicity in human cells, indicating potential safety. These findings highlight FAK as a promising target for novel anti-schistosomal therapies. However, further research, including in vivo studies, is necessary to validate efficacy and safety before clinical use. This study offers a hopeful strategy to combat schistosomiasis and reduce its global impact.

Strategies for Improved pDNA Loading and Protection Using Cationic and Neutral LNPs with Industrial Scalability Potential Using Microfluidic Technology.

In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity. The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical "post-formulation" DNA addition vs "pre" addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake. Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 µg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 µg/mL of pDNA with the "post" method, while maintaining their characteristics up to 100 µg/mL of pDNA with the "pre" addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 µg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes. In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.

Isolation and characterization of two Staphylococcus aureus lytic bacteriophages “Huma” and “Simurgh”

Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%–1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications.

Production, Characterization Purification, and Antitumor Activity of L-Asparaginase from Aspergillus niger

Cervical cancer is caused by a persistent and high-grade infection. It is caused by the Human Papillomavirus (HPV), which, when entering cervical cells, alters their physiology and generates serious lesions. HPV 18 is among those most involved in carcinogenesis in this region, but there are still no drug treatments that cause cure or total remission of lesions caused by HPV. It is known that L-asparaginase is an amidohydrolase, which plays a significant role in the pharmaceutical industry, particularly in the treatment of specific cancers. Due to its antitumor properties, some studies have demonstrated its cytotoxic effect against cervical cancer cells. However, the commercial version of this enzyme has side effects, such as hypersensitivity, allergic reactions, and silent inactivation due to the formation of antibodies. To mitigate these adverse effects, several alternatives have been explored, including the use of L-asparaginase from other microbiological sources, which is the case with the use of the fungus Aspergillus niger, a high producer of L-asparaginase. The study investigated the influence of the type of fermentation, precipitant, purification, characterization, and in vitro cytotoxicity of L-asparaginase. The results revealed that semisolid fermentation produced higher enzymatic activity and protein concentration of A. niger. The characterized enzyme showed excellent stability at pH 9.0, temperature of 50 °C, resistance to surfactants and metallic ions, and an increase in enzymatic activity with the organic solvent ethanol. Furthermore, it exhibited low cytotoxicity in GM and RAW cells and significant cytotoxicity in HeLa cells. These findings indicate that L-asparaginase derived from A. niger may be a promising alternative for pharmaceutical production. Its attributes, including stability, activity, and low toxicity in healthy cells, suggest that this modified enzyme could overcome challenges associated with antitumor therapy.

Design, synthesis, and evaluation of purine and pyrimidine-based KRAS G12D inhibitors: Towards potential anticancer therapy

Oncogenic RAS mutations, commonly observed in human tumors, affect approximately 30% of cancer cases and pose a significant challenge for effective cancer treatment. Current strategies to inhibit the KRAS G12D mutation have shown limited success, emphasizing the urgent need for new therapeutic approaches. In this study, we designed and synthesized several purine and pyrimidine analogs as inhibitors for the KRAS G12D mutation. Our synthesized compounds demonstrated potent anticancer activity against cell lines with the KRAS G12D mutation, effectively impeding their growth. They also exhibited low toxicity in normal cells, indicating their selective action against cancer cells harboring the KRAS G12D mutation. Notably, the lead compound, PU1-1 induced the programmed cell death of KRAS G12D-mutated cells and reduced the levels of active KRAS and its downstream signaling proteins. Moreover, PU1-1 significantly shrunk the tumor size in a pancreatic xenograft model induced by the KRAS G12D mutation, further validating its potential as a therapeutic agent. These findings highlight the potential of purine-based KRAS G12D inhibitors as candidates for targeted cancer therapy. However, further exploration and optimization of these compounds are essential to meet the unmet clinical needs of patients with KRAS-mutant cancers.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.