We show that Zn2+ ionophore, zinc pyrithione (ZnPy), can prevent or reverse Ca2+/CaM-mediated inhibition of Kv7.2. In the presence of both Ca2+ and Zn2+, the Kv7.2 channels lose most of their voltage dependence and lock in an open state. In addition, we demonstrate that mutations that interfere with CaM binding to Kv7.2 and Kv7.3 reduced channel membrane abundance and activity, but these mutants retained zinc sensitivity. Moreover, the relative efficacy of ZnPy to activate these mutants was generally greater, compared with the WT channels. Finally, we show that zinc sensitivity was retained in Kv7.2 channels assembled with mutant CaM with all four EF hands disabled, suggesting that it is unlikely to be mediated by CaM. Taken together, our findings indicate that zinc is a potent Kv7 stabilizer, which may protect these channels from physiological inhibitory effects of neurotransmitters and neuromodulators, protecting neurons from overactivity.Zinc (Zn) is an essential trace element; it serves as a cofactor for a great number of enzymes, transcription factors, receptors, and other proteins. Zinc is also an important signaling molecule, which can be released from intracellular stores into the cytosol or extracellular space, for example, during synaptic transmission. Amongst cellular effects of zinc is activation of Kv7  voltage-gated potassium channels. Here, we investigated relationships between Kv7 channel inhibition by Ca Plasma ns (ZnT) . In the ing 1 mM . Zn2+ reeceptors . The proocessing , 8. An e+ channels (+ currents with slow kinetics and a very negative activation threshold (negative to −60 mV). These features allow Kv7 channels to remain partially active at voltages near the resting membrane potential of a neuron and strongly influence excitability  Kchannels . The chatability , 11. Kv7tability , 10, 12.tability .via Gq/11-phospholipase C (PLC) signaling cascades. For their activity, Kv7 channels require a cofactor, membrane phosphoinositide, phosphatidylinositol 4,5-bisphoshpate (PIP2) -mediated modulation . One pre (PIP2) , 15. Accte, etc. , 10, 11.rylation  or Ca2+/dulation , 18, 19,2+  is associated with intracellular Zn2+ accumulation and also with Ca2+ influx through ionotropic receptors, such as AMPA, stabilization of Kv7 channel activity by zinc may serve a protective function preventing hyperexcitability.Interestingly, Kv7 channel activity is strongly potentiated by intracellular Zn2+  and zinc2+ . Zinc drembranes . It is yree Zn2+ . Intracehibition , 22, 23.2+ ionophores, such as zinc pyrithione (ZnPy), pyrrolidinedithiocarbamate, diiodo-8-hydroxyquinoline, and some others, augment Kv7 channel currents by delivering free Zn2+ into the cytosol (2+ affects inhibition of Kv7 channels by Ca2+. In perforated patch voltage clamp recordings from Chinese hamster ovary (CHO) cells transiently transfected with KCNQ2 complementary DNA (cDNA), bath application of ZnPy (10 μM) augmented steady-state current amplitude at 0 mV by 1.41 ± 0.04-fold . Zn2+ chelator N,N,N′,N′,tetrakis(2-pyridylmethyl)ethylenediaminepentaethylene , applied in the presence of ZnPy, characteristically reversed the augmentation without inhibiting current amplitude below the basal level (A–C). Although the reversal with 20 μM TPEN was almost complete, there was a degree of variability; at 30 μM TPEN completely reversed ZnPy augmentation (A–C). The reversibility of ZnPy effect by Zn2+ chelator suggests that the active moiety mediating Kv7.2 augmentation is indeed Zn2+. We also tested lower concentrations of ZnPy (D–F), and even at 500 nM, significant augmentation of Kv7.2 current was observed.In our recent study, we demonstrated that Zn cytosol . Here, wal level , A–C. Alentation , A–C. Th of ZnPy , D–F, an2+ elevation with ZnPy on Kv7 inhibition by Ca2+. Since CaM is required for Ca2+-dependent modulation of Kv7 channels , in the presence of 2 mM extracellular Ca2+ produced sharp, nearly complete, inhibition of Kv7.2 current amplitude from 0.95 ± 0.17 nA to 0.09 ± 0.04 nA . Application of ZnPy (still in the presence of Ca2+/ionomycin) induced rapid recovery of Ca2+/ionomycin-induced inhibition to the levels significantly higher than basal level (D–F). Compared with basal level, current amplitude was increased by 1.53 ± 0.14-fold, which was comparable with the efficacy of ZnPy to augment Kv7.2 current amplitude in control conditions (A–C). Thus, intracellular Zn2+ delivery was able to completely remove Ca2+/CaM-mediated inhibition. This was even more evident in the experiment where we applied ZnPy first, waited until Kv7.2 current amplitude