A Membrane-Active Anti-Microbial Peptide Demonstrates In Vitro Activity Against SARS-CoV-2 Infectivity

Introduction

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), put significant strains on healthy system and economies around the world, there are widespread negative social, psychological and economic effects. Up to now, millions of people had died of SARS-CoV-2 infection and new variants are still emerging [1]. Delta and Omicron variants currently pose a “double threat” to the global outbreak. And as the outbreak continues, new strains may also emerge during a pandemic. Although the vaccines including whole-virus (live-attenuated virus, inactivated virus, and viral-vector) vaccines [2], subunit vaccines [3,4], and nucleic acid vaccines (pDNA and mRNA) [5] had been designed to enable broad-spectrum protection against coronavirus pathogens, the emergence of SARS-CoV-2 variants that harbor mutations in the spike glycoprotein receptor binding domain(RBD) may resist neutralization by antibodies, comprising vaccine efficacy [6,7]. The continued emergence of SARS-CoV-2 mutations poses a major challenge to the development of antiviral drugs and vaccines.

It has been reported that the tropism of SARS-CoV-2 was determined by the S protein which binds to the membrane receptors, angiontensin-converting enzyme 2 (ACE2) and/or CD147 [8,9]. These membrane receptors on the host cells mediated the viral and cellular membrane fusion and contributed to SARS-CoV-2 invasion for host cells. S protein is divided into two subunits, S1 and S2. The RBD of S1 is responsible for recognizing ACE2 on the surface of human cells and completing the binding of virus to cells. Then S2 subunit is responsible for the fusion of virus envelope and human cell membrane to complete the invasion process. Two important conserved repeating amino acid sequences, heptad repeat 1 (HR1) and 2(HR2), are present in the S2 subunit of SARS-CoV-2. The two domains combine to form a spiral structure called six-helix bundle core fusion structure (6-HB) which could bring the viral envelop closed to the cellular membranes [10]. Recent studies have shown that this 6-HB is the key to complete SARS-CoV-2 envelope and cell membrane fusion, which is also shown to be stronger and more efficient in entering cells than SARS virus [11]. Several peptides such as AP-9, EK1 et al were identified to potentially block the infection of SARS-CoV-2 [12,13], As a broad-spectrum anti-Human Coronaviruses (HCoV) therapeutic and prophylactic agent, it has broad development prospects in the treatment and prevention of COVID-19 and other emerging HCoV diseases.

KSL can directly act on microbial lipid membranes and has amphiphilic α helix and Net positive charge, so it has strong irreversible inhibitory activity against both bacteria and fungi. What’s more, KSL has no hemolytic activity [14], so it has great potential to be developed as a new antibacterial agent. In this study, we generated a novel decapeptide, K04 (RRVVFHVKFK) derived from KSL which killed microorganisms through the action on the membrane as its primary target [15]. Our results demonstrated that K04 had higher activity against bacteria and fungi without cytotoxicity. More interesting, K04 had a strong synergism with JX0020 peptide, a membrane-fusion inhibitor of SARS-CoV-2 and significantly reduced the effective inhibition concentration of JX0020. The specific mechanism needs further study.

Materials and Methods

Cells and Peptide

Human embryonic kidney cell line 293T, Human hepatocyte cell line LO2 and human fetal lung fibroblast cell MRC-5 were purchased from Cell Bank/Stem Cell bank (Shanghai institute of Biochemistry and Cell Biology). Human epidermal keratinocyte cell line HaCaT was obtained from Biomedical Cell Resource Center (BMCR). LO2 and 293T cells were cultured in DMEM medium plus 10% FBS, MRC-5 and HaCaT cells were cultured in MEM medium plus 10% FBS and 1% NEAA in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Peptides were synthesized with Fmoc-strategy solid phase methodology by Harbin Jixianglong Biotech Co. Ltd.

Growth Inhibition Assay

This test was performed with the strains Candida albicans CMCC( B) 98001, Staphylococcus aureus CMCC(B) 26003, Streptococcus mutans UA159, Escherichia coli CMCC(B) 44102. The reference drugs used were chlorhexidine and bisabolol. Aliquots of microbial suspension in indicated culture medium were mixed with peptides or reference drugs respectively and incubated at 37°C for different time (0, 4, 8, 12, 16, 20, 24 h). Then, the viable cell count was done by diluting the suspension sample and plating aliquots of the dilutions onto an appropriate culture medium.

