Combination Of AgNPs Research Articles

In vitro and in vivo antimicrobial activity of combined therapy of silver nanoparticles and visible blue light against Pseudomonas aeruginosa.

Silver nanoparticles (AgNPs) have been used as potential antimicrobial agents against resistant pathogens. We investigated the possible therapeutic use of AgNPs in combination with visible blue light against a multidrug resistant clinical isolate of Pseudomonas aeruginosa in vitro and in vivo. The antibacterial activity of AgNPs against P. aeruginosa (1×105 colony forming unit/mL) was investigated at its minimal inhibitory concentration (MIC) and sub-MIC, alone and in combination with blue light at 460 nm and 250 mW for 2 hours. The effect of this combined therapy on the treated bacteria was then visualized using transmission electron microscope. The therapy was also assessed in the prevention of biofilm formation by P. aeruginosa on AgNP-impregnated gelatin biopolymer discs. Further, in vivo investigations were performed to evaluate the efficacy of the combined therapy to prevent burn-wound colonization and sepsis in mice and, finally, to treat a real infected horse with antibiotic-unresponsive chronic wound. The antimicrobial activity of AgNPs and visible blue light was significantly enhanced (P<0.001) when both agents were combined compared to each agent alone when AgNPs were tested at MIC, 1/2, or 1/4 MIC. Transmission electron microscope showed significant damage to the cells that were treated with the combined therapy compared to other cells that received either the AgNPs or blue light. In addition, the combined treatment significantly (P<0.001) inhibited biofilm formation by P. aeruginosa on gelatin discs compared to each agent individually. Finally, the combined therapy effectively treated a horse suffering from a chronic wound caused by mixed infection, where signs of improvement were observed after 1 week, and the wound completely healed after 4 weeks. To our knowledge, this combinatorial therapy has not been investigated before. It was proved efficient and promising in managing infections caused by multidrug resistant bacteria and could be used as an alternative to conventional antibiotic therapy.

Synergistic Effect of Silver Nanoparticles with Neomycin or Gentamicin Antibiotics on Mastitis-Causing &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt;

Objective: Mastitis is one of the most costly diseases in dairy cows, which greatly decreases milk production. Use of antibiotics in cattle leads to antibiotic-resistance of mastitis-causing bacteria. The present study aimed to investigate synergistic effect of silver nanoparticles (AgNPs) with neomycin or gentamicin antibiotic on mastitis-causing Staphylococcus aureus. Materials and Methods: In this study, 46 samples of milk were taken from the cows with clinical and subclinical mastitis during the august-October 2015 sampling period. In addition to biochemical tests, nuc gene amplification by PCR was used to identify strains of Staphylococcus aureus. Disk diffusion test and microdilution were performed to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Fractional Inhibitory Concentration (FIC) index was calculated to determine the interaction between a combination of AgNPs and each one of the antibiotics. Results: Twenty strains of Staphylococcus aureus were isolated from 46 milk samples and were confirmed by PCR. Based on disk diffusion test, 35%, 10% and 55% of the strains were respectively susceptible, moderately susceptible and resistant to gentamicin. In addition, 35%, 15% and 50% of the strains were respectively susceptible, moderately susceptible and resistant to neomycin. According to FIC index, gentamicin antibiotic and AgNPs had synergistic effects in 50% of the strains. Furthermore, neomycin antibiotic and AgNPs had synergistic effects in 45% of the strains. Conclusion: It could be concluded that a combination of AgNPs with either gentamicin or neomycin showed synergistic antibacterial properties in Staphylococcus aureus isolates from mastitis. In addition, some hypotheses were proposed to explain antimicrobial mechanism of the combination.

Combination of Silver Nanoparticles and Curcumin Nanoparticles for Enhanced Anti-biofilm Activities.

