Inoculation of heavy metal resistant bacteria alleviated heavy metal-induced oxidative stress biomarkers in spinach (Spinacia oleracea L.).

Most vegetable crops are severely affected by the uptake of heavy metals from the soil. Heavy metals in vegetable bodies generate reactive oxygen species (ROS) that unbalance the antioxidant defense system. This study was initiated to determine the physiological and biochemical characteristics of spinach plants grown on soil contaminated with heavy metals and responding to Bacillus cereus and Bacillus aerius were isolated from soil contaminated with heavy metals. Heavy metal contamination led to a significant reduction in seed germination, seedling biomass, protein, and total nitrogen content of spinach plants grown in contaminated soils compared to control soils. In contrast, a significant increase in the content of metallothioneins and antioxidant enzymes was observed. Plants inoculated with B. cereus and B. aerius significantly reduced the oxidative stress induced by heavy metals by improving seed germination (%), seedling growth, nitrogen, and protein content. The content of metallothioneins and the activities of antioxidant enzymes were reduced in spinach plants grown from seeds inoculated with bacterial strains. In addition, plants inoculated with, B. cereus and B. aerius showed greater stomata opening than plants grown on soil contaminated with heavy metals, whose stomata were almost closed. These results suggested that both bacterial strains enhanced plant growth by reducing oxidative stress caused by metals.

Nanoformulated 3'-diindolylmethane modulates apoptosis, migration, and angiogenesis in breast cancer cells.

Background: It is well-established that specific herbal plants contain natural active ingredients that have demonstrated anti-cancer potential. Therefore, they are considered highly beneficial as a potential adjuvant, alternative or complementary agent in anti-cancer therapy. However, the low chemical stability and limited bioavailability of 3, 3'-Diindolylmethane (DIM), a plant-derived compound used in clinical settings, limit its therapeutic applications. To overcome this challenge, researchers have focused on developing innovative approaches to improve DIM's biological activity, such as utilizing nanoformulations. Here, we investigated the potential benefits of coating DIM nanoparticles (DIM-NPs) with PEG/chitosan in the treatment of breast cancer. Our results demonstrate the molecular mechanism underlying the activity of DIM-NPs, highlighting their potential as an effective therapeutic strategy for breast cancer treatment. Methods: DIM-PLGA-PEG/chitosan NPs were synthesised and characterised using dynamic light scattering (DLS) and evaluated the impact of these NPs on two breast cancer cell models. Results: DIM-NPs had an average diameter of 102.3 nm and a PDI of 0.182. When treated with DIM-NPs for 48 h, both MCF7 and MDA-MB-231 cells displayed cytotoxicity at a concentration of 6.25 g/mL compared to untreated cells. Furthermore, in MDA-MB-231 cells, treatment with 2.5 µg/mL of DIM-NPs resulted in a significant decrease in cell migration, propagation, and angiogenesis which was further enhanced at 10 µg/mL. In chicken embryos, treatment with 5 µg/mL of DIM-NPs on day 2 led to a significant reduction in angiogenesis. Furthermore, this treatment induced cell death through a regulatory pathway involving the upregulation of Bax and p53, as well as the downregulation of Bcl-2. These results were supported by in-silico analysis of DIM's binding affinity to key proteins involved in this pathway, namely Bax, Bcl-2, and p53. Conclusion: Our findings show that DIM-NPs induces apoptosis, inhibit migration, and reduce angiogenesis in breast cancer. However, further research using a preclinical cancer model may be necessary to determine the pharmacokinetics of DIM-NPs and ensure their safety and efficacy in vivo.

Chitosan-PEI passivated carbon dots for plasmid DNA and miRNA-153 delivery in cancer cells.

