Enzyme-immobilized hierarchically porous covalent organic framework biocomposite for catalytic degradation of broad-range emerging pollutants in water.

Efficient enzyme immobilization is crucial for the successful commercialization of large-scale enzymatic water treatment. However, issues such as lack of high enzyme loading coupled with enzyme leaching present challenges for the widespread adoption of immobilized enzyme systems. The present study describes the development and bioremediation application of an enzyme biocomposite employing a cationic macrocycle-based covalent organic framework (COF) with hierarchical porosity for the immobilization of horseradish peroxidase (HRP). The intrinsic hierarchical porous features of the azacalix[4]arene-based COF (ACA-COF) allowed for a maximum HRP loading capacity of 0.76 mg/mg COF with low enzyme leaching (<5.0 %). The biocomposite, HRP@ACA-COF, exhibited exceptional thermal stability (∼200 % higher relative activity than the free enzyme), and maintained ∼60 % enzyme activity after five cycles. LCMSMS analyses confirmed that the HRP@ACA-COF system was able to achieve > 99 % degradation of seven diverse types of emerging pollutants (2-mercaptobenzothiazole, paracetamol, caffeic acid, methylparaben, furosemide, sulfamethoxazole, and salicylic acid)in under an hour. The described enzyme-COF system offers promise for efficient wastewater bioremediation applications.

Efficient degradation of various emerging pollutants by wild type and evolved fungal DyP4 peroxidases.

The accumulation of emerging pollutants in the environment remains a major concern as evidenced by the increasing number of reports citing their potential risk on environment and health. Hence, removal strategies of such pollutants remain an active area of investigation. One way through which emerging pollutants can be eliminated from the environment is by enzyme-mediated bioremediation. Enzyme-based degradation can be further enhanced via advanced protein engineering approaches. In the present study a sensitive and robust bioanalytical liquid chromatography-tandem mass spectrometry (LCMSMS)-based approach was used to investigate the ability of a fungal dye decolorizing peroxidase 4 (DyP4) and two of its evolved variants-that were previously shown to be H2O2 tolerant-to degrade a panel of 15 different emerging pollutants. Additionally, the role of a redox mediator was examined in these enzymatic degradation reactions. Our results show that three emerging pollutants (2-mercaptobenzothiazole (MBT), paracetamol, and furosemide) were efficiently degraded by DyP4. Addition of the redox mediator had a synergistic effect as it enabled complete degradation of three more emerging pollutants (methyl paraben, sulfamethoxazole and salicylic acid) and dramatically reduced the time needed for the complete degradation of MBT, paracetamol, and furosemide. Further investigation was carried out using pure MBT to study its degradation by DyP4. Five potential transformation products were generated during the enzymatic degradation of MBT, which were previously reported to be produced during different bioremediation approaches. The current study provides the first instance of the application of fungal DyP4 peroxidases in bioremediation of emerging pollutants.

Challenges and Recent Advances in Enzyme-Mediated Wastewater Remediation-A Review.

Different classes of artificial pollutants, collectively called emerging pollutants, are detected in various water bodies, including lakes, rivers, and seas. Multiple studies have shown the devastating effects these emerging pollutants can have on human and aquatic life. The main reason for these emerging pollutants in the aquatic environment is their incomplete removal in the existing wastewater treatment plants (WWTPs). Several additional treatments that could potentially supplement existing WWTPs to eliminate these pollutants include a range of physicochemical and biological methods. The use of enzymes, specifically, oxidoreductases, are increasingly being studied for their ability to degrade different classes of organic compounds. These enzymes have been immobilized on different supports to promote their adoption as a cost-effective and recyclable remediation approach. Unfortunately, some of these techniques have shown a negative effect on the enzyme, including denaturation and loss of catalytic activity. This review focuses on the major challenges facing researchers working on the immobilization of peroxidases and the recent progress that has been made in this area. It focuses on four major areas: (1) stability of enzymes upon immobilization, enzyme engineering, and evolution; (2) recyclability and reusability, including immobilization on membranes and solid supports; (3) cost associated with enzyme-based remediation; and (4) scaling-up and bioreactors.

Efficient Degradation of 2-Mercaptobenzothiazole and Other Emerging Pollutants by Recombinant Bacterial Dye-Decolorizing Peroxidases.