Biofilm tolerance has become a serious clinical concern in the treatment of nosocomial pneumonia owing to the resistance to various antibiotics. There is an urgent need to develop alternative antimicrobial agents or combination drug therapies that are effective via different mechanisms. Silver nanoparticles (AgNPs) have been developed as an anti-biofilm agent for the treatment of infections associated with the use of mechanical ventilations, such as endotracheal intubation. Meanwhile curcumin, a phenolic plant extract, has displayed natural anti-biofilm properties through the inhibition of bacterial quorum sensing systems. The aim of this study was to investigate the possible synergistic/additive interactions of AgNPs and curcumin nanoparticles (Cur-NPs) against both Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) microorganisms. The combination of AgNPs and Cur-NPs (termed Cur-SNPs) at 100 μg/mL disrupted 50% of established bacterial biofilms (formed on microtiter plates). However, further increase in the concentration of Cur-SNPs failed to effectively eliminate the biofilms. To achieve the same effect, at least 500 μg/mL Cur-NP alone was needed. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) revealed that combination therapy (Cur-SNPs) was the most potent to eradicate preformed biofilm compared to monodrug therapy. These agents are also nontoxic to healthy human bronchial epithelial cells (BEAS2B).

Silver nanoparticles modulate ABC transporter activity and enhance chemotherapy in multidrug resistant cancer

The emergence of multidrug resistant (MDR) cancer phenotypes dramatically attenuates the efficiency of antineoplastic drug treatments often leading to the failure of chemotherapy. Therefore there is an urgent need to engineer new therapeutically useful agents and propose innovative approaches able to defeat resistant cancer cells. Although the remarkable anti-cancer features of silver nanoparticles (AgNPs) have already been delineated their impact on MDR cancer has never been investigated. Herein, we report that AgNPs have notable anti-proliferative effect and induce apoptosis mediated cell death both in drug sensitive and in MDR cancer cells. Furthermore we show evidence that AgNPs exert an inhibitory action on the efflux activity of MDR cancer cells which feature could be exploited to enhance drug accumulation. We verified synergistic interactions of AgNPs with six different antineoplastic agents on drug resistant cells which emphasizes the excellent potential of AgNPs as combinational partners in the chemotherapy of MDR cancer. From the Clinical EditorThe treatment of cancer often fails due to the development of multidrug resistant (MDR) cancer cells. Hence, novel approaches are being investigated to combat drug resistant cancer cells. One particular method studied here uses silver nanoparticles (AgNPs). The authors showed that AgNPs had anti-proliferative effect and ?exerted an inhibitory action on ABC transporter. The findings could suggest the possible use of AgNPs in combination with other chemotherapeutic agents in the clinical setting.

Characterization of silver nanoparticles synthesized using Urtica dioica Linn. leaves and their synergistic effects with antibiotics

In continuation of the efforts for synthesizing silver nanoparticles (AgNPs) by green chemistry route, here we report a facile bottom-up ‘green’ route for the synthesis of AgNPs using aqueous leaves extract of Urtica dioica (Linn.). The synthesized AgNPs were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FTIR), Zeta-sizer and Zeta-potential, Scanning electron microscopy (SEM), Energy dispersive X-ray (EDX) spectroscopy, Transmission electron microscopy (TEM) and Selected area electron diffraction (SAED). The results obtained from various characterizations revealed that AgNPs were in the size range of 20–30 nm and crystallized in face-centered-cubic structure. The antibacterial activity against Gram-positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium) bacterial pathogens was demonstrated by synthesized nanoparticles. Further, synergistic effects of AgNPs with various antibiotics were evaluated against above mentioned bacterial pathogens. The results showed that AgNPs in combination with antibiotics have better antibacterial effect as compared with AgNPs alone and hence can be used in the treatment of infectious diseases caused by bacteria. The maximum effect, with a 17.8 fold increase in inhibition zone, was observed for amoxicillin with AgNPs against S. marcescens proving the synergistic role of AgNPs. Therefore, it may be used to augment the activities of antibiotics.