increase reached a plateau, and then applied ionomycin (still in the presence of ZnPy). Under these conditions, ionomycin failed to produce any inhibition (G–I). In our previous experiments, 5 μM ionomycin induced [Ca2+]i transients in the range of 500 nM in CHO cells under our experimental conditions  and “high” , physiological scenarios, respectively (G–J). In both scenarios, ZnPy induced a robust Kv7.2 current augmentation; expectedly smaller currents recorded with 1 μM [Ca2+]i were augmented to the level of basal currents recorded in 50 nM [Ca2+]i (I).Next, we expanded this protocol to test the effect of Znchannels , 24, 25,channels , 18, 26,al level , D–F. Conditions , A–C. Thhibition , G–I. Inectively , G–J. In [Ca2+]i I.2+ and Ca2+ ionophores, the currents lost these features almost entirely, allowing instantaneous change to voltage in either direction . We investigated this further by comparing activation kinetics and current–voltage relationships of Kv7.2 currents in control conditions, in the presence of ZnPy or in the presence of both, ZnPy and ionomycin (A and B). The remaining, slower-activating, fraction activated several-fold faster, as compared with control conditions, across the voltages tested (B and C). Consistent with earlier reports . Both, dramatic acceleration of current kinetics and loss of voltage dependence, suggest that in the presence of both Ca2+ and Zn2+, the channel is being locked in the open state and no longer controlled by its voltage sensor. Of note, as evident from D and G, the current through these constitutively open channels was completely blocked by the selective Kv7 channel inhibitor, XE991 (10 μM), confirming that it was indeed generated by the Kv7.2.An interesting observation from the experiments reported in onomycin . As noteonomycin , A and Bs tested , B and C reports , 21, ZnP2+-induced inhibition of a native neuronal M current can be rescued by Zn2+. In perforated-patch experiments, 5 μM ionomycin almost completely inhibited M-like outward XE991-sensitive current, and well over 50% of this inhibition was recovered by 10 μM ZnPy  (A) reduce CaM binding to Kv7.2 and also reduce plasma membrane expression of the channels as well as the whole cell currents generated (B and C), confirming reduced membrane trafficking of the mutants. Kv7.2 (S511D) generated sizable M-like current, which was potentiated by ZnPy to a similar extent , as compared with the WT Kv7.2 . The I340E mutant produced almost no current under basal conditions; however, when ZnPy was applied, there was a clear increase in the whole-cell current amplitude (0 mV), which was reversible by TPEN and sensitive to XE991 (E and G). This was a surprising observation since there were clearly much fewer membrane-localized I340E channels (B and C), and this mutant was early characterized as nonfunctional  , 39, 40. (2+ (A) A reduce enerated , 42. We enerated , B and Cto XE991 , E and Gchannels , B and CA). We also confirmed the effect of the mutations on Kv7.2–CaM interaction (B). In a coimmunoprecipitation (co-IP) experiment, FLAG-tagged WT Kv7.2 and Kv7.2 (I340E) or hemagglutinin-tagged Kv7.2 (S511D) were coexpressed with CaM and immunoprecipitated using the corresponding antibodies ; coprecipitated CaM was then detected using Western blot. As expected, both mutants bound CaM weaker (B), yet, CaM was still detectable. We interpret these results as that a fraction of the mutant channels does reach plasma membrane; these are likely to be still CaM bound. In the case of I340E, the number of functional channels at the plasma membrane is particularly low, and their basal activity (even at saturating voltages) is miniscule but can still be augmented by zinc. Interestingly, the relative efficacy of ZnPy to potentiate these I340E channels was significantly higher, as compared with WT Kv7.2 (E and G).Importantly, membrane abundance of either Kv7.2 channels tested  was not affected by the ZnPy treatment A. We alseraction B. In a cM weaker B, yet, CWT Kv7.2 , E and G2+ and Zn2+ elevations. Kv7.2 (S511D) behaved similarly to the WT Kv7.2 and displayed robust sensitivity to Ca2+/ionomycin, which was completely reverted by 10 μM ZnPy (A–C). Compared with basal level, Kv7.2 (S511D) current amplitude was increased by 1.46 ± 0.21-fold, which was comparable with the efficacy of ZnPy to augment WT Kv7.2 current amplitude (A–F). Current amplitude generated by Kv7.2 (I340E) with CaM was too small for meaningful quantification of inhibitory effect of Ca2+/ionomycin; hence, we first applied 10 μM ZnPy, which produced more that twofold