In recent years, concerns are being raised about the potential harmful effects of emerging pollutants (EPs) on human and aquatic lives. Extensive research is being conducted on developing efficient remediation strategies to target this new class of toxic pollutants. Studies focused on biological (enzyme-based) methods have shown potential as greener and possibly more economical alternatives to other treatment approaches, such as chemical methods. The current study focused on the use of recombinantly produced novel bacterial peroxidases, namely dye-decolorizing peroxidases (DyPs), to study their effectiveness in degrading a number of diverse EPs. In this context, a sensitive bioanalytical Liquid chromatography-tandem mass spectrometry (LCMSMS)-based method was developed to simultaneously detect a mixture of 31 EPs and to examine their degradability by a panel of seven different recombinant bacterial DyPs (rDyPs). We show that up to 9 of the 31 tested EPs could be degraded by at least one of the DyPs tested. The results also indicated that not all rDyPs behaved similarly in their abilities to degrade EPs, as some rDyPs (such as SviDyP and CboDyP) showed a promising potential to degrade EPs while others (such as ScDyP) were almost ineffective. Additionally, the role of redox mediators for effective emerging pollutant degradation by rDyPs was also examined, which showed dramatic improvement in the DyP-mediated degradation of five different EPs. Detailed analysis of 2-mercaptobenzothiazole degradation by SviDyP showed that six distinct breakdown products were generated. The present study showed for the first time that recombinant bacterial DyPs can be used for wastewater remediation by degrading a range of different EPs.

Bioremediation of various aromatic and emerging pollutants by Bacillus cereus sp. isolated from petroleum sludge.

The accumulation of toxic chemical constituents in sludge and wastewater has fuelled an interest in investigating efficient and eco-friendly wastewater remediation approaches. In this study, a set of bacterial samples were isolated from petroleum sludge and tested for their ability to degrade different aromatic pollutants, including azo dyes and emerging pollutants. Although exhibiting differential specificity, all bacterial isolates were able to degrade different classes of aromatic dyes efficiently. Ribosomal 16S rRNA sequencing of the 12 bacterial isolates showed that they belonged to two different bacterial genera: Bacillus cereus and Pseudomonas guariconensis. Of these 12 strains, MA1 (B. cereus) was the most promising and was chosen for further optimization and biochemical studies. The optimum culture and remediation conditions for MA1 was found to be at pH 7, with 100 ppm dye concentration, and under aerobic condition. In addition to efficiently degrading various aromatic dyes (e.g. Congo Red, Reactive Black 5, PBS, and Toluidine Blue), MA1 was also found to be capable of degrading various emerging pollutants (e.g. prometryn, fluometuron and sulfamethoxazole). Preliminary transcriptome analysis shows that MA1 grown on media containing a mixture of aromatic dyes appears to differentially express a number of genes. Data shown here strongly suggests that petroleum sludge is a rich reservoir of bacteria with powerful remediation abilities.

Pharmacological and Antioxidant Activities of Rhus coriaria L. (Sumac).

Rhus coriaria L. (Anacardiaceae), commonly known as sumac, is a commonly used spice, condiment, and flavoring agent, especially in the Mediterranean region. Owing to its bountiful beneficial values, sumac has been used in traditional medicine for the management and treatment of many ailments including hemorrhoids, wound healing, diarrhea, ulcer, and eye inflammation. This plant is rich in various classes of phytochemicals including flavonoids, tannins, polyphenolic compounds, organic acids, and many others. By virtue of its bioactive, Rhus coriaria possesses powerful antioxidant capacities that have ameliorative and therapeutic benefits for many common diseases including cardiovascular disease, diabetes, and cancer. This review describes the phytochemical properties of R. coriaria and then focuses on the potent antioxidant capacities of sumac. We then dissect the cellular and molecular mechanisms of sumac's action in modulating many pathophysiological instigators. We show how accumulating evidence supports the antibacterial, antinociceptive, antidiabetic, cardioprotective, neuroprotective, and anticancer effects of this plant, especially that toxicity studies show that sumac is very safe to consume by humans and has little toxicity. Taken together, the findings we summarize here support the utilization of this plant as an attractive target for drug discovery.

Never let a crisis go to waste: Repurposing independent research projects to enhance students' critical thinking skills.

The recent lockdown of laboratories in the COVID-19 era has negatively impacted many lab-based courses for undergraduate students; however, research project classes have been hardest hit. Unlike the lab-based lecture courses, where many of the lab instructors have resorted to virtual experiments and videos to substitute for actual lab experiments, senior year independent research courses, which are supposed to involve real lab-based research, are struggling to find an appropriate solution. A possible solution to cases where the coronavirus pandemic lockdown has led to no or little student-generated lab data are to shift the focus of these research project courses such that they are taught as mentor-guided critical thinking exercises, using the primary literature. As an example, the lack of complete understanding for the molecular basis of protein thermostability can be used to promote such higher-level thinking skills.