Cobweb as novel biomaterial for the green and eco-friendly synthesis of silver nanoparticles

In this study, spider cobweb as a novel biomaterial was used for the green synthesis of silver nanoparticles (AgNPs). The synthesized AgNPs were characterized using UV–Vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy. The efficacy of biosynthesized particles as antibacterial agents was evaluated using multi-drug resistant clinical bacterial isolates through sensitivity testing with AgNPs and combination of AgNPs with some selected antibiotics. In addition, the potential application of the particles as additives in paints was demonstrated using some bacterial and fungal isolates. The synthesized AgNPs which were dark brown in color displayed maximum absorbance at the wavelength of 436 nm. It was observed that the reaction mixture of 1:40 (extract:AgNO3 solution) at pH of 8.5 produced particles with maximum absorbance at 436 nm. The FTIR spectrum showed peaks at 3298, 2359, 2089, and 1635 cm−1, indicating that proteins were the capping and stabilization molecules in the synthesis of AgNPs. The particles were spherical in shape with size ranging about 3–50 nm. The energy-dispersive X-ray analysis showed the presence of silver as the most prominent metal, while the selected area electron diffraction pattern conformed to the face-centered cubic phase and crystalline nature of AgNPs. The AgNPs inhibited the growth of several bacterial isolates including S. aureus, E. coli, Klebsiella granulomatis and P. aeruginosa in the range of 10–17 mm at concentration of 100 µg/ml. It was also demonstrated that AgNPs potentiated the activities of augmentin, ofloxacin and cefixime in the AgNP–antibiotic synergy studies. Similarly, the inclusion of AgNPs as additive in white emulsion paint led to the total inhibition of growth of E. coli, P. aeruginosa, Aspergillus niger and A. fumigatus. To the best of our knowledge, this is the first report of the use of cobweb for the green synthesis of AgNPs. The immense antimicrobial activities of the particles can be explored in the creation of novel products, where it can be used as additive to protect materials against microbial attack.

Bacteriagenic silver nanoparticles: synthesis, mechanism, and applications.

Silver nanoparticles (AgNPs) have received tremendous attention due to their significant antimicrobial properties. Large numbers of reports are available on the physical, chemical, and biological syntheses of colloidal AgNPs. Since there is a great need to develop ecofriendly and sustainable methods, biological systems like bacteria, fungi, and plants are being employed to synthesize these nanoparticles. The present review focuses specifically on bacteria-mediated synthesis of AgNPs, its mechanism, and applications. Bacterial synthesis of extra- and intracellular AgNPs has been reported using biomass, supernatant, cell-free extract, and derived components. The extracellular mode of synthesis is preferred over the intracellular mode owing to easy recovery of nanoparticles. Silver-resistant genes, c-type cytochromes, peptides, cellular enzymes like nitrate reductase, and reducing cofactors play significant roles in AgNP synthesis in bacteria. Organic materials released by bacteria act as natural capping and stabilizing agents for AgNPs, thereby preventing their aggregation and providing stability for a longer time. Regulation over reaction conditions has been suggested to control the morphology, dispersion, and yield of nanoparticles. Bacterial AgNPs have anticancer and antioxidant properties. Moreover, the antimicrobial activity of AgNPs in combination with antibiotics signifies their importance in combating the multidrug-resistant pathogenic microorganisms. Multiple microbicidal mechanisms exhibited by AgNPs, depending upon their size and shape, make them very promising as novel nanoantibiotics.

Biologically synthesized silver nanoparticles enhances antibiotic activity against Gram-negative bacteria

Here we report a simple, fast, cost-effective, and nonpolluting approach for synthesis of silver nanoparticles (AgNPs) using leaf extract of Typha angustifolia. We demonstrate the dose-dependent antibacterial activity of AgNPs and different antibiotics against Escherichia coli and Klebsiella pneumoniae. Furthermore, we demonstrate the efficacy of AgNPs in combination with various broad-spectrum antibiotics against E. coli and K. pneumoniae. The results show that combinations of antibiotics and AgNPs show significant antimicrobial effects at sub-lethal concentrations of the antibiotics. These data suggest that combinations of antibiotics and AgNPs can be used therapeutically for the treatment of infectious diseases.

Co-spinning of Silver Nanoparticles with Nisin Increases the Antimicrobial Spectrum of PDLLA: PEO Nanofibers.