augmentation of current amplitude (D–F). Then, we added 5 μM ionomycin, still in the presence of ZnPy. As in the case of WT Kv7.2, there was no inhibition, and the current was fully inhibited by XE991. In combination, the experiments presented in 2+ and Zn2+, these mutant channels behave similarly to the WT Kv7.2: Ca2+ inhibits them and Zn2+ removes this inhibition. Inhibition by Ca2+ does not hamper Zn2+ efficacy to augment channel activity, whereas pretreatment with Zn2+ renders the channel insensitive to inhibition by Ca2+.Next, we coexpressed both mutants with CaM and tested their sensitivity to Ca μM ZnPy , A–C. Complitude , A–F. Cumplitude , D–F. ThPo) of Kv7.3, which is close to 1 at saturating voltages . We then mutated residues I379 and A518, which in Kv7.3 are at positions equivalent to I340 and S511 in Kv7.2, to impair CaM binding to helix A or B, respectively. We used Kv7.3 A315T/I379A and A315T/A518D mutants that were shown to have reduced CaM binding previously (B–E). The most striking was the case of the Kv7.3 A315T/A518D. Consistent with a previous report . Overall, ZnPy produced 1.08 ± 0.04-fold (n = 5) current augmentation of Kv7.3 A315T single mutant, 2.62 ± 0.4-fold  augmentation of Kv7.3 A315T/I379A, and staggering 3.99 ± 0.81-fold  augmentation of A315T/A518D. These experiments reveal that CaM binding deficiency impairs Kv7.3 channel function, but this can be rescued by intracellular Zn2+ to a significant degree. Even a channel previously characterized as nonfunctional (Kv7.3 A315T/A518D) can be “brought to life” in the presence of Zn2+.Kv7.3 was shown to have marginal sensitivity to zinc , 21. Thevoltages , owing ttages 2 , 44. Thu. ThuPo o by Zn2+ . We next by Zn2+ , 45, we affinity . As preveviously . Both doeviously , B–E. Ths report , in CHO by XE991 , C and D2+-sensitive PLCδ delta and deplete membrane PIP2   did induce robust translocation of the probe. Hence, we conclude that under our experimental conditions, 5 μM ionomycin does not produce measurable effect on PIP2 abundance, and the inhibitory effect of ionomycin on Kv7 channels is likely to be mediated by Ca2+/CaM.Interestingly, at higher concentrations, ionomycin has been shown to activate Caane PIP2 . In thisependent , 15, 43,mutated) , 48, to 2 levels . Similar2 levels , 5 μM io2+ but also Zn2+  or with CaM1234 (B–D) displayed very similar response to 10 μM ZnPy. Thus, it is unlikely that the augmenting effect of Zn2+ on Kv7 channels is mediated by CaM.Because CaM was shown to be able to bind not only Caaffinity , 50) andaffinity , we testaffinity , 52). As CaM1234 , B–D dis2+/CaM and the potentiating effect of zinc. Our main findings are as follows: (i) Zn2+ ionophore ZnPy can completely prevent inhibition of Kv7.2 by Ca2+/CaM. Likewise, ZnPy can reverse Ca2+/CaM-induced inhibition of Kv7.2. In either case, the presence of Ca2+ does not significantly affect the efficacy of the augmentation of current amplitude by ZnPy. Consistent with a previous report , indicating that Zn2+ is the active moiety mediating the effect of ZnPy. (ii) In the presence of both Ca2+ (delivered by ionomycin) and Zn2+ (delivered by ZnPy), Kv7.2 channels lose most of their voltage dependence and lock in the open state. (iii) Consistent with the previous literature  may fail to manifest.Functional relationships between Kv7 channel activity, CaM and Cachannels , 42, 53.channels , 24, 25.hibitory , 33, 54 hibitory  for revihibiting , 32, 34.B region , 57, 58,B region , 57, 59.for PIP2 , may hav2-binding sites, specifically, the ones in the HA–HB linker  Kv7 channel inhibition, makes zinc-bound Kv7 channels resistant to most physiological inhibitory modulation, for example, by acetylcholine, glutamate, bradykinin, histamine, angiotensin II, and so on. In support of this speculation, inhibition of Kv7.2, Kv7.3 ; it can2, Kv7.3 . SynaptiM levels . Signifieceptors , 5 as weI mGluRs ), could 2) and passaged about every 2 days. Rat dorsal root ganglion neurons were isolated and cultured as described before (KCNQ2 (Y15065) or KCNQ3 (NM_004519) cDNA subcloned into pCDNA3.1 (Youbio), with or without WT CaM (NP_008819.1) or CaM1234 . Point mutations in KCNQ2 and KCNQ3 were produced by Youbio. YFP-tubby (tubby-R332H-cYFP was a gift from Andrew Tinker (UCL)). Human M1 receptor cDNA was a gift from Hailin Zhang . For transfection, cells were cultured on 24-well plates and transfected with FuGENE HD Transfection Reagent (Promega), according to the manufacturer’s instructions. Twenty-four hours later, the cells were plated onto poly-l-lysine–coated coverslip chips; experiments were