Inhibition of Pseudotyped SARS-CoV-2 Infection

Plasmid pLVX-puro, psPAX2, pcDNA3.1-Spike (SARS-CoV-2), pGL-Basic were purchased from Miaoling Plasmid Sharing Platform. To generate pLVX-luciferase-puro plasmid, Luciferase gene was amplified using pGL-Basic plasmid as template and cloned into pLVX-puro plasmid. The pseudo viruses were produced using methods as previously described. Briefly, 293T cells were inoculated into the 6-well cell plate, and the cell density was controlled at 70%-90% confluence before transfected with psPAX2 (3μg), pLVX-luciferase-puro (4 μg), pcDNA3.1-spike (SARS-CoV-2) plasmid DNA mix using Lipofectamine 2000. Six hours later, the medium was changed, and the transfected cells were cultured with fresh medium containing DMEM medium plus 10% FBS for 48 h. Then, SARS-CoV-2 pseudo viruses containing culture supernatants were harvested, filtered and stored at -80°C in aliquots. To detect the inhibitory activity of a peptide on infection of SARS-CoV-2 pseudo virus, target cells (293T/ACE2) were seeded at a density of 1.5 × 104 cells per well in a 96-well plate one day prior to infection. Pseudo virus was mixed with an equal volume of a peptide which was series diluted with PBS at 37°C for 1 h. EK1 peptide was used as positive control. The mixture was transferred to the target cells and incubated for 72 h. Then, cell supernatant was collected, and luciferase activity was analyzed by the Luciferase assay system (Promega).

Cytotoxicity Assay

This test was performed with the cell lines LO2, HEK293T, MRC- 5, HaCaT using CCK-8 assay as previously described with some modifications. Cells (5 × 103 or 1.5 × 104 per well) were seeded in 96-well cell plate and cultured at 37°C in a humidified atmosphere with 5% CO2 for 12 h. Then, cell culture medium was changed with fresh medium containing a series of concentrations of peptides and further incubated for 24, 48 or 72 h respectively. Finally, 10 μL of the CCK-8 reagent was added into each well, and OD at 450 nm was measured using a multimode microplate reader after incubation for 4 h at 37°C.

Results and Discussion

K04 demonstrated antifungal and antibacterial activity. Previous study had investigated the influence of different amino acid residues of decapeptide on the antifungal activity through screening the combinatorial peptide libraries composed of seven amino acids (Lys, Leu, Val, Pro, Ser, Phe, and Gln). The positively charged amino acid at the N terminus and the Lys at sixth position confer to the highest level of antifungal activity. In this study, we constructed three analogs of KSL (Table 1) by replacing the amino acid residue at the first, second and sixth position. KSL and its analogs K02, K03, K04 (Table 1) were generated by solid-phase synthesis in high purity (> 95% by HPLC). Some physiochemical properties, such as their secondary structures, hydrophobicities (H), hydrophobic moments (μH) and net charge (at neutral pH) were listed in Table 1. Similar to KSL, these analogs did not form perfect amphipathic α-helical structure in a wheel diagram, but the mutation of Lys to His at the sixth position increases the hydrophobicities (H) and decreases hydrophobic moments (μH). To test the in vitro antifungal and anti- bacterial activity, KSL and its analogs were co-cultured with microbial cells for different times. Then, the live microbial cells were enumerated by plate count method (CFU/mL). As shown in Figure 1, among the three KSL analogs, K04 was the best anti-microbial agent and showed higher anti-microbial activity when compared to its native peptide KSL. To evaluate possible toxic effect of K04 peptide with healthy eukaryotic cells, different concentrations of K04 peptide were prepared and added into the cell culture media of human embryonic kidney cell 293T, hepatocyte cell line LO2, epidermal keratinocyte line HaCaT and fetal lung fibroblast cell MRC-5 for three days, respectively. As shown in Figure 2, the CCK-8 assays demonstrated that K04 caused almost no influence on the viability of cell lines tested at the concentration below 125 μg/mL. Above results indicated that the membrane-active peptide K04 improved the anti-microbial activity by introducing three amino acid mutation.

Table 1: Structures, predict models and physicochemical characteristics of synthetic peptides.

Figure 1: Time course of KSL and its analogs-induced killing of C. albicans (A), S. aureus (B), S. mutans (C) and E. coli (D).

Figure 2: Cell viability of HEK293T, LO2, HaCaT, MRC-7 induced by K04 after Day1, Day 2 and Day3 in CCK-8 assay.

