Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3β, is a protein kinase that phosphorylates Pah1 on serine  and threonine  residues. Enzymological characterization of Rim11 showed that its Km for Pah1 (0.4 μM) is similar to those of other Pah1-phosphorylating protein kinases, but its Km for ATP (30 μM) is significantly higher than those of these same kinases. Furthermore, we demonstrate Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased ∼2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PA phosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites  indicated that Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis.Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in  Saccharomyces cerevisiae, Pah1 is a Mg2+-dependent phosphatidate (PA) phosphatase (PAP) catalyzing the conversion of PA to diacylglycerol (DAG) (de novo synthesis of membrane phospholipids (In the yeast ol (DAG)   regulatory circuit  motif are essential for catalytic activity  lie in the same plane (B), suggesting the importance of their alignment on the membrane surface for the enzyme to recognize its substrate (B).The two conserved domains  of Pah1 , 28 are activity , 28, 60 activity . In addiactivity  located activity , 61. Trpactivity , 61. Accactivity , Trp637 me plane B, suggesubstrate B.Figure Pah1 is easily degraded by the 20S proteasome when it is dephosphorylated (or unphosphorylated) but stable against the proteasomal degradation when it is phosphorylated , 64. An A). Many of those sites have been identified as target residues for protein kinases such as Cdc28-cyclin B . The protein kinase–target site relationship is important to understand the role of a specific phosphorylation in the regulation of Pah1 for its location, PAP activity, and protein stability , its phosphorylated form  showed a decrease in electrophoretic mobility. To better determine the mobility shift, Pah1 was resolved in the gel containing Phos-tag, a molecule that traps phosphorylated proteins. In the Phos-tag gel, unphosphorylated Pah1 migrated as a single discrete band , whereas the protein phosphorylated by Rim11 showed multiple bands of slower migration , indicating heterogeneous phosphorylation of the protein. Compared with the Rim11-phosphorylated Pah1, the protein phosphorylated endogenously in yeast showed more bands of slower electrophoretic mobility , indicating that it is highly and heterogeneously phosphorylated in the cell.To examine Pah1 phosphorylation by Rim11, we utilized the protein purified from its heterologous expression in in yeast , 61. We 32P]ATP into the protein. In the assay, the 32P-labeled Pah1 was resolved from [γ-32P]ATP by SDS-PAGE and then quantified by phosphorimaging (B). Phosphopeptide mapping of the 32P-labeled Pah1 indicated that its phosphorylation by Rim11 occurs on multiple sites (C), and phosphoamino acid analysis showed that its phosphorylation occurs on the serine and threonine residues with the former being a major target site .Although the electrophoretic mobility is useful to assess the phosphorylation state of Pah1, it is not as sensitive and quantitative as enzyme assays  that mearimaging B. Phosphle sites C, and phget site D, right.D, right).The phosphorylation sites of Pah1 were identified by LC-MS/MS analysis of phosphopeptides derived from its digestion with trypsin, chymotrypsin, and Glu-C . Based oFig. 4A and B). In addition, the Rim11 activity was dependent on the concentrations of Pah1 and ATP (C and D). The Vmax and Km values of Rim11 for Pah1 were 169 units/mg and 0.4 μM, respectively, and those for ATP were 253 units/mg and 30 μM, respectively. At the point of maximum phosphorylation, Rim11 catalyzed the incorporation of 2 mol of phosphate/mol of Pah1. Overall, these enzymological properties demonstrate that Pah1 is a bona fide substrate of Rim11.The Rim11 protein kinase was further characterized with Pah1 as substrate. The enzyme activity was dependent on reaction time and the amount of Rim11, indicating that the protein kinase follows zero order kinetics , A and B and ATP , C and DE. coli-expressed Pah1 was phosphorylated by Rim11 and then measured for its PAP activity with respect to the surface concentration of PA in Triton X-100/PA-mixed micelles (n = 3.4) with respect to PA  and PAP activity with the inhibitory effect stronger at the higher concentrations of PA. The Vmax value (1.2 μmol/min/mg) of the phosphorylated Pah1 was 1.8-fold lower than that of its unphosphorylated form. However, the Km value (4.5 mol %) of the phosphorylated enzyme for the substrate PA was not significantly different from that of the unphosphorylated form. These kinetic data indicate that the Rim11-mediated inhibition of Pah1 PAP occurs by decreasing its catalytic efficiency.We examined the PAP activity of Pah1 phosphorylated by Rim11. The micelles , 76. Undmicelles , the PAPct to PA . The VmaXXXS/T by another protein kinase  Pah1 was phosphorylated first by Pho85-Pho80 with nonradioactive ATP and then by Rim11 with [γ-32P]ATP. The measurement of radioactive Pah1 showed that its phosphorylation by Pho85-Pho80 stimulated (∼2-fold) the subsequent phosphorylation by Rim11 XXXS/T byby Rim11 . In the by Rim11 . In thisby Rim11 , 75, wasby Rim11 ; phosphoE. coli-expressed Pah1 was phosphorylated by Rim11 with [γ-32P]ATP to synthesize a radiolabeled substrate. The 32P-labeled Pah1 was mixed with the Nem1-Spo7 complex reconstituted in phospholipid vesicles, and the phosphatase activity was scored by the release of 32Pi from the radioactive protein  of Pah1 phosphorylation by Rim11 and examined the physiological consequences of the phosphorylation deficiency. The phosphorylation-deficient alleles of PAH1  were expressed on a low-copy plasmid in the pah1Δ and pah1Δ nem1Δ strains. The pah1Δ nem1Δ mutant, which lacks the Nem1 protein phosphatase catalytic subunit . In addition, we examined whether the localization of Pah1 is affected by the lack of its phosphorylation by Rim11. As described previously . The TAG content was not significantly affected by the Rim11 phosphorylation-deficient mutations. When wildtype Pah1 was expressed in pah1Δ nem1Δ cells, TAG accounted for only 2% of the total lipids (B). This result confirms previous work showing that the dephosphorylation of Pah1 by the Nem1-Spo7 protein phosphatase complex is crucial for its function in TAG synthesis (pah1Δ nem1Δ cells expressing the Rim11 phosphorylation-deficient mutant forms of Pah1 was 1.4- to 2-fold higher when compared with that of the cells expressing the wildtype Pah1 (B). This result indicates that the lack of Pah1 phosphorylation on Thr163, Thr164, and Ser602 partially bypasses the requirement of the Nem1-Spo7 complex for its localization to the membrane.As the effect of Pah1 PAP activity on TAG content is pronounced in stationary phase cells , 11, 17,d lipids A. The TAl lipids B. This rynthesis , 80. Theype Pah1 B. This rRIM11 were grown in SC medium from the exponential to the stationary phase, and the cell extracts were analyzed for Rim11 abundance by Western blotting with anti-protein A antibody . Considering that one protein kinase is not sufficient to properly phosphorylate Pah1, the employment of multiple protein kinases provides a mechanism to ensure its phosphorylation for the Nem1-Spo7-dependent regulation. In this regard, Ser602 is shown to be a site whose phosphorylation is secured by Rim11, casein kinase I Km value XXXS/T XXXS/T  are involved in the control of Pah1 function is a question that needs to be addressed to better understand its regulation by phosphorylation.Rim11 phosphorylates Pah1 mainly on Ser602, which is one of the seven sites required for its membrane translocation by Nem1-Spo7 protein phosphatase complex. Accordingly, Pah1 deficient in the phosphorylation site bypasses the requirement of Nem1-Spo7 as indicated by its partial functional role in the S. cerevisiae strain BY4741-RIM11-TAP were purchased from Thermo Fisher Scientific. Phos-tag Acrylamide AAL-107 was purchased from Wako Chemicals.Chemicals were reagent grade. Lipids and silica gel GHL TLC plates, respectively, were from Avanti Polar Lipids and Analtech. Bio-Rad was the supplier of DNA size ladders, molecular mass protein standards, and reagents for electrophoresis and Western blotting. Cayman Chemical was the source of leupeptin and pepstatin. Clontech supplied carrier DNA for yeast transformations. Growth media were obtained from Difco Laboratories. Expedeon was the source of InstantBlue (Coomassie blue) protein stain. GE Healthcare was the source of Q-Sepharose, IgG-Sepharose, PVDF membrane, and the chemifluorescence Western blotting detection kit. Invitrogen supplied mouse anti-phosphoglycerate kinase antibodies . Millipore Sigma was the source of ATP, bovine serum albumin, cellulose TLC plates, chymotrypsin, phosphoamino acid standards, protease and phosphatase inhibitors, Ponceau S stain, L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin, Triton X-100, rabbit anti-calmodulin binding protein epitope tag antibody , rabbit anti-protein A antibody , and alkaline phosphatase–conjugated goat anti-mouse IgG antibodies . Scintillation counting supplies were from National Diagnostics. Q5 site-directed mutagenesis kit and other reagents for DNA manipulations were from New England Biolabs. Radiochemicals were purchased from PerkinElmer Life Sciences. Qiagen supplied DNA gel extraction and plasmid purification kits and the nickel-nitrilotriacetic acid agarose resin. Pierce mass spectrometry grade proteases and strong anion exchange spin columns, alkaline phosphatase–conjugated goat anti-rabbit IgG antibody , and S. cerevisiae strain W303-1A was the source of the RIM11 gene and used for the construction of the rim11Δ mutant SKY001 and for the expression of plasmid-borne RIM11. BY4741-RIM11-TAP was the source of the RIM11-TAP gene that was cloned into pYES2, a galactose-inducible overexpression plasmid, for the construction of pSK001. E. coli strains DH5α and BL21(DE3)pLysS were used for the propagation of plasmids and heterologous expression of His6-tagged Pah1, respectively.The yeast and bacterial strains used in this study are listed in PAH1 (T163A/T164A), PAH1 (S602A), and PAH1 (T163A/T164A/S602A) were generated from pGH315 using the Q5 Site-directed mutagenesis kit. The RIM11 gene was amplified by PCR with the addition of NotI/XhoI sites from the W303-1A genomic DNA and inserted into the NotI/XhoI sites of plasmid pRS415 to construct pSK003. Plasmid pSK001 was generated by inserting the RIM11-TAP sequence, which was PCR amplified from BY4741-RIM11-TAP DNA, into pYES2 at the KpnI/NotI sites. The rim11Δ mutant strain SKY001 was constructed from W303-1A by homologous recombination of the rim11Δ::KanMX cassette that was PCR amplified from the yeast mutant collection. Standard methods were used for isolation of chromosomal and plasmid DNA, for digestion and ligation of DNA, and for PCR amplification of DNA  using a spectrophotometer. Yeast cells carrying a plasmid were grown at 30 °C in synthetic drop-out medium, which lacks a specific amino acid from synthetic complete (SC) medium for the plasmid selection. Cells of strain W303-1A harboring plasmid pSK001 were inoculated into 250 ml of SC-Ura/2% glucose medium to a final cell density of A600 ∼ 0.1 and then grown to saturation. The saturated culture was harvested by centrifugation at 1500g for 10 min and the cell pellet was resuspended (A600 ∼ 0.4) in 2 L of SC-Ura/1% raffinose/2% galactose medium and incubated for 14 h (A600 ∼ 1.0) with shaking at 250 rpm. The E. coli cells were grown at 37 °C in lysogeny broth (LB) medium ; ampicillin (100 μg/ml) was added to select for cells carrying plasmids. For His6-tagged Pah1 expression, the bacterial cells harboring plasmid pGH313 were grown to A600 ∼ 0.5 at room temperature in 500 ml of LB medium containing ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml); expression was induced for 1 h with 1 mM isopropyl-β-D-thiogalactoside  buffer containing 0.3 M sucrose, 10 mM 2-mercaptoethanol, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin. Glass beads (0.5 mm diameter) were added to cell suspensions and then subjected to five repeats of 1-min burst and 2-min cooling using a BioSpec Products Mini-Beadbeater-16 (g for 10 min to separate unbroken cells and cell debris (pellet) from cell extracts (supernatant). The cell extract was centrifuged at 100,000g for 1 h to separate the cytosol (supernatant) from the membrane (pellet). The membrane fraction was resuspended in the same volume of cell disruption buffer.All steps were performed at 4 °C. Yeast cultures were harvested by centrifugation at 1500eater-16 . The dis6-tagged Pah1 expressed in E. coli was purified from cell extracts by affinity chromatography with nickel-nitrilotriacetic acid-agarose  buffer containing 150 mM NaCl, 1 mM EDTA, Roche EDTA-free protease inhibitors, and 2% Triton X-100 and centrifuged at 100,000g for 1 h. The supernatant was applied to a 0.5-ml IgG-Sepharose column equilibrated with 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 0.5 mM EDTA, and 0.1% Triton X-100. The column was washed with 20 column volumes of the equilibration buffer and then incubated at room temperature for 1 h with 75 units of tobacco etch virus protease in 0.5 ml 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 0.5 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol. The protein A–free Rim11 was eluted from the IgG-Sepharose resin with the chromatography buffer. The tobacco etch virus protease was removed from the Rim11 preparation by passage through a strong anion exchange spin column equilibrated with 20 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl and 10% glycerol. Rim11 was then eluted from the spin column by increasing the NaCl concentration in the equilibration buffer to 250 mM. Purified Rim11 preparations were stored at −80 °C.All steps were performed at 4 °C unless otherwise indicated. The galactose-induced yeast culture of W303-1A expressing the TAP-tagged Rim11 was harvested, and the cell pellet was resuspended in 16 ml of 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 1 mM EDTA, and Roche EDTA-free protease inhibitors and lysed with glass beads using a Mini-Beadbeater-16 (5 repeats of 1-min burst with 2-min cooling between bursts). The cell lysate was centrifuged at 15006-tagged Pho85-Pho80 complex was purified from E. coli BL21(DE3) cells that expressed plasmids EB1164 and EB1076 by affinity chromatography with nickel-nitrilotriacetic acid-agarose , 0.25 μM Pah1, 2 mM dithiothreitol, and the indicated amount of Rim11. The phosphorylation reaction was terminated by the addition of 6.7 μl 4x Laemmli sample buffer, followed by SDS-PAGE , and the extent of phosphorylation was quantified by ImageQuant software.Rim11 protein kinase activity was measured at 30 °C by following the incorporation of radioactive phosphate from ATP as described by Carman and Lin  and [γ-32P]ATP  for 2 h at 30 °C with Rim11 (300 ng) or Pho85-Pho80 (1.7 μg). The protein kinase and nucleotide were removed from the phosphorylated Pah1 by use of a strong anion exchange spin column  with the accession number MSV000088038. All other data are contained within the article or the supporting information.Raw MS phosphorylation data and database search results for Pah1, along with the Rim11 analysis data are deposited in the MassIVE repository (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


For thoracolumbar (TL) major curvatures, T improved from 37.2° ± 10.7° to 18.8° ± 9.4° (p < 0.01) and TL from 49.0° ± 6.4° to 20.1° ± 8.5° (p < 0.01). Major curve inclinometer measurements and SRS-22 domains, except activity, improved significantly (p ≤ 0.05). At the latest FU, one (2%) patient required fusion of the T curve and revision of the TL tether due to curve progression in the previously uninstrumented T curve and tether breakage (TB) in the TL. Twenty (41%) patients experienced TB. VBT in AIS patients with limited remaining skeletal growth resulted in satisfactory clinical outcomes at the latest FU.Vertebral Body Tethering (VBT) is a non-fusion surgical treatment for Adolescent Idiopathic Scoliosis (AIS) that elicits correction via growth modulation in skeletally immature patients. VBT after peak height velocity is controversial and is the subject of this study. A retrospective review of Risser 3–5 AIS patients treated with VBT, and min. 2-year FU was performed. Pre to post-op changes in clinical outcomes were compared using Student’s t-test or the Mann-Whitney test. A total of 49 patients met criteria, age 15.0 ± 1.9 years, FU 32.5 ± 9.1 months. For thoracic (T) major curvatures, T curvature improved from 51.1 ± 6.9° to 27.2° ± 8.1° ( Vertebral body tethering (VBT) is a non-fusion surgical option for the treatment of adolescent idiopathic scoliosis (AIS). It was approved by the FDA via the Humanitarian Device Exemption (HDE) pathway and is currently indicated for use in skeletally immature patients for whom growth modulation occurs via the Heuter-Volkmann principle. There is an increasing body of literature supporting the use of VBT; however clinical outcomes have been less predictable than those following posterior spinal fusion (PSF), the current gold standard for AIS. This appears due, in part, to the challenge posed in determining ideal surgical timing. In a cohort of 17 young patients (16 with open triradiate cartilage and 1 closed) Newton et al. reported a low clinical success rate of 10/17  with a 41% revision rate in all patients .Clinical success can be defined by improvement of radiographic parameters, pain reduction, improved body image and durability of outcomes. Patient and family preferences and goals must be taken into account when recommending a procedure, acknowledging attributes and drawbacks as well as gaps in knowledge of a particular procedure and approach ,4. Some Unfortunately, a paucity of research on clinical outcomes in VBT for patients who are nearing, or at, skeletal maturity means there is a need to further elucidate clinical indications, ideal surgical timing, and limits of the procedure ,10,11,12An IRB-approved retrospective chart review was performed for VBT cases performed between 2016 and 2019. Inclusion criteria included AIS patients undergoing VBT, a minimum 2-year follow-up, and Risser 3–5. The VBT procedure was performed with the Zimmer Dynesys implant. All families and patients were informed of the off-label use of the implant. The thoracic curve implantation was performed as described previously . The thoClinical, radiographic, and SRS-22 outcomes were collected at the pre-operative visit and at latest follow-up. Hand radiographs for Sanders staging were not routinely available. The proximal humerus ossification system (PHOS) was used to assess skeletal maturity at baseline from standard scoliosis PA radiographs, in addition to Risser staging ,15. CoroDescriptive statistics were calculated for all variables. Normality was assessed using a Kolmogorov-Smirnov test. Pre-operative data were compared to latest radiographic, clinical, and SRS-22 follow-up data. Student t-tests and non-parametric Mann-Whitney tests were used for comparisons.A total of 49 consecutive patients treated with VBT were included . The meaPatients with major thoracic versus thoracolumbar curvatures were assessed separately .p < 0.01). Improvement was also seen in both the compensatory proximal thoracic curvature from 25.0 ± 8.2° to 16.5 ± 7.3°  and compensatory thoracolumbar curvature from 37.2 ± 10.7° to 19.2 ± 6.8° . There were significant improvements in 3D T5-T12 kyphosis restoration from 6.3 ± 10.8° to 22.5 ± 9.1° at latest follow-up (p < 0.01). Thoracic inclinometer measurements improved from 13.5 ± 3.8° to 7.9 ± 3.5° (p < 0.01) at latest follow-up and thoracolumbar inclinometer measurements improved from 8.1 ± 4.2 to 2.7 ± 1.8 (p < 0.01). The Fulcrum Bending Correction Index (FBCI) for thoracic major curves was 92.6 ± 37.7%. When thoracic curves were separated by magnitude at latest follow-up ≤ 30° (n = 15/24 (63%)) were compared to those that were >30° (n = 9/24 [38%]), pre-operative flexibility rate was 59.1% ± 18.8% versus 55.7% ± 18.1%, respectively. Furthermore, FBCI was 102.0 ± 39.1% in the clinically successful group compared to 77.0 ± 31.3% in the non-clinically successful group.For those with thoracic major curvatures, the mean preoperative Cobb angle was 51.1 ± 