performed 48 to 96 h after transfection. As a marker for successfully transfected cells, cDNA-encoding GFP was cotransfected together with the other genes of interest.CHO cells were obtained from Kunming Institute of Zoology, Chinese Academy of Sciences. Cells were grown in T25 flasks in Dulbecco's modified Eagle's medium (DMEM)/F12 medium with 10% fetal bovine serum and 0.1% penicillin/streptomycin in a humidified incubator at 37 °C  using amphotericin B (0.5 mg/ml) as a pore-forming agent. Pipettes were pulled from borosilicate glass capillaries using a Flaming/Brown micropipette puller P-97 and had resistances of 2 to 5 MΩ. Currents were amplified by the EPC10 USB amplifier (HEKA) and recorded using the Patchmaster software . Capacitance current artifacts were cancelled, and series resistance was compensated by ∼80%. To evaluate the amplitude of Kv7 currents, CHO cells were held at 0 mV, and 1000 ms hyperpolarizing steps to –60 mV, followed by a 500 ms pulse back to 0 mV, were applied every 3 s. In dorsal root ganglion neuron recordings, similar voltage protocol was used, but cells were held at −30 mV. To investigate current–voltage relationships, CHO cells were held at −80 mV, and the currents were elicited by a series of 1500 ms steps from −70 to +50 mV in 10 mV increments. The G–V curves were fit by the Boltzmann equation:2 incubator. Cells were transfected with FLAG-tagged WT KCNQ2 and KCNQ2 (I340E) or with HA-tagged KCNQ2 (S511D) by using polyethyleneimine transfection reagent (Polysciences), at plasmid:polyethyleneimine ratio of 1:3. In experiments with ZnPy application, 10 μM ZnPy or vehicle, cells were applied for 2 min before harvest. Membrane proteins were extracted and separated from nonmembrane proteins by Minute Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent). Membrane protein fraction was collected and dissolved in Minute Nondenatured Protein Solubilization Reagent (Invent). Protein concentration was measured with Pierce BCA Protein Assay Kit (Solarbio). Samples were denatured at 95 °C for 5 min and separated on a gradient SDS-PAGE gel. Samples were transferred to a 0.2 μm polyvinylidene difluoride membrane (Millipore) at 120 V for 90 min on ice and blocked with 5% skim milk powder in Tris-buffered saline with Tween-20 on a rocking platform for 1 h at room temperature. The membranes were then incubated with following primary antibodies overnight at 4 °C: anti-DDDDK-tag monoclonal antibody ; antihemagglutinin-tag monoclonal antibody ; anti-β-actin monoclonal antibody ; and anti-CaM monoclonal antibody . Next, membranes were incubated with respective secondary antibody in 5% skim milk powder: antimouse (goat) DyLight TM 800  or anti-rabbit (goat) DyLight TM 800 . Protein signals were detected using Odyssey infrared fluorescence scanning imaging system. The intensity of each protein band was analyzed using ImageJ .Human embryonic kidney 293T cells  were grown in 100 mm cell culture dishes in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin (complete medium) at 37 C in a 5% COg at 4 °C for 5 min. The pellet was resuspended with 60 μl of loading buffer, boiled for 2 to 3 min, and then subjected to Western blot analysis.Cells were prepared and transfected as described in previous section. Cells were lysed using cell lysis buffer for Western blot and IP (Beyotime). Protein concentration was measured with Pierce BCA Protein Assay Kit. The total protein from each sample was precleaned and divided into three groups: input/immunoglobulin G (IgG)/co-IP. The “input” sample was mixed with 5× loading buffer at a ratio of 4:1, heated at 95 °C for 5 min, and stored at −20 °C until further analysis. The “co-IP” samples were incubated with anti-FLAG antibody  or antihemagglutinin-tag monoclonal antibody  at 4 °C overnight. The “IgG” samples were incubated with mouse IgG (Santa Cruz) under the same conditions. Protein A/G PLUS-Agarose beads  were added to the “co-IP” and “IgG” samples and incubated at 4 °C for 4 h with rotation. These beads were washed five times with PBS buffer and centrifuged at 12,0002, 1 MgCl2, and 10 Hepes (pH adjusted to 7.4 with NaOH). The pipette solution for perforated patch contained (in millimolar): 90 potassium acetate, 20 KCl, 1 CaCl2, 3 MgCl2, 40 Hepes, 3 EGTA, and 0.5 mg/ml amphotericin B (pH adjusted to 7.4 with KOH). In whole-cell experiments with “low” (50 nM) and “high” (1 μM) [Ca2+]I, the pipette solutions were (in millimolar): 130 KCl, 5 MgCl2, 5 EGTA, 3 K-ATP, and 5 Hepes. To obtain 50 nm and 1 μM cytosolic-free [Ca2+], 1.94 or 4.63 mM CaCl2 was added . Reagents were obtained as follows: ZnPy, TPEN, XE991, amphotericin B (Sigma–Aldrich); ionomycin ; DMEM-F12, DMEM, and fetal bovine serum (Gibco).The external solution for patch-clamp recording from the CHO cells contained (in millimolar): 160 NaCl, 2.5 KCl, 2 CaClt test, one-way ANOVA, or repeated-measures ANOVA with Tukey’s or Bonferroni’s post hoc tests as appropriate.The data were analyzed and plotted with Origin 9.1 (OriginLab) and GraphPad Prism 8 (GraphPad Software). Data are presented as means ± SEM and were statistically compared using Student’s paired or unpaired All data are available in the main text and presented as scatter plots.