Each appearance of such as Alpha, Beta, Delta, Omicron mutant sets off a new epidemic of infections. And new variants have been found that make the vaccine less effective. In order to prevent reinfection of the virus, in addition to vaccines, new drugs that can block the fusion of the virus with cells need to be developed. Since SARSCoV- 2 spike protein-mediated membrane fusion played important role on the virus infection, spike protein became an important target for the development of preventative and therapeutic drugs. As an optimized form of OC43-HR2P, EK1 peptide was reported to be a promising pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike [11]. In this study, we constructed JX0020 peptide derived from HR2 region on the basis of the 6-helix bundle structure of SARS-CoV-2 Spike protein and introduced ten mutations (N6E, A7K, V10K, N11K, D17K, R18K, V22E, N25K, S29E, L36E), at the i to i + 3 or i to i + 4 positions to form intramolecular E-K or K-E salt bridges. Then, we used the pseudo virus assay, a good model to study the process of virus entry into the target cell, to assess the inhibitory activity of JX0020 against SARS-CoV-2 pseudo virus infection with EK1 as positive control and K04 as negative control. As showed in Figure 3, EK1 demonstrated antiviral activity against pesudotyped SARS-CoV-2 infection with IC50 value of 0.143 mg/mL, while JX0020 exhibited improved fusion inhibitory activity with IC50 value of 0.049 mg/mL.

Interestingly, K04 also showed mild antiviral activity with IC50 value of 0.43 mg/mL (Fig. 3C). Then, the synergic effect between K04 and JX0020 on the inhibition of SARS-CoV-2 infection was investigated. As showed in Figure 4, JX0020 improved the inhibitory activity with the increase of concentration from 15 μg/mL to 150 μg/mL in the absence of K04. In addition, when combined with a relatively low amount of K04 peptide (10 μg/mL), JX0020 significantly decreased the effective inhibitory concentration from 150 μg/mL to 15 μg/mL by exhibiting the comparative inhibition activity. These results indicated that K04 had a strong synergism with JX0020 against SARS-CoV-2 pseudovirus infection. A number of studies have shown that KSL and its analogs have significant antibacterial activities against a variety of bacteria and fungi. However, there have been no reports on KSL or its analogs antiviral or co-antiviral activities so far, and the specific mechanism needs further study.

Figure 3: Inhibitory activity of EK1 (A), JX0020 (B), and K04 (C) peptide in pseudovirus infection assay against SARS-CoV-2.

Figure 4: K04 had synergic effect with JX0020 in pseudovirus infection assay against SARS-CoV-2.

Conclusion

In summary, our results indicated that K04, as a novel membrane- active peptide, demonstrated antifungal, antibacterial activity and synergic effect with membrane-fusion inhibitor without cytotoxicity to eukaryotic cells. Antimicrobial peptides were paid more attention as the potential substitutions for common antibiotics. Our results suggested that K04 might be a promising drug candidate in the development of multi-functional antimicrobial agent.

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Carotid Web Syndrome: Two Cases Treated with Carotid Endarterectomy

Introduction

Carotid web is pathologically defined as an intimal variant of fibromuscular dysplasia, which appears radiographically as a shelf-like filling defect on the posterior aspect at the origin of the internal carotid artery. The prevalence of these lesions is estimated to be on the order of 1%, although they may be overlooked in clinical practice as they are not typically associated with significant vascular stenosis [1,2]. Up to one-third of all patients presenting with ischemic strokes lack an identifiable cause, and the strokes are classified as cryptogenic in etiology, with most cases occurring in younger patients without traditional vascular risk factors [2]. Carotid web may be an underappreciated risk factor for stroke: available literature indicates that carotid web leads to ischemic stroke in young patients (aged <60 years) without the typical vascular risk factors. There is an association between carotid artery web and ischemic stroke in patients who lack an alternative cause of stroke. [1,3]. Hemodynamic changes such as blood flow stasis and turbulence in the rostral aspect of the carotid web result in thrombus formation and dislodgement, which can provoke artery-to-artery embolism to the anterior circulation. [1] Depending of the clinical presentation and the anatomical features, patients with symptomatic carotid web can be managed with different methods; in the literature the treatment include fibrinolysis, thromboaspiration, mechanical thrombectomy, carotid endarterectomy and carotid stenting. We present two cases of symptomatic carotid web associated carotid thrombosis treated with carotid endarterectomy.