Silver nanoparticles (AgNPs), synthesized using N,N-dimethylformamide (DMF), were electrospun with nisin in a 50:50 blend of 24% (w/v) poly(D,L-lactide) (PDLLA) and poly(ethylene oxide) (PEO). Addition of AgNPs decreased the average diameter of the nanofibers [silver nanofibers (SF)] from 588±191 to 281±64nm, or to 288±63nm when nisin was co-spun with AgNPs. Nanofibers containing AgNO3 (SF) had a beads-on-string structure, whereas nanofibers with AgNPs and nisin [silver plus nisin nanofibers (SNF)], nanofibers with only nisin [nisin nanofibers (NF)], and nanofibers without AgNPs and nisin [control nanofibers] had a uniform structure. The irregular topography was confirmed by atomic force microscopy. No interactions occurred between silver, nisin, PDLLA, and PEO, as confirmed with Fourier transform infrared spectroscopy. Most of the AgNPs (18±2.8ppm) and nisin (78.1±1.2µg/ml) were released within the first 2h. SF and SNF inhibited the growth of gram-positive and gram-negative bacteria, whereas NF failed to inhibit gram-negative bacteria. A wound dressing with broad-spectrum antimicrobial activity may be developed by the incorporation of nanofibers containing a combination of AgNPs and nisin.

Enhanced antibacterial and anti-biofilm activities of silver nanoparticles against Gram-negative and Gram-positive bacteria.

Silver nanoparticles (AgNPs) have been used as antibacterial, antifungal, antiviral, anti-inflammtory, and antiangiogenic due to its unique properties such as physical, chemical, and biological properties. The present study was aimed to investigate antibacterial and anti-biofilm activities of silver nanoparticles alone and in combination with conventional antibiotics against various human pathogenic bacteria. Here, we show that a simple, reliable, cost effective and green method for the synthesis of AgNPs by treating silver ions with leaf extract of Allophylus cobbe. The A. cobbe-mediated synthesis of AgNPs (AgNPs) was characterized by ultraviolet-visible absorption spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Furthermore, the antibacterial and anti-biofilm activity of antibiotics or AgNPs, or combinations of AgNPs with an antibiotic was evaluated using a series of assays: such as in vitro killing assay, disc diffusion assay, biofilm inhibition, and reactive oxygen species generation in Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus, and Streptococcus pneumonia. The results suggest that, in combination with antibiotics, there were significant antimicrobial and anti-biofilm effects at lowest concentration of AgNPs using a novel plant extract of A. cobbe, otherwise sublethal concentrations of the antibiotics. The significant enhancing effects were observed for ampicillin and vancomycin against Gram-negative and Gram-positive bacteria, respectively. These data suggest that combining antibiotics and biogenic AgNPs can be used therapeutically for the treatment of infectious diseases caused by bacteria. This study presented evidence of antibacterial and anti-biofilm effects of A. cobbe-mediated synthesis of AgNPs and their enhanced capacity against various human pathogenic bacteria. These results suggest that AgNPs could be used as an adjuvant for the treatment of infectious diseases.

Catalytic and synergistic antibacterial potential of green synthesized silver nanoparticles: Their ecotoxicological evaluation on Poecillia reticulata

In the present study, stable silver nanoparticles (AgNPs) were fabricated at a rapid rate from leaf extract of medicinally important plant Alstonia macrophylla. Biosynthesized AgNPs are of spherical shape and narrow size (70nm), exhibiting a surface plasmon resonance peak at 435nm, and a zeta potential of -30.8 mV and have a crystalline nature. A diverse biochemical consortium of protein, terpenoids, phenolics, and flavonoids in leaf extract of A. macrophylla was found to be responsible for AgNP synthesis as evidenced from qualitative-quantitative chemical analysis and Fourier transform infrared spectroscopy studies. Nitroaromatic compounds are anthropogenic pollutants with long-lasting environmental persistence and are needed to transform into less toxic derivatives. 4-Nitrophenol and p-nitroaniline were reduced to less hazardous and commercially useful 4-aminophenol and p-phenylenediamine by phytosynthesized AgNPs. Rate constants of 0.052 and 0.040Min(-1) were calculated for 4-nitrophenol and p-nitroaniline reduction, respectively. Thin-layer chromatography also confirms the reduction of these nitroaromatic compounds. Combinational studies could be one of the strategies to overcome microbial resistance to antibiotics. In synergistic antibacterial assay, the highest increase in a fold area of 3.84 was reported against Staphylococcus aureus using a combination of AgNPs with penicillin. Biosynthesized AgNPs were found to be less toxic (LC50 = 9.13 ppm) than chemically synthesized AgNPs having a LC50 value of 2.86 ppm against nontarget fish Poecillia reticulata. Our green nanosynthesis method offers a faster rate of formation of stable AgNPs having antibacterial and catalytic potential with lower environmental toxicity.