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The strain was identified based on phenotypic and genotypic analysis, and deposited in the GenBank with accession number OK090864.1. Cultivation of the selected strain on rice, chromatographic purification, and structural elucidation led to the isolation of two compounds C1: umbelliferone, and C2: 1-methoxy-3-methyl-8-hydroxy-anthraquinone. The antimicrobial activity of the obtained compounds showed that C1 and C2 have low antibacterial activity toward S. aureus and E. coli with no pronounced activity toward P. aeruginosa, C. albicans, and A. niger. Additionally, the antioxidant activity of C1 and C2 revealed that C2 has a good antioxidant activity, with DPPH scavenging activity reaching (55.25%), followed by C1 (30.20%). Moreover, both compounds displayed anti-Gyr-B enzyme activity with IC50 value of (3.79 ± 0.21 µM) for C1, and (IC50 = 13 ± 0.71 µM) for C2. The ADME-related physicochemical properties of the obtained compound were predicted using SwissADME web tools and the ProToxii webserver was used to estimate in silico toxicity.This study was designed to evaluate the antimicrobial, antioxidant, and cytotoxic potentials of the marine actinomycetes spp. isolated from the Red Sea water, Hurghada, Egypt. Out of 80 actinomycetes isolates, one isolate AW6 was selected based on its antioxidant activity (ICThe online version contains supplementary material available at 10.1007/s00203-022-03092-5. Staphylococcus aureus is a well-known organism that causes illnesses such as pneumonia and recently has gained resistance to the majority of antibiotic groups , but unfortunately, Vancomycin-resistant strains have emerged clinically (Gardete and Tomasz S. aureus (VRSA) is considered a critical problem to doctors not only because of its vancomycin and methicillin resistance but also due to resistance to a variety of other antibiotics, including aminoglycosides, fluoroquinolones, and macrolides . As a result of this phenomenon, many strains of bacteria have developed antibiotic resistance, and in many cases, multi-resistant, to these known therapeutic medicines, and to address this issue, a new antibiotic with a novel mechanism of action is required  was added afterward. 1 g of sediment was taken from each sample and mixed separately with 9 mL of sterile saltwater. In 100 mL of sterile starch nitrate agar medium, five milliliters of the suspension were added. Each sterilized plate received approximately 20 mL of medium with no air bubbles. The inoculated plates were incubated for 7 days at 30 °C. After incubation, the actinomycete colonies were picked and purified using the streaking method. The purified strains were preserved on starch nitrate slants and 25% glycerol stocks at − 20 °C : 20 starch; 0.5 KBacillus subtilis (ATCC66), Staphylococcus aureus (ATCC6538-P), and Methicillin-resistant Staphylococcus aureus (MRSA) (ATCC25923); Gram-negative bacteria Escherichia coli (ATCC14169), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae, Salmonella typhi, and Salmonella enterica; yeasts Candida albicans (ATCC10231), and (ATCC9080) and fungus Aspergillus niger (NRRL A-326). In each plate, 6 mm diameter wells were drilled with a sterile cork borer and approximately 20 µL of each extract was put into each well against each of the test organisms. After that, plates were incubated for 24 h at 37 °C. The diameter of the inhibition zones was used to determine antimicrobial activity. Only isolates with broad-spectrum activity were chosen for further study  , the mycelium structure, color, and arrangement of conidiophores were observed. The studying of spore-bearing hyphae, spore chain structure, spore color, aerial mass color, and substrate mycelia color were studied according to Bergey  and the −3 primers, 5 μL of 2 mmol dm−3 dNTP, 0.3 μL Taq DNA polymerase, and 0.5 μL of template DNA). The PCR reaction ran under the following conditions: 94 °C for 45 s, 55 °C