These days carbon dots have been developed for multiple biomedical applications. In the current study, the transfection potential of synthesized carbon dots from single biopolymers such as chitosan, PEI-2kDa, and PEI-25kDa (CS-CDs, PEI2-CDs, and PEI25-CDs) and by combining two biopolymers (CP2-CDs and CP25-CDs) through a bottom-up approach have been investigated. The characterization studies revealed successful synthesis of fluorescent, positively charged carbon dots <20 nm in size. Synthesized carbon dots formed a stable complex with plasmid DNA (EGFP-N1) and miRNA-153 that protected DNA/miRNA from serum-induced degradation. In-vitro cytotoxicity analysis revealed minimal cytotoxicity in cancer cell lines (A549 and MDA-MB-231). In-vitro transfection of EGFP-N1 plasmid DNA with PEI2-CDs, PEI25-CDs and CP25-CDs demonstrated that these CDs could strongly transfect A549 and MDA-MB-231 cells. The highest EGFP-N1 plasmid transfection efficiency was observed with PEI2-CDs at a weight ratio of 32:1. PEI25-CDs polyplex showed maximum transfection at a weight ratio of 8:1 in A549 at a weight ratio of 16:1 in MDA-MB-231 cells. CP25-CDs exhibited the highest transfection at a weight ratio of 16:1 in both cell lines. The in-vitro transfection of target miRNA, i.e., miR-153 in A549 and MDA-MB-231 cells with PEI2-CDs, PEI25-CDs, and CP25-CDs suggested successful transfer of miR-153 into cells which induced significant cell death in both cell lines. Importantly, CS-CDs and CP2-CDs could be tolerated by cells up to 200 µg/mL concentration, while PEI2-CDs, PEI25-CDs, and CP25-CDs showed non-cytotoxic behavior at low concentrations (25 µg/mL). Together, these results suggest that a combination of carbon dots synthesized from chitosan and PEI (CP25-CDs) could be a novel vector for transfection nucleic acids that can be utilized in cancer therapy.

Identification of natural antiviral drug candidates against Tilapia Lake Virus: Computational drug design approaches.

Tilapia Lake Virus (TiLV) is a disease that affects tilapia fish, causing a high rate of sudden death at any stage in their life cycle. Unfortunately, there are currently no effective antiviral drugs or vaccines to prevent or control the progression of this disease. Researchers have discovered that the CRM1 protein plays a critical function in the development and spreading of animal viruses. By inhibiting CRM1, the virus's spread in commercial fish farms can be suppressed. With this in mind, this study intended to identify potential antiviral drugs from two different tropical mangrove plants from tropical regions: Heritiera fomes and Ceriops candolleana. To identify promising compounds that target the CRM1 protein, a computer-aided drug discovery approach is employed containing molecular docking, ADME (absorption, distribution, metabolism and excretion) analysis, toxicity assessment as well as molecular dynamics (MD) simulation. To estimate binding affinities of all phytochemicals, molecular docking is used and the top three candidate compounds with the highest docking scores were selected, which are CID107876 (-8.3 Kcal/mol), CID12795736 (-8.2 Kcal/mol), and CID12303662 (-7.9 Kcal/mol). We also evaluated the ADME and toxicity properties of these compounds. Finally, MD simulation was conducted to analyze the stability of the protein-ligand complex structures and confirm the suitability of these compounds. The computational study demonstrated that the phytochemicals found in H. fomes and C. candolleana could potentially serve as important inhibitors of TiLV, offering practical utility. However, further in vivo investigations are necessary to investigate and potentially confirm the effectiveness of these compounds as antiviral drugs against the virus TiLV.

Modulation of the Tumor Microenvironment by Ellagic Acid in Rat Model for Hepatocellular Carcinoma: A Potential Target against Hepatic Cancer Stem Cells.

The resistance to therapy and relapse in hepatocellular carcinoma (HCC) is highly attributed to hepatic cancer stem cells (HCSCs). HCSCs are under microenvironment control. This work aimed to assess the systemic effect of ellagic acid (EA) on the HCC microenvironment to decline HCSCs. Fifty Wistar rats were divided into six groups: negative control (CON), groups 2 and 3 for solvents (DMSO), and (OVO). Group 4 was administered EA only. The (HCC-M) group, utilized as an HCC model, administered CCL4 (0.5 mL/kg in OVO) 1:1 v/v, i.p) for 16 weeks. HCC-M rats were treated orally with EA (EA + HCC) 50 mg/kg bw for five weeks. Biochemical, morphological, histopathological, and immunohistochemical studies, and gene analysis using qRT-PCR were applied. Results revealed elevated liver injury biomarkers ALT, AST, ALP, and tumor biomarkers AFP and GGT, and marked nodularity of livers of HCC-M. EA effectively reduced the biomarkers and restored the altered structure of the livers. At the mRNA level, EA downregulated the expression of TGF-α, TGF-ß, and VEGF, and restored p53 expression. This induced an increase in apoptotic cells immunostained with caspase3 and decreased the CD44 immunostained HCSCs. EA could modulate the tumor microenvironment in the HCC rat model and ultimately target the HCSCs.