6.9° corrected to 27.2 ± 8.1° at latest follow-up  were compared to those with bilateral instrumentation (n = 9). Compared to patients with bilateral instrumentation, patients with only their major thoracic curve instrumented saw similar improvement in curve magnitudes  and similar rates of major curve correction . Preoperatively, those with bilateral instrumentation had significantly larger compensatory thoracolumbar curves than their single-curve counterparts  and a subsequently larger percent correction . Thoracolumbar compensatory curve magnitude at latest follow-up was similar .Patients with major thoracic instrumentation only (p < 0.01). There was also significant improvement in the compensatory thoracic curvature from 37.2 ± 10.7° to 18.8 ± 9.4° . T5-T12 kyphosis did not significantly change, correcting from 23.3 ± 12.1 to 27.3 ± 16.4 (p = 0.34). Similarly, T12-S1 lumbar lordosis was maintained remaining at 55° between baseline and follow-up (p = 0.89). Thoracic inclinometer measurements were relatively stable  but thoracolumbar rotation decreased from 16.3 ± 4.4° to 4.6 ± 2.7° (p < 0.01). When thoracolumbar curves were separated by magnitude at latest follow-up ≤ 30° (n = 23/25 (92%)) compared to those that were > 30° (n = 2/25), the pre-operative flexibility rate was 78.8 ± 15.2% versus 72.0 ± 17.4%. Furthermore, FBCI was 76.4 ± 2.7% in the clinically successful group compared to 69.2 ± 9.1% in the non-clinically successful group.For those with thoracolumbar major curvatures, the mean preoperative Cobb angle was 49.0 ± 6.4°corrected to 20.1 ± 8.5° at latest follow-up  were compared to patients with bilateral instrumentation (n = 7). Compared to patients with bilateral instrumentation, patients with only their major thoracolumbar curve instrumented saw similar pre-operative to post-operative curve magnitudes  and similar rates of correction in their main thoracolumbar curves . Those with bilateral instrumentation had significantly larger compensatory thoracic curves than their single-curve counterparts preoperatively  but with similar percent correction  and magnitude of the final thoracic compensatory curve .Patients with major thoracolumbar instrumentation only (p = 0.87) or Risser staging distribution (p = 0.06). Those in the clinically unsuccessful group had significantly larger preoperative major Cobb magnitude  as well as less preoperative flexibility of the major curve although this did not reach statistical significance .There were 37/49 (76%) patients who were deemed clinically successful and had residual major curves ≤ 30°. Of those, 22/25 (88%) had thoracolumbar major curves while 15/24 (63%) had thoracic major curves. Sub-analysis was performed comparing clinically successful patients to those who were not. There was no difference between groups in age (There were two major complications in this cohort. One patient developed late onset superior mesenteric artery syndrome (SMAS) 1 year following her VBT procedure requiring Ladd’s derotation surgery. The second patient required revision to posterior fusion of the compensatory thoracic curvature 3.1 years after the index procedure due to curve progression in the uninstrumented thoracic curve and tether breakage in the thoracolumbar curve. The tether was revised with a second row of screws and double cord.There were 20 (41%) patients who showed evidence of cord breakage. Of those 20 patients, three individuals showed evidence of cord breakage at two levels .p = 0.01). While one patient with a broken tether was revised to PSF and counted amongst the treatment failure cohort, the other 19/20 (95%) patients with tether breakage still performed at the same rate as those with tethers that did not break; that is, of the broken tethers, 4/19 (21%) did not meet criteria for clinical success which was similar to the 7/29 (24%) of patients with intact tethers .When stratified by major curve, thoracolumbar curves with broken tethers demonstrated a greater decline in percent correction, from 63.6 ± 15.7% to 46.6 ± 12.6%, than thoracic curves, which declined from 48.9 ± 13.5% to 41.6% ± 18.6%  of participants. In those participants, there were significant improvements in all SRS domains except for activity scores .p < 0.01). Patient-reported outcomes improved with all SRS-22 domains except activity. When analyzing groups by success or failure, the age and Risser staging did not differ; however, the major Cobb angle in the unsuccessful group was about 55°, while it was closer to 49° in the successful group, which suggests that for slightly larger curves, extra care must be taken to assure sufficient tension is applied to the construct. Differences in curve flexibility were seen but did not reach statistical significance. The successful group had about 12% greater flexibility, and given the biological plausibility, one can imagine that with a larger sample size, flexibility may be a significant determinant of clinical success [The performance of VBT in patients after peak height velocity is controversial, and is the subject of this study. Our data provide preliminary support for its use in select skeletally mature patients. Of the 49 patients in this study, 37 (76%) remain clinically successful with major curvature less than 30 degrees at mean latest follow-up of 32.5 months (range 23–64). This includes 19 patients with broken tethers at the time of latest FU. For patients with both major thoracic  and major thoracolumbar  curves, there was a significant improvement in major Cobb magnitude (n = 11/29 patients) and found a 74% clinical success rate  and a 7% conversion to fusion rate [VBT is indicated for skeletally immature individuals with the potential for growth modulation based on health regulatory organization guidance. However, ideal candidates must not be too skeletally immature, as this has been associated with an increased incidence of overcorrection and reduced outcome predictability ,11. On tion rate . Comparan = 7) and, as noted by the authors, the need for longer follow-up. Furthermore, they did not report on Sanders 8 patients. Unfortunately, we did not have hand X-rays of many of our patients, preventing an analysis by Sander stage. All our patients were Risser 3–5, with 75% being Risser 4 or 5 and 98% had PHOS stage of 3B-5. The PHOS system is more granular than Risser and spans the entire continuum of growth, as opposed to Risser staging. Our patients, thus, can be assumed to largely have surpassed their peak height velocity. Similarly, others have reported intraoperative correction rates ranging from 40% to 76% [p = 0.80). At the senior author’s institution, all patients and their families were advised of the risks, benefits, and unknown outcomes associated with VBT for all patients including the mature patient. The following points are emphasized for all patients considering VBT: spinal arthrodesis is the standard of care, good and reliable intermediate outcomes are achieved, the long-term outcomes of VBT are unknown, the procedure has been predicated on remaining growth, the likelihood that tether breakage will occur and may result in significant loss of correction along with back pain, and the possible need for revision. Families often state they do not want a procedure that permanently alters their child’s spine, and prefer one that leaves options for future innovation. One such innovation is the development of stronger tethers that have the potential for prolonged durability. Furthermore, the controversial nature of performing VBT in skeletally mature individuals is also highlighted. Only after extensive discussion did the families proceed with the procedure. Until additional guidance is available from basic science and preclinical research, patients and providers will need to base their decisions on knowns and unknowns alike.It has been previously reported that significant correction is achievable intraoperatively at the index operation via VBT ,21. In a% to 76% . Given tWhether or not holding the scoliotic spine straight for a prolonged period can result in sufficient spinal remodeling, to maintain correction even with tether breakage, is currently unknown, but is not without some evidence. Earlier studies in small mammals showed that remodeling of both immature and mature vertebrae is achievable through mechanical loading. For example, McBride et al. imposed a 30° scoliotic curve in rat tails and reported that the resultant vertebral wedging in immature rats was largely through asymmetric growth, exhibiting the Hueter-Volkmann law. They also reported that wedging was observed in mature rat vertebrae, and that this was largely due to diaphyseal remodeling, similar to Wolff’s Law ,23. MoreUltimately, families are counseled in a shared-decision-making paradigm about the fact that the basic science case for performing this procedure and subsequent regulatory clinical approval are based on remaining growth, and that long term outcomes are lacking. All patients and families are encouraged to consider the gold standard fusion and, in appropriately indicated cases, posterior dynamic correction .Limitations of the current study included the lack of Sanders staging as it is the most commonly reported skeletal maturity indicator spanning the period prior to the adolescent growth spurt through skeletal maturity. This was not routinely performed for our patients previously given it requires an additional hand radiograph that was not routinely collected to reduce radiation exposure. However, to address this, PHOS staging was provided for granular skeletal maturity assessment. Numbers were not sufficient to stratify outcomes based on Risser stage 3, 4, or 5. This will be the subject of future studies. Future study is also required to better elucidate predictors of clinical success in VBT for skeletally mature patients. Our sub-analysis was limited due to the sample size of those who were unsuccessful but best results were seen in smaller and more flexible curves.While the VBT procedure is currently indicated for skeletally immature patients for whom growth modulation occurs via the Hueter-Volkmann principle, we provide preliminary data for its use in patients past their peak height velocity.