for 60 s, and 72 °C for 60 s. The purified product was sequenced at Macrogen Company, South Korea. The similarity and homology of the 16S rDNA sequences were investigated by comparing the obtained sequences with similar known sequences in the NCBI database using online BLAST alignment search tools (http://www.ncbi.nlm.nih.gov/BLAST). MEGA-X software was used to create the phylogenetic tree  was used to extract the DNA, which was done according to the manufacturer’s recommendations. Two universal primers were used in the PCR amplification . The final volume of the PCR amplification reaction was as follows: 50 μL  at 30 °C for 7 days under shaking conditions. After incubation, the starch broth culture medium was used as a seed culture to inoculate the rice media for large-scale fermentation  to solute (crude extract). A total of 100 fractions of 10 mL each were collected and analyzed using thin-layer chromatography (TLC) to identify the fractions with compounds of interest. The most potent fraction was further purified using the size-exclusion chromatography technique and performed using Sephadex LH-20 sub-column. The separation was based on molecular weight. The purified compounds were subjected to different spectroscopic analyses such as NMR and ESI for structure elucidation.Actinomyces sp. AW6 compounds was carried out using an antimicrobial assay and MIC as described by Hamed et al.  was measured by the decoloration of an ethanolic solution of DPPH radical spectrophotometrically at 517 nm following Brand-Williams et al  techniqu®, London, UK) on streptavidin-coated 96-well microtiter plates , according to the manufacturer’s protocol  as the basal medium. xylose, Arabinose, mannose, glucose, lactose, cellobiose, glycerol, raffinose, glycogen, salicin, mannitol, rhamnose, starch, inositol, and sorbitol were tested. The used tests in the biochemical study are performed as described in the report of the International Subgroup on the Taxonomy of Microaerophilic Actinomycetes . The cultural properties of the AW6 strain after cultivation on different ISP media were summarized in Supplementary (2). Additionally, the physiological and biochemical characteristics for the selected isolate AW6 include nitrate reduction, catalase, methyl red (MR), indole production, Voges–Proskauer (V–P), starch hydrolysis, hydrogen sulfide production, and gelatin hydrolysis were also assessed and represented in Supplementary (3). The production of acid from carbohydrates was investigated with es Slack .http://www.blast.ncbi.nlm.nih.gov/Blast). The obtained result showed a very close similarity of the obtained sequence with 100% homology of the isolate AW6 with Actinomyces sp. Based on the analysis of the DNA sequence and the morphological characteristics of the AW6 isolate was identified as Actinomyces sp. AW6 and deposited in GenBank with accession no. OK090864.1. The maximum likelihood approach and the Tamura–Nei model were used to infer the evolutionary history  tool to measure the similarity score and calculate the statistical significance of the matches (Actinomyces sp. AW6 was cultivated on rice medium and extracted with ethyl acetate. The ethyl acetate evaporated using a rotary evaporator till complete evaporation of ethyl acetate. The extract (13 g) was primarily fractionated using Flash column chromatography into 100 fractions. Based on the chemical screening using TLC and biological screening, the most active fractions were collected together further and purified using Sephadex LH-20 column with a gradient mobile phase DCM: Methanol. The TLC chromatogram showed that fraction no. (8) contains the peaks of interest. The structural elucidation of the isolated compounds was performed using nuclear magnetic resonance (NMR) spectroscopy.The strain Compound (1) was obtained as white needles. 1H NMR spectra showed characteristic signals for ABX system of three aromatic protons at δH (ppm): 6.72 , 7.28 , and 6.75  indicating to the presence of 1,2,4-trisubstituted benzene ring. Moreover, 1,2-disubstituted olefinic protons were resonated at δH (ppm): 6.19 , and 7.61  indicating the presence of α,β-unsaturated ketone of a coumarin ring. 13C NMR spectra revealed the presence of characteristic signals for aromatic, olefinic and carbonyl carbons at δC (ppm): 162.0 (C-2), 113.7 (C-3), 145.0 (C-4), 129.89 (C-5), 112.1 (C-6), 160.97 (C-7), 102.94 (C-8), 156.2 (C-9), and 111.69 (C-10). Therefore, the compound could be identified as umbelliferone based on its chromatographic properties, proton and carbon spectra, and available reported data  was obtained as a pale orange powder. 