Case 1

A forty-eight years old woman, with no cardiovascular risk factors and no history of cardiovascular events, presented for strength deficit of the right upper limb (NIHSS = 2). No brain acute lesions were observed on the CT scan, at the CT angiography the left carotid bifurcation presented an 85% stenosis caused by the presence of a floating thrombus (Figure 1). The same day the patient was treated with thromboaspiration with percutaneous right femoral artery access; the procedure had no complications and it was effective in removing most of the carotid thrombosis. However part of the thrombus remained in place. Meanwhile the neurological symptom had regressed.After 6 days the patient was treated with left carotid endarterectomy with bovine patch angioplasty that allowed to remove the thrombus and an underlying carotid web (Figure 2). Two days after surgery the patient was discharged without symptoms or complications with dual antiplatelet therapy and lipid-lowering therapy. No complications or neurological symptoms were detected in the 1-year follow-up.

Figure 1: Case 1, left carotid floating thrombus at CT angiography.

Figure 2: Case 1, carotid bifurcation during endarterectomy.

Case 2

A fifty-five-year-old man, with no cardiovascular risk factors and no history of cardiovascular events, presented with left hemisome motor deficit. The brain CT scan and CT angiography detected M1-middle cerebral artery occlusion with left thalamic-insular ischemic lesion. Patients underwent mechanical thrombectomy. An improvement in symptoms was observed in the following days. After the exclusion of other embolic causes, patient was studied with a carotid doppler ultrasonography that detected the presence of a thrombus in right internal carotid artery (Figure 3); a new CT angiography was then performed (Figure 4). Ten days after the thrombectomy, patients was treated with right eversion carotid endarterectomy (Figure 5), the thrombus was removed and a carotid web was found and corrected.Five days after surgery the patient was discharged in stable conditions, mild weakness of the left upper limb without significant disability, no new neurological symptoms, with single antiplatelet therapy and lipid-lowering therapy. No complications or other neurological symptoms were detected in the 1-year follow-up.

Figure 3: Case 2, doppler ultrasonography with thrombus in right internal carotid artery.

Figure 4: Case 2, right carotid thrombus at CT angiography.

Figure 5: Case 2, eversion carotid endarterectomy.

Discussion

We presented two cases of carotid web associated with floating thrombus in young patient without cardiovascular risk factors. Even after extensive diagnostic workup, up to 30% of stroke cases are classified as undetermined, leaving these patients without specific treatment. [4] In their case-control study, Coutinho JM et Al. found an association between carotid artery web and ischemic stroke in patients who lack an alternative cause of stroke [3]. In a retrospective review performed by Hu H. et Al, incidence of carotid web in patients with transient ischemic attack (TIA) was 8.9% [5]. It seems clear that carotid web can be an important cause of cerebral ischemic events especially in young patients.While there is no evidence of a potential benefit in treating asymptomatic carotid web, literature suggest that medical management alone may not provide sufficient protection for patients with carotid web and ipsilateral ischemic stroke or TIA without other causes. A comparative cohort study that used data from the MR CLEAN trial (2010-2014) and MR CLEAN Registry (2014-2017) identified 30 patients with carotid web and ipsilateral stroke. These patients were not treated with carotid endarterectomy or carotid stenting, 93% received medical management and were compared with 168 patients without carotid web in the same registry: 17% of patients with carotid web had a recurrent stroke compared with 3% of patients without carotid web [6].

Although different treatments such as carotid stenting were proposed, carotid endarterectomy seems to be the most used approach to this pathology with good results. We described two cases of symptomatic carotid web treated with carotid endarterectomy plus bovine patch angioplasty and eversion carotid endarterectomy without complications and with no recurrence of cerebrovascular events during the follow up (one year for each patient). This may help to provide more evidence in order to figure out the best management of these patients.More data and studies are needed to understand the best treatment of symptomatic carotid web and weather a treatment of asymptomatic cases can give a benefit.

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Phytoremediation of Organophosphorus Pesticides from Aqueous Media Using Azolla Filiculoides (Case study: Anzali Wetland)

Introduction

Organic compounds released in the environment by anthropogenic activities. This has led to serious consequences on the environment due to their toxicity, hydrophobic nature and persistence in the environment for a longer period of time. Furthermore, organic compounds can enter the food chain, and due to their toxicity they can cause mutagenicity and carcinogenicity in animals and humans [1]. These days, different kinds of pesticides containing various groups of insecticides, herbicides, fungicides, nematocysts, acaricides, etc. are applied in agricultural activities. Due to pesticides complex chemical structures they are extremely stable and resistant to decomposition. Toxic pesticides have potential hazards for their consumers, environment, and chemosphere at a larger scale and can have negative impacts on the society and economics [2]. Insecticides are one of the most frequent water contaminants that can be found in different water resources. Therefore, the application of organochlorine insecticides is prohibited. Recently, organophosphorus pesticides are widely utilized all over the world [3]. Diazinon [O,O-diethyl O-(2-isopropyl-6-methylpyrimidin-4-yl) thiophosphate] is one of the organophosphorus insecticide that is applied in considerable quantities in agricultural and nonagricultural industries. Toxicity of diazinon is caused by its inhibition of acetyl cholinesterase. The studies on animal metabolism have demonstrated that diazinon affects their bodies by restricting acetyl cholinesterase metabolism [4]. Diazinon has been classified by the World Health Organization (WHO) as class II having a moderate hazard. This insecticide has log kow, vapor pressure, and Henry’s law constant of 3.3, 1.4*10−4 mm Hg at 20°C, and 1.4*10−6 mm3 mol−1, respectively.