Apoptosis induction in human hepatoma cell line HepG2 cells by trans- Anethole via activation of mitochondria-mediated apoptotic pathways.

trans-Anethole a valuable compound derived from star anise widely used by ethnic tribals to manage numerous human diseases. In this study antiproliferative activities of trans-Anethole towards human liver cancer (HepG2), cervical cancer (HeLa) and breast cancer (MCF-7) cells were explored. trans-Anethole showed free radical scavenging potential as assessed by DNA nicking assay. trans-Anethole exhibited strong antiproliferative potential towards HepG2 cells compared to other cell lines. trans-Anethole strongly induced apoptosis in HepG2 cells by significantly upregulating the protein expressions of p53, Caspase-3 and Caspase-9 were assessed by western blotting analysis which highlighted apoptosis-inducing capacity of trans-Anethole against HepG2 cells. Rt-qPCR analysis revealed that trans- Anethole upregulated p53, caspase - 3 and - 9 in comparison to untreated HepG2 cancer cells. Moreover, trans-Anethole provoked the generation of ROS and disruption of MMP. Our research suggests that trans-Anethole may have a significant anticancer therapeutic potential for treating liver cancer.

Impact of nanomaterials on sustainable pretreatment of lignocellulosic biomass for biofuels production: An advanced approach.

Biomass to biofuels production technology appears to be one of the most sustainable strategies among various renewable energy resources. Herein, pretreatment is an unavoidable and key step to increase free cellulose availability and digestibility to produce green fuels. Various existing pretreatment technologies of lignocellulosics biomasses (LCBs) face distinct challenges e.g., energy consuming, cost intensive, may lead partial removal of lignin, complex inhibitors production as well as may cause environmental pollutions. These, limitations may be overcome with the application of nanomaterials, employed as nanocatalysts during the pretreatment process of LCBs. In this prospect, the present review focuses and summarizes results of numerous studies and exploring the utilizations of magnetic, carbon based nanostructure, and nanophotocatalysts mediated pretreatment processes along with their possible mechanisms to improve the biofuels production compared to conventional chemical based pretreatment approaches. Furthermore, different aspects of nanomaterials based pretreatment methods with their shortcomings and future prospects have been discussed.

Effect of Co-culturing both placenta-derived mesenchymal stem cells and their condition medium in the cancer cell (HepG2) migration, damage through apoptosis and cell cycle arrest.

Human placental-derived mesenchymal stem cells (hPMSCs) are a promising candidate to inhibit the proliferation of hepatocellular carcinoma (HCC) cell lines such as HepG2. The effects of hPMSCs and their conditioned media on HepG2 are, however, still a mystery. As a result, the goal of this study was to look into the effects of hPMSCs and their conditioned media on HepG2 and figure out what was going on. Fluorescence-activated cell sorting and the MTT test were used to determine the percentage of cells that died (early apoptosis, late apoptosis). The DIO and DID colors were used to detect cell fusion and cell death in both cells. HepG2 cells were co-treated with hPMSCs or hPMSCs-conditioned medium (hPMSCs-CM) to reduce growth and promote apoptosis. Morphological changes were also seen in the 30 percent, 50 percent, and 60 percent cases. The secretion of cytokine was determined by the ELISA. Flow cytometry, caspase 9 immunofluorescence, qPCR (detection of Bax, Bcl-2, and ß-catenin genes), western blot, and immunophenotyping revealed that treatment with hPMSCs or hPMSCs-CM caused HepG2 cell death through apoptosis (detection of caspase 9, caspase 3 protein). HepG2 cell cycle arrest could be induced by hPMSCs and hPMSCs-CM. Following treatment with hPMSCs or hPMSCs-CM, HepG2 cell development was stopped in the G0/G1 phase. These treatments also inhibited HepG2 cells from migrating, with the greatest effect when the highest ratio/concentration of hPMSCs (70%) and hPMSCs-CM were used (90%). Our findings indicated that hPMSCs and hPMSCs-CM could be promising treatment options for liver cancer. To elucidate the proper effect, more research on liver cancer-induced rat/mice is needed.