1H NMR spectra showed characteristic signals for aromatic protons were resonated at δH (ppm): 7.10 , 7.34 , 7.33 , 7.74 , and 7.51  indicating the presence of quinone nucleus. Additionally, methyl and methoxy groups were resonated at δH (ppm): 2.38  and 3.71 . 13C NMR spectra revealed the presence of characteristic signals for aromatic, carbonyls, aromatic methyl, and methoxy carbons at δC (ppm): 157.0 (C-1), 115.7 (C-2), 118.9 (C-3), 122.36 (C-4), 118.7 (C-5), 137.1 (C-6), 122.9 (C-7), 160.93 (C-8), 185.0 (C-9), 182.5 (C-10), 132.6 (C-11), 113.9 (C-12), 138.9 (C-13), 128.2 (C-14), 56.1 (–OCH3-1), and 21.2 (–CH3-3) supporting the presence of anthraquinone skeleton. Based on its chromatographic properties, proton and carbon spectra, and available reported data, the compound could be identified as 1-methoxy-3-methyl-8-hydroxy-anthraquinone  based on 2,2-diphenyl-1-picrylhydrazyl radical  revealed that 1-methoxy-3-methyl-8-hydroxy-anthraquinone (C2) is the most antioxidant agent, showed maximum DPPH scavenging activity (55.25%), followed by umbelliferone)C1) (30.20%). Also, the two compounds showed DPPH antioxidant activity with IC50 values of 5.47 and 3.84 µg/mL for C1 and C2, respectively , while compound C2 was the least potent (IC50 = 13 ± 0.71 µM).DNA gyrase is a bacterial enzyme that catalyzes the ATP-dependent negative supercoiling of closed-circular double-stranded DNA. Gyrase is a member of the topoisomerase enzyme family, which is involved in the control of DNA topological transitions (Reece and Maxwell ) C1) and 1-methoxy-3-methyl-8-hydroxy-anthraquinone (C2) ester were estimated using SwissADME online server  passed the Lipinski, Veber, and Ghose rules filter with no violations. Both compounds exhibited 0.55% oral bioavailability and could be used as an oral medication  showed log Po/w values below 5, (1.50 and 2.48) suggesting a good permeability and absorption across the cell membrane. Additionally, solubility is one of the most important parameters influencing compound absorption in any formulation process , as it has one violation for this rule, while compound 2 passed the rule of three (RO3), with no violation. For synthetic accessibility score (SAscore) that estimated on the similarity of fragments and complexity penalties, both compounds (C1 and C2) showed moderate synthetic accessibility with values (2.56 and 2.57), respectively.The ADME-related physicochemical properties of umbelliferone C2 Fig. a and b.  C2 Fig. . Both te(C1 and C2) pharmacokinetic parameters measured using the vector machine algorithm (SVM) model  demonstrated high human gastrointestinal absorption (GI). The compounds are non-P-gp substrates , while their blood–brain barrier (BBB) permeant (TPSA < 75 Å2), suggesting the presence of effects on the central nervous system (CNS) (Daina and Zoete Kp) of the obtained compound was done as described by Potts and Guy (Potts and Guy (C1 and C2) showed log (Kp) (− 6.17 and − 5.52 cm/s), while the more negative log Kp, the less skin permeant is the compounds.Table C1 and C2) was performed using ProTox ii webserver  for C1, and (IC50 = 13 ± 0.71 µM) for C2. The ADME-related physicochemical properties of the obtained compound were predicted using SwissADME web tools and the ProToxii webserver was used to estimate in silico toxicity.Supplementary file1 (DOCX 1926 KB)Below is the link to the electronic supplementary material.


Trypanosoma brucei rhodesiense, a parasite causing the lethal form of Human African Trypanosomiasis. CD24 is a recently developed synthetic inhibitor of rhodesain, characterized by a nanomolar affinity towards the trypanosomal protease (Ki = 16 nM), and acting as a competitive inhibitor. In the present work, we carried out a combination study of CD24 with curcumin, the multitarget nutraceutical obtained from Curcuma longa L., which we demonstrated to inhibit rhodesain in a non-competitive manner. By applying the Chou and Talalay method, we obtained an initial additive effect at IC50 , while for the most relevant fa values, ranging from 0.6 to 1, i.e., from 60% to 100% of rhodesain inhibition, we obtained a combination index < 1, thus suggesting that an increasingly synergistic action occurred for the combination of the synthetic inhibitor CD24 and curcumin. Furthermore, the combination of the two inhibitors showed an antitrypanosomal activity better than that of CD24 alone , thus suggesting the use of the two inhibitors in combination is desirable. Rhodesain is a cysteine protease that is crucial for the life cycle of  T. brucei gambiense, which