Natural killer (NK) cells are a subset of lymphocytes that offer great potential for cancer immunotherapy due to their natural anti-tumor activity and the possibility to safely transplant cells from healthy donors to patients in a clinical setting. However, the efficacy of cell-based immunotherapies using both T and NK cells is often limited by a poor infiltration of immune cells into solid tumors. Importantly, regulatory immune cell subsets are frequently recruited to tumor sites. In this study, we overexpressed two chemokine receptors, CCR4 and CCR2B, that are naturally found on T regulatory cells and tumor-resident monocytes, respectively, on NK cells. Using the NK cell line NK-92 as well as primary NK cells from peripheral blood, we show that genetically engineered NK cells can be efficiently redirected using chemokine receptors from different immune cell lineages and migrate towards chemokines such as CCL22 or CCL2, without impairing the natural effector functions. This approach has the potential to enhance the therapeutic effect of immunotherapies in solid tumors by directing genetically engineered donor NK cells to tumor sites. As a future therapeutic option, the natural anti-tumor activity of NK cells at the tumor sites can be increased by co-expression of chemokine receptors with chimeric antigen receptors (CAR) or T cell receptors (TCR) on NK cells can be performed in the future. Solid tumors are considered as complex tissues that develop through genetic and epigenetic changes in tumor cells and restructuring of the tumor microenvironment (TME) ,2,3. TheUnmodified, blood-borne, cytokine-activated NK cells have been used for adoptive transfer in patients suffering from melanoma, renal cell carcinoma and other cancer types for many years . NK cellTo enhance the cytotoxic potential and redirect NK cells more efficiently towards tumor cells, NK cells have been previously equipped with CARs ,29. HoweTo alter the migration of NK cells, some studies overexpressed the NK cell chemokine receptor CXCR4 or variants thereof on NK cells to enhance migration to the bone marrow ,45. ExprHowever, whether receptors that are typically not found on the NK cell lineage, such as CCR2B or CCR4, can be expressed on NK cells and redirect them towards CCL2 or CCL17/22, respectively, has not been investigated before. These further modifications can potentially improve the anti-tumor properties of NK cell therapies, especially for the treatment of solid malignancies.The chemokine receptors CCR4 and CCR2B were cloned from human peripheral mononuclear cells into a lentiviral expression vector. Thereby, the CCR2 isoform CCR2B was isolated and used throughout all experiments, as CCR2B is the predominant isoform expressed on monocytes ,53. β-actin as reference gene. At the mRNA level, CCR4 expression was increased 2.5-fold compared to T regs and CCR2B expression was increased 3-fold compared to monocytes -enveloped lentiviral particles encoding CCR4 or CCR2B. Naturally, NK-92 cells express neither CCR4 nor CCR2 A,D. Howeonocytes B,E, in lonocytes C,F. The chemotactic activity of the genetically modified cells was examined using a Boyden chamber migration assay . ChemotaNext, we tested if the transduced receptor may have stimulatory or adverse effects on NK-92 cell receptor expression or cytotoxicity. To this end, we incubated CCL2 with CCR2B-NK-92 and control cells, as well as CCL22 with CCR4-NK-92 and control cells, for 16 h and then analyzed an array of NK cell receptors A. We didTherefore, we concluded that the overexpression of CCR4 and CCR2B selectively changes migratory behavior of NK-92 cells without affecting their ability to lyse tumor cells.γ-retroviral instead of a lentiviral system for the gene delivery of the chemokine receptors into the IL-2 and IL-15 activated peripheral blood NK cells. Viral supernatants were used either fresh or from frozen stocks for the different NK cell donors. Transduction efficiencies are shown in Although NK-92 cells are under clinical investigation and FDA-approved, we investigated whether the genetic modification with these two chemokine receptors could also be applied to peripheral blood NK cells, as these have superior properties for later clinical application . Neither+/CD16− NK cells within transduced and untransduced NK cells  were grown in DMEM  with 10% fetal calf serum (FCS) . NK-92 cells  were cultured in RPMI1640 , supplemented with 20% FCS  and 500 U/mL IL-2 .All cells were grown at 37 °C with 5% COg for 30 min using a Ficoll-Paque  gradient. The PBMC fraction was transferred in a sterile 50 mL tube with a 10 mL serological pipette. Primary NK cells were then purified by negative selection using NK cell isolation kit according to the manufacturer’s instructions . After isolation, 0.5 × 106 cells were cultured per well in a 24-well plate in 500 µL NK Medium , 5% Human Serum , 500 U/mL IL-2 and 140 U/mL IL-15 ). The purity of NK cells after isolation was determined by flow cytometry. All NK cells that were used had a purity greater than 80% on the day of isolation.PBMCs were isolated from buffy coats of five healthy donors bought from the Institute for Transfusion Medicine of the University Clinic in Leipzig. The buffy coat was diluted 1:2 with PBS and centrifugation was carried out at 400× Isolated T regs were kindly provided as frozen aliquots from a single donor by Dr. Anna Dünkel . Monocytes were freshly isolated from three different donors and kindly provided by Dr. Claire Fabian .The sequences of the chemokine receptors CCR2 and CCR4 and their ligands CCL2 and CCL22 were amplified from PMBC cDNA using following primers : CCR2_fw (ATTCTAGAGCCGCCACCATGCTGTCCACATCTCGT), CCR2_VarB_rev (ATGAATTCGAGACGTTATAAACCAGCCGAGACTTCC), CCR4_fw (ATTCTAGAGCCGCCACCATGAA-CCCCACGGATATAG), CCR4_rev (ACTATGAATTCCTACAGAGCATCATGGAGAT), CCL2_fw (ATTCTAGAGCCGCCACCATGAAAGTCTCTGCCGCC), CCL2_rev (ACTATGAATTCTCAAGTCTTCGGAGTTTGGG), CCL22_fw (ATTCTAGAG-CCGCCACCATGGATCGCCTACAGACT), CCL22_rev (ACTATGAATTCTCATTGGCTCAGCTTATTGAG)E. coli TOP10  and incubated on ice for 30 min followed by a 30 s heat shock at 42 °C and 5 min on ice. Then, 150 µL of SOC medium was added and incubated at 37 °C, 750 rpm for 1 h. Afterwards, 50 µL were plated on LB agar plates containing 50 µg/mL Kanamycin. Colonies were grown overnight at 37 °C. Plasmids were isolated using the ZymoPURE MiniPrep Kit according to the manufacturer’s instructions .PCR products were purified using a Zymoclean Gel DNA Recovery kit according to the manufacturer’s instructions . CCR4/CCR2B sequences were cloned in the lentiviral expression vector hEF1α-H2B-mVenus-IRES-mCherry-PGK-Puromycin  and third generation lentiviral plasmids  and γ-retroviral plasmids  containd #99278  protein d #99278  and #122d #99278 , kindly t vector   to the cells. Spinfection was performed at 400× g at 37 °C for 60 min. Afterwards, 0.5 mL of NK-92 or NK medium was added. Transgene expression was determined by flow cytometry 3 days post transduction. NK-92 cells were selected 3 days post transduction using 2 µg/mL puromycin for 2 weeks. Then, cells were used for experiments and aliquots were frozen at different time points. After thawing, cells were cultured for one week, before they were used for experiments. Peripheral blood NK cells were transduced 7 to 11 days after isolation.For transduction of NK-92 cells or peripheral blood NK cells, 2.5 × 10+, CD3/14/19−, CD56+ and CD16+/− cells. NK-92 cells were analyzed for CD56, NTB-A, NKG2D, 2B4, DNAM-1, NKp44, NKp46 and NKp30 expression  and data were analyzed using MACSQuantify  and FlowJo10 . CCR4 and CCR2B expression were determined by cell labeling with anti-CCR4 antibodies see  in a 1:1β-actin as reference gene. At first, RNA was isolated using the Quick-RNA MiniPrep kit according to the manufacturer’s instructions . DNaseI treatment was carried out as recommended. We diluted 1 µg of RNA in 16 µL H2O and 4 µL iScript Reverse Transcription Supermix (NEB) was added to synthesize the cDNA. The following primer pairs were used:To quantify RNA expression, qPCR was performed using bActin_fw_qPCR (CACCATTGGCAATGAGCGGTTC), bActin_rev_qPCR (AGGTCTTTGCGGATGTCCACGT), CCL2_fw_qPCR (AGAATCACCAGCAGCAAGTGTCC), CCL2_rev_qPCR (TCCTGAACCCACTTCTGCTTGG), CCL22_fw_qPCR (TCCTGGGTTCAAGCGATTCTCC), CCL22_rev_qPCR (GTCAGGAGTTCAAGACCAGCCT), CCR2_fw_qPCR (CAGGTGACAGAGACTCTTGGGA), CCR2_VarB_rev_qPCR (CTTCTGAACTTCTCCCCAACGA), CCR4_fw_qPCR (CTCTGGCTTTTGTTCACTGCTGC), CCR4_rev_qPCR (AGCCCACAGTATTGGCAGAGCA) .5 NK-92 (2 × 105 NK) cells in the cell culture inserts at a total volume of 100 µL RPMI (without phenol red) . The inserts were placed in 24-well dishes containing 50 ng/mL of the respective chemokine ) diluted in 600 µL RPMI (without phenol red). After 2 h (NK-92) or 4 h (NK) of incubation at 37 °C and 5% CO2, cells and medium of the lower chamber were harvested and the number of migrated cells was quantified by flow cytometry. Primary NK cells were used 7 days post transduction.A migration assay utilizing PET cell culture inserts with a 6.4 mm diameter and 5 µm pore size was used to assess the migration potential of NK cells. We loaded 2.5 × 104 transduced HEK293T cells into a single well of a 24-well plate in 600 µL RPMI (without phenol red) . The next day, cell culture inserts containing 2.5 × 105 cells in 100 µL RPMI (without phenol red)  were placed in each well. After 4 h of incubation at 37 °C, cell culture inserts were removed and the migrated NK-92 cells were stained with anti-CD56 antibody (1:600) and quantified by flow cytometry.To stimulate the migration towards cells expressing the cognate ligand, HEK293T cells were stably transduced with the chemokines CCL22 or CCL2, respectively. We seeded 2.5 × 10g for 5 min and resuspended in the appropriate volume of RPMI (without phenol red) to a final concentration of 4.2 × 104 cells/mL. Then, 2.5 × 104 transduced HEK293T cells were seeded into a single well of a 24-well plate in 600 µL RPMI (without phenol red). The next day, 33.3 × 104 NK-92 cells in 100 µL RPMI (without phenol red) were added. After 20 h, cells were harvested by pipetting and propidium iodide (PI) was added as a dead cell marker in a 1:200 dilution. Afterwards, living cells were counted by flow cytometry . Specific lysis was calculated by diving each cell count with the count obtained in a control well containing HEK293T cells only.Transduced and non-transduced HEK293T cells were stained with CellTrace Violet  at a working concentration of 1 µM in 1 mL PBS for 20 min at 37 °C protected from light. Then, 5 mL of RPMI (10% FCS) was added and incubated for 5 min to remove remaining dye. Cells were centrifuged at 300× t-test as indicated. For t-test was applied. *: p ≤ 0.05. **: p ≤ 0.01. ***: p ≤ 0.001. ****: p ≤ 0.0001.Statistical analyses were performed with GraphPad Prism 6 software . Statistical significance was determined by applying the unpaired two-tailed