Enhancement in functional stability of microbial endoglanases produced using paddy straw via treatment with manganese oxide based porous nanocomposite synthesized from mixed fruit waste.

Microbial cellulases are the enzymes used in numerous industrial biotechnological applications. Efficiency of celluloytic cocktails plays a key role in the conversion of biomass into biofuels, but limited production, high cost and low efficiency are the main obstacles to sustainable biorefining. The current work aims to establish a feasible approach for boosting the production of fungal endoglucanse (EG) and its functional stability utilizing nanocomposite materials based on manganese oxide. Herein, aqueous extract from mixed fruit waste was used to synthesize the nanocomposite sample, which was subsequently subjected to several characterization techniques for analysis. Following the solid-state fermentation of paddy straw, and by employing 75 mg nanocomposite, 192 IU/gds EG was produced under the optimal conditions, while 19 IU/gds FP and 98 IU/gds BGL production were recorded. The crude EG enzyme treated with nanocomposite also shows complete stability at pH 5.0 for 3.5 h while retaining thermal activity at 70 °C for 4 h.

Pharmacophore-based virtual screening approaches to identify novel molecular candidates against EGFR through comprehensive computational approaches and in-vitro studies.

Alterations to the EGFR (epidermal growth factor receptor) gene, which primarily occur in the axon 18-21 position, have been linked to a variety of cancers, including ovarian, breast, colon, and lung cancer. The use of TK inhibitors (gefitinib, erlotinib, lapatinib, and afatinib) and monoclonal antibodies (cetuximab, panitumumab, and matuzumab) in the treatment of advanced-stage cancer is very common. These drugs are becoming less effective in EGFR targeted cancer treatment and developing resistance to cancer cell eradication, which sometimes necessitates stopping treatment due to the side effects. One in silico study has been conducted to identify EGFR antagonists using other compounds, databases without providing the toxicity profile, comparative analyses, or morphological cell death pattern. The goal of our study was to identify potential lead compounds, and we identified seven compounds based on the docking score and four compounds that were chosen for our study, utilizing toxicity analysis. Molecular docking, virtual screening, dynamic simulation, and in-vitro screening indicated that these compounds' effects were superior to those of already marketed medication (gefitinib). The four compounds obtained, ZINC96937394, ZINC14611940, ZINC103239230, and ZINC96933670, demonstrated improved binding affinity (-9.9 kcal/mol, -9.6 kcal/mol, -9.5 kcal/mol, and -9.2 kcal/mol, respectively), interaction stability, and a lower toxicity profile. In silico toxicity analysis showed that our compounds have a lower toxicity profile and a higher LD50 value. At the same time, a selected compound, i.e., ZINC103239230, was revealed to attach to a particular active site and bind more tightly to the protein, as well as show better in-vitro results when compared to our selected gefitinib medication. MTT assay, gene expression analysis (BAX, BCL-2, and ß-catenin), apoptosis analysis, TEM, cell cycle assay, ELISA, and cell migration assays were conducted to perform the cell death analysis of lung cancer and breast cancer, compared to the marketed product. The MTT assay exhibited 80% cell death for 75 µM and 100µM; however, flow cytometry analysis with the IC50 value demonstrated that the selected compound induced higher apoptosis in MCF-7 (30.8%) than in A549.

Optimizing the catalytic activities of methanol and thermotolerant Kocuria flava lipases for biodiesel production from cooking oil wastes.

In this study, two highly thermotolerant and methanol-tolerant lipase-producing bacteria were isolated from cooking oil and they exhibited a high number of catalytic lipase activities recording 18.65 ± 0.68 U/mL and 13.14 ± 0.03 U/mL, respectively. Bacterial isolates were identified according to phenotypic and genotypic 16S rRNA characterization as Kocuria flava ASU5 (MT919305) and Bacillus circulans ASU11 (MT919306). Lipases produced from Kocuria flava ASU5 showed the highest methanol tolerance, recording 98.4% relative activity as well as exhibited high thermostability and alkaline stability. Under the optimum conditions obtained from 3D plots of response surface methodology design, the Kocuria flava ASU5 biocatalyst exhibited an 83.08% yield of biodiesel at optimized reaction variables of, 60 â—‹C, pH value 8 and 1:2 oil/alcohol molar ratios in the reaction mixture. As well as, the obtained results showed the interactions of temperature/methanol were significant effects, whereas this was not noted in the case of temperature/pH and pH/methanol interactions. The obtained amount of biodiesel from cooking oil was 83.08%, which was analyzed by a GC/Ms profile. The produced biodiesel was confirmed by Fourier-transform infrared spectroscopy (FTIR) approaches showing an absorption band at 1743 cm-1, which is recognized for its absorption in the carbonyl group (C=O) which is characteristic of ester absorption. The energy content generated from biodiesel synthesized was estimated as 12,628.5 kJ/mol. Consequently, Kocuria flava MT919305 may provide promising thermostable, methanol-tolerant lipases, which may improve the economic feasibility and biotechnology of enzyme biocatalysis in the synthesis of value-added green chemicals.