Origanum majorana Essential Oil Triggers p38 MAPK-Mediated Protective Autophagy, Apoptosis, and Caspase-Dependent Cleavage of P70S6K in Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most common type of cancer in terms of incidence and mortality worldwide. Here we have investigated the anti-colon cancer potential of Origanum majorana essential oil (OMEO) and its underlying mechanisms of action. We showed that OMEO significantly inhibited the cellular viability and colony growth of human HT-29 colorectal cancer cells. OMEO induced protective autophagy, associated with downregulation of the mTOR/p70S6K pathway, and activated caspase-8 and caspase-9-dependent apoptosis. Blockade of autophagy with 3-methyladenine (3-MA) and chloroquine (CQ), two autophagy inhibitors, potentiated the OMEO-induced apoptotic cell death. Inversely, inhibition of apoptosis with the pan-caspase inhibitor, Z-VAD-FMK, significantly reduced cell death, suggesting that apoptosis represents the main mechanism of OMEO-induced cell death. Mechanistically, we found that OMEO induces protective autophagy and apoptotic cells death via the activation of the p38 MAPK signaling pathway. Pharmacological inhibition of p38 MAPK by the p38 inhibitors SB 202190 and SB 203580 not only significantly decreased apoptotic cell death, but also reduced the autophagy level in OMEO treated HT-29 cells. Strikingly, we found that OMEO also induces p38 MAPK-mediated caspase-dependent cleavage of p70S6K, a protein reported to be overexpressed in colon cancer and associated with drug resistance. Our findings suggest that OMEO inhibits colon cancer through p38 MAPK-mediated protective autophagy and apoptosis associated with caspase-dependent cleavage of p70S6K. To the best of our knowledge, this study is the first to report on the implications of the p38 MAPK signaling pathway in targeting p70S6K to caspase cleavage.

Origanum majorana Ethanolic Extract Promotes Colorectal Cancer Cell Death by Triggering Abortive Autophagy and Activation of the Extrinsic Apoptotic Pathway.

Colorectal cancer is considered as the third leading cause of cancer death. In the present study, we investigated the potential anticancer effect and the molecular mechanism of Origanum majorana ethanolic extract (OME) against human colorectal cancer cells. We showed that OME exhibited strong anti-proliferative activity in a concentration- and time-dependent manner against two human colorectal cancer cell lines (HT-29 and Caco-2). OME inhibited cell viability, colony growth and induced mitotic arrest of HT-29 cells. Also, OME induced DNA damage, triggered abortive autophagy and activated a caspase 3 and 7-dependent extrinsic apoptotic pathway, most likely through activation of the TNFα pathway. Time-course analysis revealed that DNA damage occurred concomitantly with abortive autophagy after 4 h post-OME treatment while apoptosis was activated only 24 h later. Blockade of autophagy initiation, by 3-methyladenine, partially rescued OME-induced cell death. Cell viability arose from 37% in control group to 67% in group pre-treated with 3-MA before addition of OME. Inhibition of apoptosis, however, had a minimal effect on cell viability; it rose from 37% in control group to 43% in group pre-treated with Z-VAD-FMK. We also found that OME downregulated survivin in HT-29 cells. Our findings provide a strong evidence that O. majorana extract possesses strong anti-colon cancer potential, at least, through induction of autophagy and apoptosis. These finding provide the basis for therapeutic potential of O. majorana in the treatment of colon cancer.

Breast cancer cells exhibits specific dielectric signature in vitro using the open-ended coaxial probe technique from 200 MHz to 13.6 GHz.

Here we investigated the feasibility of using microwave spectroscopy for characterization of normal and breast cancer cell lines cultured in vitro. Healthy non-tumorigenic, MCF-10A and breast cancer, MDA-MB-231, Hs578T, T47D and MCF-7 cell lines were electrically characterized using the open-ended coaxial probe technique from 200 MHz to 13.6 GHz. The dielectric constant, dielectric loss and conductivity between breast non-tumorigenic and breast cancer cells lines were analyzed and their differences determined. Our results showed that the four breast cancer cell lines analyzed exhibited higher dielectric properties when compared to healthy cells. Interestingly, we found that breast and colon cancer cells have different dielectric properties as well, thus suggesting that each type of cancer has a unique microwave signature. This study shows that microwave characterization of breast cancer cell lines is reliable with potential in biomedical applications such as designing electromagnetic models for detection of tumorous cells in healthy tissues.