Scientific Reports, 10.1038/s41598-022-12042-6, published on 27 May 2022Correction to: The original version of this Article contained errors in the author list, where Francesco Sirano was incorrectly listed as an author of the original Article, and has subsequently been removed.The Author contributions section now reads:“L.B., G.N., designed research; G.N., I.R.P. and L.B. performed research; P.P. collected samples and cured the bioanthropological and taphonomic aspects of the conceptualization; G.N., I.R.P. and L.B. analysed data; F.D.P., G.M., E.C. and L.B. supervised data analysis; G.N., P.P, G.M., E.C. and L.B. wrote the paper.”Furthermore, the Acknowledgements section in the original version of this Article was omitted. It now reads:“The authors would like to thank Meaghan Mackie at Globe Institute, University of Copenhagen, for her help with sample preparation and LC-MS/MS. Special thanks to the Archaeological Park of Herculaneum and the Archaeological Park of Pompeii  for allowing the study of the bone samples used in this manuscript .”In addition, the Supplementary Information file that accompanies the original Article contained errors, where permission details were omitted. Under the heading ‘Description of skeletal elements’,“The skeletal elements of 15 individuals from the archaeological sites of Pompeii (7), Herculaneum (5) and Baia Scalandrone (3) were analysed. Table S1 shows each specimen and its related information. All necessary permits were obtained for the study of the human specimen from the Ethics Committee for Biomedical Activities, AOU Federico II, Naples, Italy. Protocol N. 101/17.”now reads:“The skeletal elements of 15 individuals from the archaeological sites of Pompeii (7), Herculaneum (5) and Baia Scalandrone (3) were analysed. Table S1 shows each specimen and its related information. All necessary permits were obtained from the Archaeological Park of Herculaneum and the Archaeological Park of Pompeii   and for the study of the human specimen from the Ethics Committee for Biomedical Activities, AOU Federico II, Naples, Italy. Protocol N. 101/17.”Furthermore, in the section ‘Experimental procedure’ of the Supplementary Information file, under the subheading ‘Protein extractions and digestion’,34) with slight modifications.”“Bone samples were prepared as described in (now reads:34) with modifications based on (91).”“Bone samples were prepared as based partly on a method described in  2M extraction solution, 0.5M Tris (2-carboxyethyl) phosphine, 0.5M chloroacetamide, 100 mM Tris (pH 8.0) and water. Finally, the membrane was washed with 300 μl of GuHCl 2M extraction solution that were added to the protein pellet. The pH was adjusted to 8.0 and the mixture of proteins was heated at 80°C for 3 hours under agitation and cooled to room temperature. Without separating supernatants and the residual bone powder, samples were heated to 80 °C for 2 h, and cooled to room temperature LysC-Trypsin mix, 1/100 by amount of protein, was added to the samples for protein digestion. Mixed samples were incubated at 25 °C for 30 min then diluted to 2 M GuHCl with 25 mM Tris (pH 8.0), followed by incubation at 37 °C overnight under agitation. Digestion was terminated with 10% trifluoroacetic acid to a final concentration of 1%. After centrifugation at 14 000 g for 10 min, the tryptic peptides in the supernatant were immobilized on C18 stage tips as previously described (13).”now reads:13).”“The EDTA and Tris fraction were concentrated on an Amicon 3000 Da MWCO spin column, the flow through was discarded, and the retained sample was washed with 3ml of guanidinium hydrochloride (GuHCl) 2M extraction solution containing 0.5M Tris (2-carboxyethyl) phosphine, 0.5M chloroacetamide, 100 mM Tris (pH 8.0) and water. Finally, the membrane was washed but not spun through with 300 μl of the above GuHCl 2M extraction solution which was subsequently added to the protein pellet. The pH was adjusted to 8.0 and the mixture of proteins was heated to 80 °C for 2 h and cooled to room temperature. Protein concentration was estimated by Bradford Assay. LysC, was added (0.2 µg) to the samples for initial protein digestion. Mixed samples were incubated at 37 °C for 90 min then diluted to 0.6 M GuHCl with 25 mM Tris (pH 8.0) in 10% acetonitrile (ACN), followed by incubation at 37 °C overnight under agitation with Trypsin (0.8 µg). Digestion was terminated with 10% trifluoroacetic acid (TFA) to a final concentration of 1%. After centrifugation at 14 000 g for 10 min, the tryptic peptides in the supernatant were immobilized on C18 stage tips as previously described , and detailed below. Bone samples were eluted from the stage tips using 20 μL 40% ACN and then 10 μL 60% ACN into a 96 well MS plate.”“Samples were processed as reported in (now reads:“Samples were processed based on (91) and detailed below. Bone samples were eluted from the stage tips using 30 μL 40% ACN into a 96 well MS plate.”And,“Samples were then separated on a 15 cm column (75 μm inner diameter) in-house laser pulled and packed with 1.9 μm C18 beads  on an EASY-Nlc 1000  connected to a Q-Exactive HF  on a 77 min gradient. Buffer A was milliQ water.”now reads:“5 μl of each sample was then separated on a 15 cm column (75 μm inner diameter) in-house laser pulled and packed with 1.9 μm C18 beads  on an EASY-nLC 1200  connected to a Q-Exactive HF-X  on a 77 min gradient. Buffer A was milliQ water.”Finally,“The Q-Exactive HF was operated in data dependent top 10 mode. Spray voltage was 2 kV, S-lens 162 RF level at 50, and heated capillary to 275 °C. Full scan mass spectra were recorded at a resolution of 120,000 at m/z 200 over the m/z range 350–1400 with a target value of 3e6 and a maximum injection time of 25 ms. HCD-generated product ions were recorded with a maximum ion injection time set to 108 ms and a target value set to 2e5 and recorded at a resolution of 60,000. ”now reads:“The Q-Exactive HF-X was operated in data dependent top 10 mode. Spray voltage was 2 kV, S-lens RF level at 50, and heated capillary to 275°C. Full scan mass spectra were recorded at a resolution of 120,000 at m/z 200 over the m/z range 350–1400 with a target value of 3e6 and a maximum injection time of 25 ms. HCD-generated product ions were recorded with a maximum ion injection time set to 118 ms and a target value set to 2e5 and recorded at a resolution of 60,000 at a scan range of 200-2000 m/z.”The original Article and its accompanying Supplementary Information file have been corrected.Supplementary Information.


CuO/Fe3O4/PANI transmission electron microscopy (TEM) images revealed that most nanoparticles were in a CuO/Fe3O4 hybrid form, with a narrow size distribution uniformly dispersed in a polymer background. The TEM and scanning electron microscopy (SEM) images of the MWCNT/CuO/Fe3O4/PANI nanocomposite revealed that the MWCNT was uniformly coated with CuO/Fe3O4/PANI. All three nanocomposites samples demonstrated superior microwave attenuation performance in terms of reflection loss and absorption bandwidth. The minimum reflection losses for MWCNT/CuO/Fe3O4/PANI nanocomposites (1:3), (1:4), and (1:5) were 45.7, 58.7, and 85.4, 87.4 dB, respectively. The absorption bandwidths (RL ≤ −10 dB) of MWCNT/CuO/Fe3O4/PANI nanocomposites (1:3), (1:4), and (1:5) were 6, 7.6, and 6 GHz, respectively.In this study, novel quaternary MWCNT/CuO/Fe Magnetic materials can also absorb the microwave’s magnetic field by an analogous process. Among the wide range of magnetic materials, magnetite (Fe3O4) is highly attractive for its usage as a magnetic dissipater and improver of the microwave attenuation property of carbon nanotubes or carbon-based materials because of its low toxicity, good compatibility, high unpaired spin density, and strong spin polarization at room temperature5. Carbon/ferrite-based composites exhibit a better balance of electrical conductivity and magnetic susceptibility than individual magnetic or carbonaceous absorbents, making them promising as electromagnetic wave absorbers6. Additionally, numerous reports confirm that combining other metal oxides with magnetite enhances the absorption properties of the resulting hybrid nanoparticles. The incorporation of metal oxide hybrids into magnetic materials results in the development of conduction loss mechanisms, residuals, interfacial polarization, electron spin resonance, and a resonant domain wall, all of which contribute directly to improving microwave absorption properties9. Many reports indicate that hybridized magnetite/metallic or magnetite/semiconductor nanoparticles embedded in a conductive polymer or loaded on carbon materials, especially carbon nanotubes, is an effective strategy in producing microwave absorber composites with sound absorption performance13. Copper oxide (CuO), a semiconductor with a narrow gap, was used to decorate carbon materials such as carbon fibers, carbon black, carbon nanotubes, and graphene to fabricate efficient microwave absorbing composites11. Although the microwave absorption properties of copper oxides (CuO and Cu2O) as a hybrid with magnetite have not been studied, we hypothesized that it could improve the final CuO/Fe3O4 hybrid’s dielectric and magnetic absorption. On the other hand, coating the magnetic surface with conductive polymers with high microwave absorption properties results in improved impedance matching and inhibits the aggregation, corrosion, and magnetic phase transformation of nanoparticles by producing heat during the microwave absorption process.Carbon is widely used in many applications due to its good electrical and thermal conductivity, low density, good corrosion resistance, low thermal expansion, and low elasticity. Carbon is used as a resistive element to convert input microwaves into heat. Due to the electric dipoles’ response within them to the alternating electric field of