Mycosynthesis and characterization of silver nanoparticles and their activity against some human pathogenic bacteria

The aim of this study was to biosynthesis silver nanoparticles from the fungus Nigrospora sphaerica isolated from soil samples and to examine their activity against five human pathogenic strains of bacteria viz. Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus using disc diffusion method. The synergistic effect of silver nanoparticles in combination with commonly used antibiotic Gentamycin against the selected bacteria was also examined. The synthesized silver nanoparticles from free-cell filtrate were characterized by using UV-Vis spectrophotometer analysis, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). UV-Vis spectrophotometer analysis showed a peak at 420 nm indicating the synthesis of silver nanoparticles, FTIR analysis verified the detection of protein capping of silver nanoparticles while SEM micrographs revealed that the silver nanoparticles are dispersed and aggregated and mostly having spherical shape within the size range between 20 and 70 nm. The synthesized silver nanoparticles exhibited a varied growth inhibition activity (15-26 mm diam inhibition zones) against the tested pathogenic bacteria. A remarkable increase of bacterial growth inhibition (26-34 mm diam) was detected when a combination of silver nanoparticles and Gentamycin was used. A significant increase in fold area of antibacterial activity was observed when AgNPs in combination with Gentamycin was applied. The synthesized silver nanoparticles produced by the fungus N. sphaerica is a promising to be used as safe drug in medical therapy due to their broad spectrum against pathogenic bacteria.

Synthesis of Silver Nanoparticles and Incorporation with Certain Antibiotic Using Gamma Irradiation

Aims:&nbsp;The study was explaining that silver nanoparticles (AgNPs) were synthesized biologically by&nbsp;Bacillus megaterium&nbsp;culture supernatant (as reducing and stabilizing agents) by the optimization of medium components for nitrate reductase production to enhance the synthesis of AgNPs. And use of gamma irradiation for the synthesis and incorporation of AgNPs with selected antibiotics at distinct dose.Place and Duration of Study:&nbsp;The study was carried out in 2012 in the Department of Drug Radiation Research, Egyptian Atomic Energy Authority.Methodology:&nbsp;The optimized conditions for AgNPs formation by&nbsp;B. megaterium&nbsp;culture supernatant were as follows; media containing: (%) yeast extract: 0.15, peptone: 0.25, KNO3:0.1 temp: 30ºC and incubation period 24 h with maximum nitrate reductase activity of (680.89U/ml). Physical synthesis of AgNPs and incorporation with antibiotics such as (Sodium Cefotaxime, Gentamycin sulphate and Amoxicillin) by γ-rays doses such as (0.5, 1, 1.5, 2, 2.5 and 3 kGy) were studded. AgNPs were characterized by (UV-Vis.), (DLS), (FT-IR) and (TEM) analysis. Combined and individual antibacterial activities of Amoxicillin and AgNPs were investigated against different pathogenic bacterial species by measuring the (ZOI) and by determining the (MIC).Results:&nbsp;This method shows that Aqueous Ag+&nbsp;ions were reduced to AgNPs when added to the cell-free supernatant of&nbsp;B. megaterium&nbsp;this is indicated by the color change from whitish yellow to brown and the control showed no color change. In physical method Amoxicillin was incorporated with AgNPs perfectly at 2.5kGy. The decreasing order of the average antibacterial activity against bacterial group was observed to be AgNPs &gt; Amoxicillin &gt; Amoxicillin + AgNPs.Conclusion:&nbsp;The radiation-induced AgNPs synthesis is a simple, clean which involves radiolysis of aqueous solution that provides an efficient method to reduce metal ions.&nbsp;B. megaterium&nbsp;was found to be an effective biological tool for the extracellular biosynthesis of AgNPs. The bactericidal activity have proved that AgNPs in combination with amoxicillin kill bacteria at such low concentrations (units of ppm), which do not reveal acute toxic effects on human cell, in addition to overcoming resistance, and lowering cost when compared to conventional antibiotics.