It is non-polar and moderately mobile, which raise a concern when involving groundwater and surface-derived drinking water [5]. Fenitrothion [O,O‑dimethyl O‑(4‑nitro‑m‑tolyl) phosphorothioate], is mainly used in agriculture, to control chewing and sucking insects on rice, cereals, fruits, vegetables, and etc. In addition, due to photolysis and hydrolysis it undergoes degradation. This insecticide can be stable in water only when sunlight and microbial contamination do not exist. The major reason for degradation in soil can be attributed to biodegradation, although photolysis can also be another determinant. In World Health Organization (WHO), this insecticide is classified as moderate hazardous of class II. Fenitrothion has vapor pressure of 18mPa at 20°C and 14mg·L−1 solubility in water at 30°C [6,7]. Some common and efficient treatment methods to remove diazinon and fenitrothion from polluted water resources include chemical adsorption, coagulation, membrane process, bioremediation, and Advanced Oxidation Process (AOP). In addition, physical methods, such as activated carbon adsorption, nano-filtering, and Ultra-Low Pressure Reverse Osmosis (ULPRO) can be mentioned [8-10]. Phytoremediation mention to innovations including the application of living plants to elimination of pollution from contaminated media such as water [11]. Due to A. filiculoides rapid growth rate, ability to live in contaminated waters, and the high potential in absorbing various pollutants, it was proposed as a suitable plant with high ability to use in the phytoremediation processes [8]. The main goal of this study was to estimate the efficiency of A. filiculoides to eliminate diazinon and fenitrothion concentration in water. In addition, we attempted to intensify the phytoremediation potential of this plant by changes in some factors including initial concentration of pesticides, treatment time and plant fresh weight.

Materials and Methods

The aim of the cross-sectional study is to assess the possibility of evaluating the levels of healthiness literateness assistances about vaccination in the Sindh general population; through a questionnaire from October to December 2021 a total of 500 participants shall complete the survey. The attitudes towards COVID-19 vaccination were collected via questionnaire and analyzed using descriptive and inferential statistics. The questionnaire were prepared, distributed, and collected by ‘based surveys and shared through other services. The sampling approach was convenient and nonprobability. Participants were given complete freedom to complete the survey and were encouraged to forward and share it with others. On the first page, participants were given information on the survey’s rationale and scope, which included gathering perceptions as well as methods and abilities for collecting, understanding, and using information about vaccination, including possible COVID-19 vaccines. They were assured that moving on to the second page of the survey and filling out the questionnaire meant they had given their consent.

Materials and Methods

Chemicals and Reagents

Azolla filiculoides and water that we need for the experiments were collected from the Anzali wetland, Gilan, Iran. Diazinon and fenitrothion (95% technical grade) were purchased from Caron co., China.

Experimental Procedures and Methods

Preparation of A. Filiculoides to Remove Diazinon and Fenitrothion: Healthy and matured A. filiculoides, used here were collected from the Anzali wetland, Gilan, Iran. According to the purpose and time of sampling, the smaller plants with light green in color were selected. In addition plants rinsed with distilled water and sterilized with Mercuric chloride (0.1%) for 30s, then washed with deionized water several times. Experiments for diazinon and fenitrothion elimination were carried out separately in the laboratory at ambient temperature of 25-28°C. To examine the ability of proposed method 4.5, 9.5 and 13.5 g of the water fern were laid on the surface of 12 glass aquariums with 1000 cm3 volumes which filled with real water. Then the various concentrations of pesticides (30, 50, 70 and 100 mg L-1) were prepared from the pesticides stock solution with initial concentration of 1000 mg L-1 and add them in to the glass containers. All the experiments were run for 15 day period under fluorescent lamp as a light source and greenhouse conditions. During the incubation the evaporated water was compensated by adding deionized water when needed. Sampling was done every five days (0, 5, 10 and 15 days) until the 15th day of the experiment. All experiments were conducted triplicates and the data were statistically analyzed by Microsoft Excel 2010 and SPSS [12].