Phytotoxic evaluation of neonicotinoid imidacloprid and cadmium alone and in combination on tomato (Solanum lycopersicum L.).

Neonicotinoids and heavy metals pollution exist simultaneously in agro ecosystem. However, little is known about their combined ecotoxicological effects on non-target crop plants. We have selected imidacloprid (IMI) and cadmium (Cd), applied alone and in combination, to evaluate their effect on growth, physiological and biochemical parameters of tomato. Results showed that the single application of contaminants (IMI and/or Cd) adversely affected both the growth and chlorophyll pigment, and Cd alone application was more phytotoxic than IMI. However, their combined action aggravated the inhibitory effect and indicate a synergistic effect, but it exerted antagonistic effects on chlorophyll pigment inhibition compared with IMI and Cd alone treatments. Both chemicals increased hydrogen peroxide level and generated lipid peroxidation, and the co-contamination exacerbates oxidative stress by their synergistic effect. Those results implicate that disturbance of cellular redox status is the plausible mechanism for IMI and Cd induced toxicity. In conclusion, the single or combined IMI and Cd cause negative effects on tomatoes.

Nanoencapsulated dietary polyphenols for cancer prevention and treatment: successes and challenges.

Many dietary polyphenols have been investigated for their therapeutic potential either as single agents or in combinations. Despite the significant anticancer potential of these polyphenols in in vitro cell culture and in vivo animal models, their clinical applications have been limited because of challenges such as ineffective systemic delivery, stability and low bioavailability. Nanoencapsulation of these polyphenols could prolong circulation, improve localization, enhance efficacy and reduce the chances of multidrug resistance. This review summarized the use of various polyphenols especially epigallocatechin gallate, quercetin, curcumin and resveratrol as nanoformulations for cancer prevention and treatment. Despite some success, more research is warranted to design a nanoencapsulated combination of polyphenols, effective in in vitro, in vivo and human systems.

Evaluation of Stem Cell Marker Expression in Canine B-Cell Lymphoma Cell Lines, B-Cell Lymphoma-generated Spheres and Primary Samples.

BACKGROUND: Canine lymphoma has lately drawn focus as a model of human non-Hodgkin's lymphoma due to its spontaneous occurrence and similar biological behavior. Cells with stem cell-like characteristics are believed to play a key role in therapeutic failure. Thus, an initial characterization and the possibility of specific detection of such cells could bear significant value. MATERIALS AND METHODS: Expression of 12 stem cell markers were analyzed in two canine B-cell lymphoma cell lines, their generated spheres, and in primary lymphoma samples by quantitative real-time polymerase chain reaction and partially by flow cytometry and immunocytochemistry. RESULTS: Expression of maternal embryonic leucine zipper kinase (Melk) was significantly higher in CLBL-1, CLBL-1M and in primary B-cell lymphoma samples compared to non-neoplastic lymph nodes. Spheres displayed a higher expression of v-myc myelocytomatosis viral oncogene homolog (Myc) and lower expression of Cd44 compared to original cell lines and primary B-cell lymphoma samples. CONCLUSION: The results suggest a potential interesting role of Melk in canine B-cell lymphoma. Furthermore, the up-regulation of Myc in serum-free-generated spheres offers interesting possibilities for functional assays characterizing the specific generated sub-population.

Comparative characterization of stem cell marker expression, metabolic activity and resistance to doxorubicin in adherent and spheroid cells derived from the canine prostate adenocarcinoma cell line CT1258.