input waves, most carbon allotropes exhibit excellent microwave absorption capabilities15. Polyaniline can also significantly optimize the impedance matching of EW. Thus, polyaniline can enhance electromagnetic wave absorption via various mechanisms22.The most significant polymer in the category of conductive polymers is polyaniline. It has high electrical conductivity with high polarizability , polarization relaxation, and conductive loss and is used to synthesize highly effective microwave absorber composites. Polymers can also cause a better dispersion of NPs and enhance the interfacial area and multiple interfacial reflections between individual nanoparticles. Polyaniline can uniformly conjugate with CNT’s surface and other substrates by representing surface tension and reactive chemical sites to bind with functional groups of CNTs, which results in a further increment in the interfacial area3O4/PANI nanocomposite using a step-by-step approach, ensuring that the CuO/Fe3O4 hybrid nanoparticles were prepared using the optimal protocol to achieve the desired magnetic properties and size distribution. Then, aniline was polymerized on the surface of CuO/Fe3O4 hybrid NPs via in situ polymerization, forming a CuO/Fe3O4/PANI nanocomposite. The CuO/Fe3O4/PANI nanocomposite was then loaded onto the MWCNT surface in weight ratios of three, four, and five times that of MWCNT, resulting in MWCNT/CuO/Fe3O4/PANI (1:3), (1:4), and (1:5) nanocomposites. The MWCNT/CuO/Fe3O4/PANI composites were characterized via the following methods: An X-ray diffraction pattern was used to identify the crystalline phases of CuO and Fe3O4. Fourier transform infrared (FTIR) confirmed the successful polymerization of aniline on the surface of nanoparticles. The morphology of CuO/Fe3O4/PANI and MWCNT/CuO/Fe3O4/PANI nanocomposites were investigated by SEM and TEM. To this end, SEM and TEM images indicated a uniform distribution of CuO/Fe3O4 nanoparticles embedded in the polymer and a uniform loading of MWCNT with CuO/Fe3O4/PANI nanocomposite. Vibrating sample magnetometer (VSM) analysis presented the superparamagnetic properties of MWCNT/CuO/Fe3O4/PANI nanocomposites with high saturation magnetizations (40–60 emu/g).The present study synthesized a novel quaternary MWCNT/CuO/FeElectromagnetic parameters of nanocomposites were calculated in the range of 8–18 GHz through a vector network analyzer. The reflection losses versus frequency curves were plotted based on electromagnetic parameters for all samples with different thicknesses. The results showed that all samples had a high reflection loss ranging from -50 to -87 dB and a broad absorption band exceeding 6 GHz.3O4/PANI composite with multiple interfaces and weight ratio optimization of each composite component results in a significant improvement in the composite’s microwave absorption property. MWCNT/CuO/Fe3O4/PANI composites have different dielectric permittivity and magnetic permeability interfaces (conductor/semiconductor or conductor/insulator or semiconductor/insulator and non-magnetic/magnetic). The accumulation of localized surface charge at the boundary of two materials with different dielectric constants leads to attenuation of the electric component of the incident wave. Moreover, the skin depth decreases due to creating current density at a non-magnetic/magnetic interface, and the incident wave’s magnetic field deteriorates23.In addition to the usual mechanisms of microwave absorption in magnetic and dielectric materials, the unique design of the MWCNT/CuO/Fe3O4/PANI quaternary composite using a controllable method involving a three-step process. The composite’s constituents were prepared under controlled conditions and then combined to form the final nanocomposite at an optimal weight ratio. First, a hybrid CuO/Fe3O4 nanoparticle was synthesized using the optimal protocol to achieve the best superparamagnetic property. Afterward, nanoparticles were used as an initiator to polymerize aniline and form a CuO/Fe3O4/PANI nanocomposite. Finally, the MWCNT was used as a substrate to carry and stabilize the CuO/Fe3O4/PANI nanocomposite.This study prepared a novel MWCNT/CuO/Fe2.4H2O, FeCl3.6H2O, CuNO3 (purity > 98%), HCl, and NaOH (≥ 97%), were acquired from Sigma Aldrich. Aniline and ammonium persulfate (APS) were obtained from Merck.The MWCNTs were supplied with diameters and lengths ranging from 20 to 40 nm and 5–15 μm, respectively, and a minimum purity of 95%. Other raw materials, including FeClTo obtain a homogeneous solution, 1.51 g copper nitrate and 2.31 g iron oxide were mixed with 40 mL of deionized water and stirred. Then, 0.1 mol NaOH was added dropwise until the pH of the solution reached 13. Finally, the product was collected with a magnet, washed, and dried at 40 °C.3O4/PANI nanocomposite was prepared via in situ polymerization of aniline on the functional groups of CuO/Fe3O4 NPs at a temperature lower than 4 °C in a 20 mL aqueous HCl solution (an oxidizing agent).The CuO/Fe3O4 NPs and 0.75 g of aniline monomer (1:2 ratio of aniline to CuO/Fe3O4) were dissolved in 20 mL aqueous HCl solution. Then, the mixture was exposed to ultrasonic waves for 30 min to disperse the NPs (separate nanoparticles in the clusters). The polymerization reaction must be performed at a temperature less than 4 °C. Afterward, 0.8 g of ammonium persulfate (oxidizing agent) was dissolved in 30 mL of deionized water and added dropwise to the mixture. Adding the first oxidizing droplets leads to the appearance of a green–blue color, indicating the beginning of polymerization. When the polymerization was complete, the products were collected by a magnet, and the solution was washed to remove the residual aniline and reach neutral pH18.First, 1.5 g of prepared CuO/Fe3O4/PANI nanocomposite and 0.1 g of MWCNT were dispersed separately in 100 mL of deionized water (the weight ratio of MWCNT to CuO/Fe3O4/PANI was (1:5)). Subsequently, two suspensions were added together and sonicated for 30 min. The sediment was then separated from the liquid by centrifugation. Finally, the black precipitate was dried in an oven at 50° C. Similar nanocomposites were prepared using weight ratios of MWCNT to CuO/Fe3O4/PANI of 1:4 and 1:316.To prepare the final nanocomposite, 0.5 g of CuO/Fe3O4/PANI nanocomposite was verified by determining its chemical bonding and crystalline planes using FT-IR (Nicolet IS10) and X-ray diffraction (XRD) , respectively. The magnetic behavior of products was investigated at room temperature in the presence of a magnetic field using a vibrating sample magnetometer (VSM JDM-13). The TEM technique was used to study the morphology of CuO/Fe3O4/PANI and MWCNT/CuO/Fe3O4/PANI nanocomposites using MIRA3 TESCAN and Tecnai. The composite’s electromagnetic properties and microwave absorption capability were determined using the transmission/reflection waveguide method on an Agilent vector network analyzer (VNA) E8364B over 8.2–18 GHz.The successful synthesis of MWCNT/CuO/Fe3O4/PANI and MWCNT/CuO/Fe3O4/PANI nanocomposites were prepared by the KBr tablet method. According to Fig. −1 in the CuO/Fe3O4/PANI spectrum is due to the presence of O–H groups and the carboxylic acid (C = O and C–O) functional groups of CNT at 1562 and 1150 cm−1. The absorption band at 1562 cm−1 also corresponds to the tensile vibration of C = N corresponding to the quinoid ring in the polyaniline structure. The characteristic peak of carbon nanotubes at 1638 cm−1 is related to the symmetrical expansion vibration of the C = C bonds. The absorption band at 1479 cm−1 is attributed to the C = C functional groups in the benzenoid rings in the polyaniline structure24. The absorption band at 1296 cm−1 demonstrates the tensile vibration of the C–H bonds, while the absorption band at 1131 cm-1 exhibits the bending vibration of the C-H bonds, which is a characteristic of polyaniline bonds25. The absorption band at 800 cm−1 is associated with the Fe–O functional groups, while the absorption band at 559 cm−1 is linked with the Cu–O functional groups. Peaks in the range of 800 cm−1 are characteristic of the substitution of aromatic rings, which indicates the formation of the polymer26. The absorption band at 3435 cm-1 is related to the tensile vibration of N–H bonds in polyaniline. The absorption band at 1570 cm−1 is due to the tensile vibration of the C = N bonds corresponding to the quinoid ring. The absorption bands at 1119 cm−1 and 1030 cm−1 represent the expansion state of C–N bonds and the bending vibration of C-H bonds, respectively.The FT-IR spectra of CuO/Fe3O4/PANI nanocomposite was determined by the X-ray diffraction pattern. As illustrated in Fig. 3O4). According to JCPDS Card 19–0629, this pattern corresponds to the face center cubic structure (FCC) of Fe3O4, and no additional peaks are observed to indicate the presence of impurities27. The peak of the carbon nanotube was observed at an angle of 25.9°, corresponding to the (002) of graphite’s hexagonal structure26. The peak at 39.4° is associated with the (200) and (111) planes, while the peak at 32.5° is attributed to the (110) plane, which is indexed to CuO’s monoclinic structure (JCPDS Card 48–1548)29.The crystalline structure of MWCNT/CuO/Fe3O4/PANI (1:3) and (1:5). FESEM images of MWCNT/CuO/Fe3O4/PANI (1:3) and (1:5)  and (1:5) nanocomposite, respectively  was performed to determine the type of elements present in a 9 µm2 surface area of MWCNT/CuO/Fe3O4/PANI (1:4) nanocomposites’ thin film  was used to study the morphology of the synthesized powder of MWCNT/CuO/Fe3O4/PANI nanocomposite . Additionally, a few isolated CuO NPs (gray) and Fe3O4 NPs (black) were observed. TEM images of MWCNT/CuO/Fe3O4/PANI nanocomposite (1:3) and (1:5)  in the range of -15,000 to 15,000 Oe and in the saturation magnetization (Ms) range of ± 60 emu/g. A closed-loop of hysteresis was observed for MWCNT/CuO/Fe3O4/PANI nanocomposites (remanences are lower than 5 emu/g and coercivities are below 15 Oe), indicating the presence of superparamagnetic CuO/Fe3O4 NPs. MWCNT/CuO/Fe3O4/PANI saturation magnetization (1:3), (1:4), and (1:5) are 38.61, 47.5, and 60 emu/g, respectively. Magnetic saturation was expected to increase as the weight ratio of magnetic CuO/Fe3O4 NPs increased31.As shown in Fig. 3O4/PANI nanocomposites were calculated to determine their absorption capacity. To this end, each nanocomposite was homogeneously