Rhus coriaria increases protein ubiquitination, proteasomal degradation and triggers non-canonical Beclin-1-independent autophagy and apoptotic cell death in colon cancer cells.

Colorectal cancer is the fourth leading cause of cancer-related deaths worldwide. Here, we investigated the anticancer effect of Rhus coriaria extract (RCE) on HT-29 and Caco-2 human colorectal cancer cells. We found that RCE significantly inhibited the viability and colony growth of colon cancer cells. Moreover, RCE induced Beclin-1-independent autophagy and subsequent caspase-7-dependent apoptosis. Blocking of autophagy by chloroquine significantly reduced RCE-induced cell death, while blocking of apoptosis had no effect on RCE-induced cell death. Mechanistically, RCE inactivated the AKT/mTOR pathway by promoting the proteasome-dependent degradation of both proteins. Strikingly, we also found that RCE targeted Beclin-1, p53 and procaspase-3 to degradation. Proteasome inhibition by MG-132 not only restored these proteins to level comparable to control cells, but also reduced RCE-induced cell death and blocked the activation of autophagy and apoptosis. The proteasomal degradation of mTOR, which occurred only 3 hours post-RCE treatment was concomitant with an overall increase in the level of ubiquitinated proteins and translated stimulation of proteolysis by the proteasome. Our findings demonstrate that Rhus coriaria possesses strong anti-colon cancer activity through stimulation of proteolysis as well as induction of autophagic and apoptotic cell death, making it a potential and valuable source of novel therapeutic cancer drug.

Rhus coriaria suppresses angiogenesis, metastasis and tumor growth of breast cancer through inhibition of STAT3, NFκB and nitric oxide pathways.

Recently, we reported that Rhus coriaria exhibits anticancer activities by promoting cell cycle arrest and autophagic cell death of the metastatic triple negative MDA-MB-231 breast cancer cells. Here, we investigated the effect of Rhus coriaria on the migration, invasion, metastasis and tumor growth of TNBC cells. Our current study revealed that non-cytotoxic concentrations of Rhus coriaria significantly inhibited migration and invasion, blocked adhesion to fibronectin and downregulated MMP-9 and prostaglandin E2 (PgE2). Not only did Rhus coriaria decrease their adhesion to HUVECs and to lung microvascular endothelial (HMVEC-L) cells, but it also inhibited the transendothelial migration of MDA-MB-231 cells through TNF-α-activated HUVECs. Furthermore, we found that Rhus coriaria inhibited angiogenesis, reduced VEGF production in both MDA-MB-231 and HUVECs and downregulated the inflammatory cytokines TNF-α, IL-6 and IL-8. The underlying mechanism for Rhus coriaria effects appears to be through inhibiting NFκB, STAT3 and nitric oxide (NO) pathways. Most importantly, by using chick embryo tumor growth assay, we showed that Rhus coriaria suppressed tumor growth and metastasis in vivo. The results described in the present study identify Rhus coriaria as a promising chemopreventive and therapeutic candidate that modulate triple negative breast cancer growth and metastasis.

Rhus coriaria induces senescence and autophagic cell death in breast cancer cells through a mechanism involving p38 and ERK1/2 activation.

Here, we investigated the anticancer effect of Rhus coriaria on three breast cancer cell lines. We demonstrated that Rhus coriaria ethanolic extract (RCE) inhibits the proliferation of these cell lines in a time- and concentration-dependent manner. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, downregulation of cyclin D1, p27, PCNA, c-myc, phospho-RB and expression of senescence-associated ß-galactosidase activity. No proliferative recovery was detected after RCE removal. Annexin V staining and PARP cleavage analysis revealed a minimal induction of apoptosis in MDA-MB-231 cells. Electron microscopy revealed the presence of autophagic vacuoles in RCE-treated cells. Interestingly, blocking autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death and senescence. RCE was also found to activate p38 and ERK1/2 signaling pathways which coincided with induction of autophagy. Furthermore, we found that while both autophagy inhibitors abolished p38 phosphorylation, only CQ led to significant decrease in pERK1/2. Finally, RCE induced DNA damage and reduced mutant p53, two events that preceded autophagy. Our findings provide strong evidence that R. coriaria possesses strong anti-breast cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against breast cancer.