BACKGROUND: Canine prostate cancer represents a spontaneous animal model for the human counterpart. Cells with stem cell-like character are considered to play a major role in therapeutic resistance and tumor relapse. Thus, the identification of markers allowing for recognition and characterization of these cells is essential. MATERIALS AND METHODS: Expression of 12 stem cell marker genes in the canine prostate cancer cell line CT1258 and spheroid cells generated from these was analyzed by quantitative real-time PCR. In CT1258 and the generated spheroid cells, CD44 and CD133 expression was analyzed by flow cytometry, as well as proliferation and doxorubicin resistance. RESULTS: Integrin alpha-6 (ITGA6) expression and metabolic activity were significantly up-regulated in CT1258-derived spheroid cells, while doxorubicin resistance remained comparable. CONCLUSION: ITGA6 de-regulation and metabolic activity appear to be characteristic of the generated spheres, indicating potential intervention targets.

Generation and characterisation of a canine EGFP-HMGA2 prostate cancer in vitro model.

The architectural transcription factor HMGA2 is abundantly expressed during embryonic development. In several malignant neoplasias including prostate cancer, high re-expression of HMGA2 is correlated with malignancy and poor prognosis. The let-7 miRNA family is described to regulate HMGA2 negatively. The balance of let-7 and HMGA2 is discussed to play a major role in tumour aetiology. To further analyse the role of HMGA2 in prostate cancer a stable and highly reproducible in vitro model system is precondition. Herein we established a canine CT1258-EGFP-HMGA2 prostate cancer cell line stably overexpressing HMGA2 linked to EGFP and in addition the reference cell line CT1258-EGFP expressing solely EGFP to exclude EGFP-induced effects. Both recombinant cell lines were characterised by fluorescence microscopy, flow cytometry and immunocytochemistry. The proliferative effect of ectopically overexpressed HMGA2 was determined via BrdU assays. Comparative karyotyping of the derived and the initial CT1258 cell lines was performed to analyse chromosome consistency. The impact of the ectopic HMGA2 expression on its regulator let-7a was analysed by quantitative real-time PCR. Fluorescence microscopy and immunocytochemistry detected successful expression of the EGFP-HMGA2 fusion protein exclusively accumulating in the nucleus. Gene expression analyses confirmed HMGA2 overexpression in CT1258-EGFP-HMGA2 in comparison to CT1258-EGFP and native cells. Significantly higher let-7a expression levels were found in CT1258-EGFP-HMGA2 and CT1258-EGFP. The BrdU assays detected an increased proliferation of CT1258-HMGA2-EGFP cells compared to CT1258-EGFP and native CT1258. The cytogenetic analyses of CT1258-EGFP and CT1258-EGFP-HMGA2 resulted in a comparable hyperdiploid karyotype as described for native CT1258 cells. To further investigate the impact of recombinant overexpressed HMGA2 on CT1258 cells, other selected targets described to underlie HMGA2 regulation were screened in addition. The new fluorescent CT1258-EGFP-HMGA2 cell line is a stable tool enabling in vitro and in vivo analyses of the HMGA2-mediated effects on cells and the development and pathogenesis of prostate cancer.

Evaluation of stem cell marker gene expression in canine prostate carcinoma- and prostate cyst-derived cell lines.

BACKGROUND/AIM: In human prostate cancer cells with a stem cell-like character (cancer stem cells, CSC) are considered to play a major role in disease development, progression and relapse. Aim of the study was to evaluate if similar cells are present and active in canine prostate cancer providing a naturally-occurring mammalian model for the development of therapeutic approaches targeting CSC. MATERIALS AND METHODS: Stem cell marker expression of CD133, CD44, C-KIT, CD34, ITGA6, OCT4, DDX5 and MELK in canine prostate carcinomas and prostate cyst cell lines were screened by Polymerase Chain Reaction (PCR), quantitative Polymerase Chain Reaction (qPCR) and partially analysed by flow cytometry. RESULTS: Marker analyses by PCR and qPCR, revealed a complex expression pattern for the analysed marker genes, providing a characteristic marker pattern for the studied cell lines. Thereby CD44, CD133, ITGA6 and DDX5 showed the most prominent expression in the analysed cell lines. CONCLUSION: The results revealed a characteristic stem cell marker expression in the analysed cell lines, indicating the presence of CSC in canine prostate cancer.