Chromatographic Conditions

To assess the efficiency of proposed method in the elimination of diazinon and fenitrothion from real water samples, the water samples were collected from three different parts of International Anzali Wetland. Before evaluating the plants efficiency, all the mentioned water samples were analyzed to examine the available pesticides concentrations in the water. Based on the results there were no concentrations of diazinon and fenitrothion in the samples. At the beginning of the procedure, the water samples were passed through a membrane filter with a pore size of 0.45 μm to separate all the suspended solid particles. Real samples analysis was carried out on an Agilent 8790 system through Gas Chromatography-Mass Spectrometry equipped with nitrogen-phosphorus detection (NPD) and a 30 m* .53 mm i.d., 1.5 μm film thickness, HP1 (polydimethylsiloxane) capillary column. Nitrogen was used as carrier gas (linear velocity 34 cm s−1). The detector gas (hydrogen) flow was 4 mL min−1, the air flow was 93.8 mL min−1 and nitrogen was used as detector make-up at 32.3 mL min−1. The injector temperature was 180°C. The column temperature was programmed from 155°C to 250°C at 3° min−1. The detector temperature was 320°C and the injection volume was 1 μL [7].

Evaluation of the Removal Ability of A. Filiculoides

To evaluate the elimination potential of A. filiculoides, the effective parameters in the process, including initial concentration, contact time and the adsorbent dosage were examined. To investigate the adsorption potential of A. filiculoides to eliminate diazinon and fenitrothion the removal percentage (%R) were determined based on the following equation [13,14]:

where C0 and Ce are the initial and final concentrations of diazinon and fenitrothion in the solution, respectively. To analyze of numerical results statistically, ANOVA factorial experiments were conducted. In most environmental experiments, the researcher seeks to compare and examine two or more factors, and this issue can be realized in factorial experiments, because in this method, the effect of one or more independent variables on dependent variables can be measured and examined separately or simultaneously. Also, among the advantages of this method, we can mention economic and time saving, more information generation and more accuracy. In this method, the effect of each of the investigated factors (concentration of pollutant, time and amount of adsorbent) on the removal of diazinon and fenitrothion by Azolla has been studied.

Results and Discussion

Phytoremediation of Diazinon and Fenitrothion by Azolla Filiculoides

Table 1: Evaluation of the diazinon and fenitrothion removal ability of A. filiculoides.

To evaluate the accuracy of the proposed method, replicate analysis of real water samples was performed in different days of retention time. As displayed in Table 1, a good compliance with the added and found diazinon and fenitrothion concentrations has been accrued by the proposed procedure. Concentrations of residual diazinon and fenitrothion in the water samples were measured through gas chromatography with nitrogen phosphorus detection (NPD) (an Agilent 8790A GC system). As a consequence, high removal efficiencies were observed in all treatments with more than 95% the remaining concentrations of all the samples were less than 20 μg L-1. Thus, the suggested method was suitable to remove noticed pesticides from the real water samples.

Statistical Analysis of the Removal Efficiencies of the Azolla Filiculoides

Due to the multifactorial nature of the experiment, to analyze the information obtained from the evaluation of the residual concentration of diazinon and fenitrothion the factorial ANOVA test has been used. In addition the effect of contact time, initial concentration of pesticides, the mass of A. filiculoides and the interaction of these parameters on the removal process was investigated. As demonstrated in Tables 2 & 3 the parameters of contact time, concentration of pesticides and amount of plants have a significant interaction with the elimination process. Based on the results the influence factor of the parameters was 0- 0.05. The interaction of the effective parameters showed a significant relationship between these variables with the removal process of diazinon and. However as demonstrated in the results just the contact time has a significant relationship with the removal process of fenitrothion, which means that the only controlling factor of the removal process of fenitrothion by Azolla is the contact time.

Table 2: The interaction of effective parameters with diazinon removal process.

Table 3: The interaction of effective parameters with fenitrothion removal process.

Conclusion

We conclude by nominating Azolla filiculoides as a phytoremediation agent that can be used for the organophosphorus pesticides such as diazinon and fenitrothion. This technology offers efficient, easy, cost-effective and environmentally-sustainable solution to the concern of pesticides accumulation in public reservoirs and waterways. A. filiculoides which is abundantly used as a nitrogen-infuser for rice cultivation provides a vehicle for purifying water bodies infested with diazinon and fenitrothion. Based on pervious papers and this research it is also recommended that artificial pond-based systems cultivated with A. filiculoides can easily be employed as a biological filter to eliminate pesticides, heavy metals, dyes and etc. from agricultural and industrial effluents and to let the resultant flow-through exit from the drainage system.