dispersed in paraffin with a 25 wt% filler content. Complex permittivity (ε) and complex permeability (µ) are electromagnetic parameters that are intrinsic to each absorbent material and determine a matter's ability to absorb waves were achieved by a network vector analyzer (Anritsu.37269D) in the 8.2–18 GHz frequency range, presented in Fig. The electromagnetic (EM) parameters of MWCNT/CuO/Fe31. εʺ and μ″, the imaginary parts, are related to polarization relaxation and spin rotation relaxation , respectively. εʺ originated from different polarization such as the electron, nuclei, dipoles, and interfacial polarization relaxation (space accumulated charge polarization). Here, space-charge polarization is the primary factor in the dielectric loss. This is due to additional polarizations at higher frequency ranges32. Interfacial polarization is caused by space-charge accumulation at the interface of materials with different dielectric constants. For conductive materials, εʺ typically originates from conduction loss as well as polarization relaxation33. From Fig. 3O4 in hybrid form, CuO or Fe3O4 in single form and polyaniline, CuO/Fe3O4 and polyaniline, polyaniline and CNT35. Dispersion of nanoparticles within the polymer results in a significant increase in the nanoparticles’ joint surface with the polymer. Additionally, the nanotube’s surface is coated with a polymer layer. The nanotubes’ high surface-to-volume ratio creates a large interface between the nanotubes and the polymer. It is well established that the larger the interface area, the more space charge, and polarization relaxation occurs36.The real part of permittivity and permeability εʹ and µʹ, which show the degree of the polarizability of absorbent material, is a measure of the ability to store electrical and magnetic energy. εʺ and µ″, also known as dielectric and magnetic loss, refer to the attenuation of the incident wave’s electric and magnetic energy within the material, respectively3O4 NPs, causing more incident wave dissipation38. As a result, the nanocomposite (1:5) with the highest amount of CuO/Fe3O4/PANI nanocomposite represents the highest εʺ and dielectric tangent loss (tanδε) at a higher frequency range.The presence of a conductive loss in polyaniline and CNT is another phenomenon that significantly contributes to the εʺ of nanocomposites. The polarization relaxation of the space charge at the NPs/PANI interface, which occurs at high frequencies (> 14 GHz), and the conductive loss of PANI appear to be the dominant mechanisms. In addition, multiple reflections occurred between separated CuO/Fe3O4 NPs in nanocomposites (1:4) and (1:5) than (1:3). µʹ demonstrates the possibility of magnetic dipoles in this regard. Since magnetic dipoles can only occur in magnetic materials with unpaired spin, by increasing the amount of magnetite, µʹ values increased. μ″ is related to spin rotation relaxation that initially corresponds to natural resonance in the ferromagnetic domain wall resonance, occurring at lower frequencies (< 100 MHz), exchange resonance, and eddy current39. our nanocomposites contain CuO/Fe3O4 with single domain superparamagnetic nanoparticles. Hence, exchange resonance and eddy current loss are involved in magnetic dissipation, occurring at high frequencies (> 13 GHz)33. Since ferrites have a relatively high electrical resistance40, they have a low dielectric loss and low eddy current loss. However, by incorporating CuO, a semiconductor with a narrow bandgap, to form a CuO/Fe3O4 hybrid, conductivity and eddy current loss may be increased. An increment in μ″ values and magnetic tangent loss (tanδμ) of nanocomposite (1:5) over 13 GHz was expected. Generally, tanδε is higher than tanδμ for all samples, indicating dielectric loss makes a larger contribution to microwave dissipation which has often been observed41. The relaxation  is an essential dielectric loss mechanism in the absorption of EM waves. The Cole–Cole model can be used to describe the relaxation process .As illustrated in Fig. ess Fig. . The Deb44. The Cole–Cole curve of nanocomposite (1:3) is composed of large and small semicircles and indicates the presence of three relaxation resonances, two of which have long relaxation times and one of which has a short relaxation time. Nanocomposite (1:4) exhibits two large semicircles (with a higher radius than that of nanocomposite (1:3)) and a minute one which indicates two long relaxation times and a short one. Nanocomposite (1:5) shows two medium semicircles and two equal relaxation times. As per the statements above, polarization relaxation is a significant factor in the incident wave’s attenuation. The resonance peaks in the RL diagram depend on these relaxation frequencies. Thus, in nanocomposite (1:4) with the highest relaxation frequency, the minimum reflection loss is located at the lowest frequency, whereas in nanocomposite (1:3) to (1:5) with increasing relaxation frequency, the minimum reflection loss is located at higher frequencies.In all three composites, one or two semicircles were observed, indicating multiple relaxation processes occur for the composites, proving the contribution of the Debye relaxation in increasing the dielectric dispersion of the compositesZin is the input impedance of the microwave absorbing material, Z0 denotes the impedance of free space, j denotes the imaginary unit, d represents the thickness of the absorbent, f is the microwave frequency, c denotes the velocity of light, and RLmin is the reflection loss.The reflection loss values for various sample thicknesses were calculated using the EM parameters:3O4/PANI (1:3), (1:4), and (1:5) versus frequency in the 8.2–18 GHz range. The results imply that all MWCNT/CuO/Fe3O4/PANI samples exhibit excellent MA behavior. The thickness of the absorber has a significant effect on the microwave absorption properties. The RLmin peak gradually moves to a lower frequency with increasing absorbent thickness, which follows the formula min, and d is the sample thickness. For all samples, two strong absorption peaks were observed at a thickness of 3 mm. Except for the thickness of 2.2 and 3 mm, both reflection loss and bandwidth decrease as the sample thickness increases in the nanocomposite (1:3) RLmin curve. A minimum reflection loss of −47.5 dB is achieved at 14.3 GHz with an absorbent thickness of 2.2 mm and an absorption bandwidth of approximately 6 GHz (12.2–18 GHz). For nanocomposite (1:4), the reflection loss decreases by increasing the sample thickness. For thicknesses of 3 mm, minimum reflection losses reach -85.4 and -58.7 dB at 10.6 and 9.8 GHz, respectively, with an effective bandwidth of 3 GHz (8.8–11.8 GHz). The broadest bandwidth is 7.6 GHz (10.4–18 GHz) for 2.2 mm absorbent. RLmin is greater than 87.4 dB at 16.2 GHz in nanocomposite (1:5), with the broadest absorption band of 6 GHz (12–18 GHz) for a 2.2 mm absorbent thickness. Two strong peaks with RLmin −45 and −43 dB appeared at 10.3 and 11.8 GHz, respectively, with the absorption band of 4.4 GHz (9.8–14.2) for a 3 mm absorbent thickness. The absorption bandwidths are greater than 3 GHz for all absorbent thicknesses.Figure Figure The higher values (α) indicate the more powerful the magnetic and dielectric loss is if the microwave can reach the inner space of the absorber. The magnetic and dielectric loss tangent are two key factors influential in the absorption efficiency of an absorber. The higher values of loss tangent indicate the high ability to convert electromagnetic waves to other forms of energy. The attenuation constant was significantly increased in the 10–18 GHz frequency range. Meanwhile, the (1:5) exhibited a larger attenuation constant, which was also well consistent with the results of the RL curves.3O4/PANI nanocomposites, significantly greater than comparable composites reported in Table Several absorption mechanisms discussed in Sect. 3O4/PANI nanocomposite.A multi-step method was prepared for a new quaternary MWCNT/CuO/Fe3O4 hybrid NPs with a narrow size distribution were prepared via a simple co-precipitation route.CuO/Fe3O4 NPs served as initiators for the polymerization of aniline on the surface of the CuO/Fe3O4 NPs, resulting in the formation of a CuO/Fe3O4/PANI nanocomposite.The O–H functional groups on the surface of the CuO/Fe3O4/PANI nanocomposite via simple physical agitation.MWCNTs were loaded with different weight ratios (1:3), (1:4), and (1:5) of CuO/Fe3O4 nanoparticles were dispersed in the polyaniline background with an almost uniform distribution. Moreover, the images demonstrated that MWCNTs were coated with a nearly uniform thickness of CuO/Fe3O4/PANI nanocomposite.The FESEM and TEM images revealed that the CuO/Fe3O4/PANI samples had a superparamagnetic property with a high saturation magnetization ranging from 38 to 60 emu/g.All MWCNT/CuO/Fe3O4/PANI samples exhibited superior absorption properties in terms of reflection loss and absorption bandwidth. Minimum reflection losses were 45, 58.7 and 85.4, 87.4 dB (2.2 mm absorbent) and 80 dB (3 mm absorbent) for MWCNT/CuO/Fe3O4/PANI (1:3), (1:4), and (1:5), respectively. The broadest absorption bandwidths were 6, 7.6 GHz (2.2 mm absorbent), and 6 GHz (2.6 mm absorbent) for MWCNT/CuO/Fe3O4/PANI (1:3), (1:4), and (1:5), respectively.All MWCNT/CuO/Fe