Enhanced effect of silver nanoparticles combined with thermotherapy on rat C6 glioma cells

Objective To investigate the effect of silver nanoparticles (AgNPs) on rat glioma C6 cells upon the combination of hyperthermia treatment (HTT).Methods The polyol process and transmission electron microscopy (TEM) were used to synthesize the AgNPs.Methylthiazol tetrazolium (MTT) assay was used to analyze the cytotoxicity of AgNPs.The enhance effect of AgNPs in combination with HTT was detected by using colony-forming assay and TEM.Results AgNPs were synthesized and the mean size was 15 nm.The particles showed dose-dependent cytotoxicity on C6 cells from the experimental data [50％inhibitory concentration (IC50):4 mg/L].Survival rate in heating (44℃C 15 min) combined with AgNPs group was 10.5％,and that in the control group was up to 39.7％.Conclusion AgNPs could increase the effect of thermotherapy on rat C6 glioma cells by inducing cytotoxicity and enhancing the thermosensitivity. Key words: Gliomas ; Silver nanoparticles; Hyperthermia

Silver nanoparticles: a novel radiation sensitizer for glioma?

Malignant gliomas are the most common primary intracranial tumors with a dismal prognosis. Previous investigations by our group demonstrated the radiosensitizing effect of silver nanoparticles (AgNPs) on glioma cells in vitro. The goal of the present study was to evaluate the efficacy of intratumoral administration of AgNPs in combination with a single dose of ionizing radiation at clinically relevant MV energies for the treatment of C6 glioma-bearing rats. AgNPs (10 or 20 μg/10 μl) were stereotactically administered on day 8 after tumor implantation. One day after AgNP injection, rats bearing glioma received 10 Gy radiation. The mean survival times were 100.5 and 98 days, the corresponding percent increase in life spans was 513.2% and 497.7%, and the cure rates were 41.7 and 38.5% at 200 days for the 10 and 20 μg AgNPs and radiation combination groups, respectively. In contrast, the mean survival times for irradiated controls, 10 and 20 μg AgNPs alone, and untreated controls were 24.5, 16.1, 19.4, and 16.4 days, respectively. Furthermore, a cooperative antiproliferative and proapoptotic effect was obtained when gliomas were treated with AgNPs followed by radiotherapy. Our results showed the therapeutic efficacy of AgNPs in combination with radiotherapy without apparent systemic toxicity, suggesting the clinical potential of AgNPs in improving the outcome of malignant glioma radiotherapy.

Murraya koenigii-mediated synthesis of silver nanoparticles and its activity against three human pathogenic bacteria

Synthesis of silver nanoparticles (Ag NPs) by the leaf extract of Murraya koenigii (Indian curry leaf tree) is reported in this study. The colour of the leaf extract prepared by grinding turned from green to brown after treatment with AgNO3 (1 mM). The UV–visible spectroscopic analysis showed the absorbance peak at about 420 nm, which indicates the synthesis of Ag NPs. Further characterisation by Fourier transform infrared spectroscopy showed the presence of proteins as capping agents, which increase the stability of Ag NPs in the colloids. Scanning electron microscopy demonstrated the presence of spherical Ag NPs in the range of 40–80 nm. The bactericidal activity of the standard antibiotics was significantly increased in the presence of Ag NPs against pathogenic bacteria, viz., Escherichia coli-JM-103 (ATCC 39403), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (MTCC 424). Ag NPs in combination with gentamicin showed the maximum activity against E. coli (increase in fold area −4.06), foll...