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How Very Low Intensity Magnetic Torque Can Overcome Angular Momentum from Molecular Thermal Agitation in Biological Systems: A Possible Classical Approach to the kT Problem

Mini Review

For several decades we have been discussing possible harmful effects of manmade electromagnetic fields (EMFs) on human health, without being able to accurately highlight the existence of an intensity threshold (or frequency, or both) of EMFs not to be exceeded. However, it is a matter of fact that a huge number of studies on several effects on biological systems of EMFs emitted by human activities at very low intensities and regardless of the frequency of the field have been published so far. Furthermore, the fact that these effects have been significantly detected and quantified even on very simple organic systems such as proteins and cells leave no doubt as to the existence of such effects [1- 5]. Nevertheless, some researchers have pointed out that these effects do not find any theoretical explanation despite having been observed experimentally. In this regard, I would like to begin by citing the paper of Pierre and Dobson [6] in which it is expressly reported that the Lorentz force exerted on ions passing through cells membrane channels due to an applied EMF is negligible with respect to the forces that are needed to the activation of ions gating channel. In that paper the authors considered Na+ ion moving in cell channel at velocity v under a magnetic field (MF) B. Following Lorentz’s law, this charge should be subjected to the force F = qv× B .

The authors highlighted that a MF of 3 x 108 T is needed to induce a force of 1 pN and even considering ion velocity of 102 m s-1, a MF of 105 T would be necessary, which is several orders of magnitude larger than any magnetic field on Earth! Another study on the apparent contradiction between experimental observations of the effects of EMFs and negative theoretical results is that of Adair [7]. He made a simulation in which Hemoglobin (HB), chosen as protein prototype, is exposed to an EMF with amplitude E = 100 kV/m for Δt = 1 ns (the time integration from Sandia electric pulse, corresponding to the frequency of 900 MHz). The EMF will generate a torque on the protein because its significant dipole moment [8], inducing an angular impulse which has to compete with the mean angular momentum from thermal agitation to which the protein is subjected too. In this simulation, despite of the large value of amplitude of the EMF which was used, the mean angular momentum from thermal agitation resulted significantly larger than the impulse from EMF. Why do theoretical simulations contradict experimental observations? On the other hand, how is it possible that all the experimental results observed in recent years concerning the effects of EMFs are wrong? This apparent contradiction has been named the “kT problem”.

Let we consider macroparticles with a large dipole moment such as charged dust particles, mitochondria and some cells under exposure to an EMF. They will align towards the direction of the applied field: this phenomenon was named in literature “pearl chains” [9-13]. It can be easily explained by classical theory because the large mass of the organic particle gives rise to a large angular impulse that will overhead mean angular momentum from thermal agitation, as it can be easily shown. In contrast, kT problem arises when dealing with particles at the microscopic level such as molecules and ions. In this case, classical physics cannot longer be used, but a quantum approach should be used [14-23]. Indeed, light particles such electrons should have large wavelengths so that they can move from one side of a potential-energy barrier to the other side, despite of not having enough kinetic energy to overhead the barrier. However, in the second simulation above reported a macromolecule was taken in account so that even using classical physics a correct explanation should be found.

That is, it is a situation which is a cross between an atomic particle and a macroparticle, so that a semiclassical approach could apply. Instead, in the simulation reported above it seems that this cannot be done only because an adequate exposure time to EMF was not taken in account. For instance, in the first simulation reported above the integration time was not really considered and a constant intensity of MF was assumed. Nevertheless, even if the MF intensity has been assumed to be constant, the effects due to the applied MF should also depend on exposure time. Even if the torque induced by the applied MF is not intense enough to produce a displacement of the organic particle, however it will produce a very small displacement that will change the initial conditions from which the subsequent action of the force will begin in the following instants of time, In such a way that these imperceptible shifts will add up as the particle is exposed. An illustrative example of this situation can be represented by a (constant) force produced by the impact of a drop of water on a rock that does not produce any visible effect. However, the persistence of this action over time will produce considerable macroscopic effects on the rock that seem inexplicable by the single event, but which are instead produced by the sum of many events of this type. Regarding the second simulation above reported, instead, a time interval given by Sandia electric pulse Δt = 1 ns was taken in account, corresponding to the EMF frequency of 900 MHz. This choice depends on having considered a typical high frequency (HF) EMF. In this scenario the protein exposed to the EMF rotates from one side to the other following the frequency of the field, returning to the initial position after 1 ns, so that the average displacement is zero and no effect induced by the EMF could be explained.