Ocimum basilicum L.) cultivated in the net-house in Vietnam during four months. Ten samples of basil aerial parts were hydrodistilled to obtain essential oils which had the average yields from 0.88 to 1.30% . The oils analyzed using GC-FID and GC-MS showed that the main component was methyl chavicol (87.4–90.6%) with the highest values found in the oils of basil under lighting conditions of 6 h/day and 150–200 µmol·m−2·s−1. Additional lighting conditions caused the significant differences (p < 0.001) in basil biomass and oil production with the highest values found in the oils of basil under two conditions of (1) 71% Red: 20% Blue: 9.0% UVA in at 120 μmol·m−2·s−1 in 6 h/day and (2) 43.5% Red: 43.5% Blue: 8.0% Green: 5.0% Far-Red at 100 μmol·m−2·s−1 in 6 h/day. The oils of basil in some formulas showed weak inhibitory effects on only the Bacillus subtilis strain. Different light spectra affect the biomass and essential oil production of basil, as well as the concentrations of the major components in the oil.This study was performed to investigate the effects of different supplemental light spectra and doses (duration and illuminance) on the essential oil of basil ( Ocimum basilicum L.) is an herb, probably native to the tropical and subtropical regions of India, well known for its aromatic and medicinal properties. This herb contains a distinctive essential oil and is used in both fresh and dried culinary dishes, providing a variety of positive health benefits when consumed. Some of the reports demonstrated that basil has pharmacological effects such as anti-inflammatory D20 = [+] 0.26°  and retention index (RI) data. A total of 20 to 27 compounds representing from 99.1% to 99.9% (by mass intensity) of the compositions were identified in the essential oils of basil cultivated under 10 different supplemental light conditions. These were comprised of dominant benzenoid aromatics ranging from 88.4 to 91.6% of the oils. While monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes of the oils were at very low concentrations.−2·s−1 (formulas F7 and F8), the methyl chavicol concentrations were higher than under the other conditions of lighting time and light intensity ; the main component ranged from 87.4 to 90.6%. The order of methyl chavicol content in basil oils is as follows: F7 = F8 > F10 > F2 = F5 > F3 > F4 = F9 > F6 > F1 . Additiontensity .−2·s−1, which is comparable to the formulas F2 and F5 in the present study with supplemental lighting conditions of 6 h/day  at PPFD 120 µmol·m−2·s−1. In the formulas F7 and F8, the highest methyl chavicol content in basil can be attributed, in addition to lighting duration and illuminance, which is 6 h/day  at PPFD 150–200 µmol·m−2·s−1 -equivalent to DLI 14.45–15.53 mol·m−2·d−1, the composition of the light spectra has a higher ratio of B (43.5%). Its value is in agreement with the results reported by Hosseni (2018) [−2·d−1 (16 h/day and PPFD 250 µmol·m−2·s−1).The high content of methyl chavicol in basil in the formulas F2 and F5 could be influenced by both lighting duration and illuminance. Siposs et al. (2021) and Ahmed et al. (2020) ,31 suggei (2018)  where th−2·s−1 PPFD, day/night cycles of 16/8 h after 30 days), methyl chavicol content in purple basil oil was highest (71.87%) [−2·s−1 reached the highest value (90.6%).Basil oil compositions have been divided into 7 chemotypes: (1) high linalool, (2) linalool/eugenol, (3) methyl chavicol without linalool, (4) methyl chavicol/linalool, (5) methyl eugenol/linalool, (6) methyl cinnamate/linalool and (7) bergamotene chemotypes . Many ea(71.87%) . The preStaphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum, Salmonella enterica, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. The essential oils of basil in 6 formulas from F2 to F7 showed weak inhibitory effects on B. subtilis with respective IC50 values of 6223, 6144, 12,698, 13,995, 5660, and 6349 µg/mL. The IC50 values of the oils on other 6 microorganism strains and all the 7 MIC values of the oils exceeded 16,384 µg/mL. The essential oils of basil grown in 10 different formulas were evaluated for antimicrobial activity against 7 strains: The presence of high concentration of methyl chavicol and low concentration of linalool in the Basil oils in the present study may explain for the weak antimicrobial activity of the oils. A previous study showed that methyl chavicol is devoid of antimicrobial activity . LikewisNo relationship was found between supplemental lighting conditions and the antimicrobial activity of basil essential oil in this study. However, previous studies showed that the antimicrobial activity of essential oils depends on their main compounds. On the other hand, supplemental lighting for basil with different light spectra can alter the content of its oil main compounds. Therefore, in the future, it is possible to find a certain light spectrum/mixture of light spectra that drastically changes the contents of the main compounds in basil essential oil, thereby increasing the oil’s antimicrobial activity.−2·d−1 was determined for experimental time . The supplemental LED lighting was applied after sunset and before sunrise with ten different light formulations as described in The experiments were set up in a net-house in Hanoi, VN  with 40% diffused light transmission being determined using relating photosynthetic photon flux density (PPFD) (400–700 nm) inside to outside the net-house. The basil seeds, purchased from Duc Thang Company in Hanoi, were sown in March 2021, then the seedlings were transplanted in April and a supplemental light experiment was held from May to September 2021. Daylight illuminance in a net-house was measured by the DigiSense Data Logging Light Meter (Model 20250-00), which was converted into Daily Light Integral (DLI) using conversion factors as described in the literature ,44. An aEach basil sample was determined the water content using A&D Weighing AD-4714A General purpose moisture determination balance.Each basil sample consisting of aerial biomass of 15 plants (4.3–7.7 kg) was shredded and hydrodistilled for 4 h using a Clevenger type apparatus according to the previously published procedure  , as well as by comparison of their mass spectral fragmentation patterns with those stored on the MS library NIST08, Wiley09, HPCH1607 [The essential oils were analyzed by GC/MS-FID using an Agilent GC7890A system with Mass Selective Detector (Agilent 5975C). An HP-5MS fused silica capillary column (60 m × 0.25 mm i.d. × 0.25 μm film thickness) was used. Helium was the carrier gas with a flow rate of 1.0 mL/min. The inlet temperature was 250 °C and the oven temperature program was as follows: 60 °C to 240 °C at 4 °C/min. The split ratio was 1:100, the detector temperature was 270 °C, and the injection volume was 1 μL. The MS analysis was carried out at interface temperature 270 °C, MS mode, E.I. detector voltage 1258 V, and mass range 35–450 Da at 1.0 scan/s. FID analysis was carried out using the same chromatographic conditions. The FID temperature was 270 °C. Essential oil constituents were identified by their relative retention indices, determined by co-injection of a homologous series of HPCH1607 ,48  bacteria including Staphylococcus aureus (ATCC 13709), Bacillus subtilis (ATCC 6633) and Lactobacillus fermentum (VTCC N4), 3 strains of Gram (−) bacteria including Salmonella enterica (VTCC), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 15442), 1 strain of yeast Candida albicans (ATCC 10231). The ATCC strains were obtained from American Type Culture Collection; the VTCC strains were obtained from Vietnam Type Culture Collection, Institute of Microbiology and Biotechnology, Vietnam National University, Ha Noi.The MIC and IC50 of the essential oils were measured by the microdilution broth susceptibility assay [14, 213, 212, 211, 210, 29, 27, 25, 23, 21 μg/mL) in sterile distilled water in micro-test tubes, from where they were transferred to 96-well microtiter plates. Bacteria grown in double-strength Mueller-Hinton broth or double-strength tryptic soy broth, and fungi grown in double-strength Sabouraud dextrose broth were standardized to 5 × 105 and 1 × 103 CFU/mL, respectively. The last row, containing only the serial dilutions of sample without microorganisms, was used as a negative control. Sterile distilled water and medium served as a positive control. After incubation at 37 °C for 24 h, the MIC values were determined at the well with the lowest concentration of agents that completely inhibited the growth of microorganisms. The IC50 values were determined by the percentage of microorganisms inhibited growth based on the turbidity measurement data of EPOCH2C spectrophotometer  and Rawdata computer software  according to the following equations:OD: optical density; control (+): only cells in medium without antimicrobial agent; test agent: corresponds to a known concentration of antimicrobial agent; control (−): culture medium without cells. ConcHigh/ConcLow: voncentration of test agent at high concentration/low concentration; Inh%High/Inh%Low: % inhibition at high concentration/% inhibition at low concentration.MIC and ICty assay . Stock s50 and MIC values in the ranges of 0.02–3.62 µg/mL and of 0.125–32.0 µg/mL, Cefotaxime for Gram (−) bacteria with IC50 and MIC values in the range of 0.07–4.34 µg/mL and of 0.5–32.0 µg/mL, Nystatin for fungal strains with IC50 and MIC values of 1.32 µg/mL and 8.0 µg/mL.Reference materials: Ampicillin for Gram (+) bacteria with ICp ≤ 0.05 using IRRISTAT ver. 5.0 .The measurements of physiological parameters and essential oil contents of basil were analyzed by a single factor completely randomized analysis of variance (ANOVA), which compared the lighting treatments. For the significant values, means were separated by the least significant difference (LSD) test at v/w), calculated on a DW basis, were obtained. Methyl chavicol (=estragole) was the unique main component of the oils ranging from 87.4 to 90.6%. Additional lighting gave out effects on the growth and essential oil content of basil, as shown by the significant differences (p < 0.001) in biomass, oil yield and oil production between the lighting formulas. Supplemental light conditions of formulas F2 (71% R:20% B:9.0% UVA at 120 µmol·m−2·s−1 in 6 h/day) and F9 (43.5% R:43.5% B:8.0% G:5.0% FR at 100 µmol·m−2·s−1 in 6 h/day) were the most suitable for the growth of basil that produced the highest biomass  and essential oil productivities (44.03 L/ha and 46.11 L/ha) among all supplementary light conditions of the present study. Additional lighting time and light intensity may have influenced the concentration of the major component of basil essential oil with higher contents of methyl chavicol under conditions of 6 h/day (formulas F2 and F5) and 150–200 µmol·m−2·s−1 (formulas F7 and F8) than under the other conditions. The essential oils from basil in formulas from F2 to F7 showed weak inhibitory effects on only Bacillus subtilis. However, no effect of supplemental lighting conditions on the antimicrobial activity of basil essential oil was recorded. The results of present study can be the basis for future research to promote the increase in biomass yield, production and quality of essential oils of plants.This is the first study to provide the information on the various aspects of basil cultivated under ten different supplemental light spectra and dosages in Vietnam. The average yields of basil essential oils from 0.88 to 1.30% (