However, this reasoning is correct if the particle rotates in a vacuum, whilst if it is embedded in a medium it cannot follow the EMF HF oscillation due to the inertia of particle and to the friction induced by the viscous damper of the medium in which the molecule is embedded. Hence, the particle exposed to the EMF will undergo a non-zero displacement correlated to the exposure time and a time interval larger than that of Sandia electric pulse should be taken in account. In the study published in [24] a macrodipole represented by the α-helix of typical proteins was considered to test this model, explaining the rotation of proteins α-helices and their alignment along the direction of the applied EMF observed by FTIR spectroscopy. This scenario can explain all the effects of exposure to EMFs of biological systems observed so far. Indeed, α- helix is present in all biological membrane channels and constitutes the walls of these channels [25-27], so that an applied EMF at very low intensity could not induce the rotation of these α-helices considering an integration time of the order of Sandia pulse, due to the significant binding forces in membrane channels.

In contrast, the impulse of the magnetic force due to an applied EMF calculated for the duration of exposure time can induce a torque on the α-helix causing a little increase in the diameter of cellular membrane channel [28-30]. This increase doesn’t have to be huge. A small variation is enough to produce an alteration in the flow of ions through cell membrane channel, causing alterations in cellular functions and the consequent effects that researchers have so far observed. Otherwise, it is true that the α-helix displacement must also compete with thermal molecular agitation, as Adair showed in his study [7]. However, even in this sense, considering an integration time comparable to a typical exposure time, it is easy to demonstrate that the magnetic torque exceeds the angular momentum from molecular thermal agitation [29,30]. A further study to explain experimental results of exposure to EMF by classical theory was carried out by the author and reported in [31], in which the competition between the mechanical moment due to a MF applied to HB protein and the diffusion quantity which is due to thermal molecular agitation was quantified. The complete equation of motion of a molecule subjected by both actions is given by

in which f(φ) is a distribution function and Φ is the rotational diffusion coefficient. The first two terms of this equation are due to the equation of diffusion process, the last term represents the quantity due to the mechanical moment and ω is the angular velocity of the molecule that can be calculated by the term

in which mH is the mechanical moment, FH is the force induced by the MF, r is the radius of the molecule and d = r sinφ is the distance between the application point of FH and the center of the molecule [32]. At the equilibrium, the torque of MF should balance thermal agitation so that we should have

The solution of this equation is given by

and the number of molecules that are rotated by an angle φ with respect to the direction of the field is given by

in which N is the total amount of paramagnetic molecules per unit of mass, μ is the molecular magnetic permeability and H is the applied MF [32].

The ratio of the numbers of molecules rotated with respect to the direction of the MF by two different angles φ1 and φ2 at the corresponding temperatures T1 and T2 is given by:

The parameters in the study of [31] were used to test the modeling above reported. As a result, the terms 2kBT1 and 2kBT2 can be neglected in comparison to μH and it can be assumed that cos φ1 = cos φ2 given that the displacement of molecules at the temperature T1 should be close to that at the temperature T2.

Finally, Eq. (6) can be written 

so that the ratio between the number of molecules that are oriented along the direction of the applied MF by an angle φ is inversely proportional to the ratio between the temperatures. Such as showed in previous literature concerning FTIR spectroscopy, the intensity of the vibration band Amide I should be proportional to the number of proteins α-helices aligned with the direction of the applied MF. The ratios between the Amide I integrated area ratios of exposed/unexposed HB samples and some couple of temperatures T1 and T2 were computed in [31], providing the result that these values coincide with those obtained from Eq. (7), proving that the alignment of α-helix with the direction of an applied EMF is inversely proportional to the temperature. Above all, it was proved that the rotation of α-helix is not impeded by the Brownian motion due to thermal molecular agitation despite of the low intensity of MF used in [31], in agreement with the theoretical classical approach above reported.

Conclusion

Previous studies have highlighted that the effects caused by exposure of some organic systems to man-made electromagnetic fields at low intensities cannot be explained theoretically because angular momentum from molecular thermal agitation of surrounding medium is found to exceed the torque induced by EMF. Some authors assumed that a different approach by quantum physics is needed to solve this problem, in particular if we consider particles at atomic or subatomic size scales such as electrons or ions crossing cell membrane. However, if larger scales are considered, such as macromolecules like proteins, then classical physics can also be able to explain experimental results observed after exposure to man-made electromagnetic fields.

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