is able to cause the chronic form of HAT and widespread in western and central Africa, and T. b. rhodesiense, which is common in eastern and southern Africa, and responsible for the rapid-onset high death rate HAT 1/m ,34.CD24 is a competitive inhibitor of rhodesain  = 0 μM, ymin as the minimum value, obtained at high inhibitor concentrations, and s as the Hill coefficient.Fluorescence of the product AMC of the substrate hydrolyses was measured using an Infinite 200 PRO microplate reader  at room temperature, with a 380 nm excitation filter and a 460 nm emission filter. Results are expressed as ICCD24 and curcumin were tested in combination using 5 data points: 0.25 × IC50F1+F2, 0.50 × IC50F1+F2, IC50F1+F2, 2 × IC50F1+F2, 4 × IC50F+F2, where F1 = CD24 and F2 = curcumin.As a second step, T. b. brucei 449, descendants of the Lister strain 427 [CD24 with curcumin in 1:1 molar ratio against T. b. brucei was determined using the ATPlite assay, as described previously [CD24 and curcumin in DMSO was prepared by mixing the compounds in a 1:1 molar ratio, for a final concentration of 10 mM. This stock was then used to perform serial dilutions in culture media. The final concentrations applied on the cells were 33.33 µM, 16.66 µM, 8.33 µM, 4.16 µM, 2.08 µM, 1.04 µM, 0.52 µM, 0.26 µM, 0.13 µM, and 0.06 µM. The parasites used in this study were culture-adapted rain 427 . Cytotoxeviously ,36,41,422. Cytotoxic activities of CD24 and CD24/curcumin combination (in 1:1 molar ratio) were assayed using resazurin staining, as described previously [HEK293 cells were cultured in high glucose DMEM medium with L-glutamine, supplemented with 10% FCS, 20 U/mL penicillin, and 20 µg/mL streptomycin, at 37 °C and 5% COeviously . Brieflyp < 0.05 with respect to the percentage of rhodesain inhibition of curcumin, CD24, and curcumin + CD24. The analyses were performed with GraphPAD Prism 6 . Results are expressed as the arithmetic mean ± standard deviation (SD).The statistical analysis of the data was performed using the one-way test (ANOVA) with Dunnett’s multiple comparison test, considering significant differences of CD24 and curcumin against rhodesain, we investigated the activity of their combination, concluding that at IC50 an initial additive effect was observed (CI = 1), while for the most significant fa values, i.e., those ranging from 0.6 to 1 (corresponding to the range 60–100% of rhodesain inhibition), an increasingly synergistic action was observed. Moreover, at cellular level we obtained, with the combination synthetic inhibitor + nutraceutical, an antitrypanosomal activity in the low micromolar range and a selectivity index better than that exhibited by CD24 alone; thus, for all the described reasons, their use in combination is desirable.In summary, in this study, starting from the single activities of


S. pneumoniae, which is mainly responsible for meningitis and community-acquired pneumonia (CAP), highlighting the need for new anti-pneumococcal agents. We have identified a potential anti-pneumococcal agent, enol 3, which acts by hindering the cell division process by perturbing Z-ring dynamics inside the cell. Enol 3 was also shown to inhibit FtsZ polymerization and induce its aggregation in vitro but does not affect the activity of tubulin and alkaline phosphatase. Docking studies show that 3 binds near the T7 loop, which is the catalytic site of FtsZ. Similar effects on Z-ring and FtsZ assembly were observed in B. subtilis, indicating that 3 could be a broad-spectrum anti-bacterial agent useful in targeting Gram-positive bacteria. In conclusion, compound 3 shows strong anti-pneumococcal activity, prompting further pre-clinical studies to explore its potential.Multi-drug resistance is increasing in the pathogenic bacterium  E. coli and B. subtilis have resulted in a greater understanding of bacterial cell division +, 349.1440; Found: [M + H]+, 349.1434. Cyclohexanone 1  was dissolved and stirred in methanol (10 mL) with 10% Pd/C (1.5 mol%) and cyclohexene  was added dropwise. The reaction was heated under reflux for two hours and monitored by TLC. The resulting solution was filtered through celite to obtain the crude product which was purified by flash column chromatography (ethyl acetate:hexane—1:4 to 2:3) to produce 3 as yellow oil . HPLC: 96.1% (360 nm); IR νmax/cm−1: 3350 (OH), 1715 (C=O), 1650 (C=C); 1H NMR  δH 2.16–3.35 , 3.35 , 6.69 , 7.00 ; 13C NMR  δC 29.5 , 30.7 , 31.4 , 35.0 , 37.8 , 40.3 , 41.5 (C-6), 116.1 , 130.4 , 130.9 , 132.6 , 133.1 , 156.6 , 161.3 , 208.1 ; HRMS (ESI/FT-MS/ ve) m/z: Calc. for C22H24O4: [M + Na]+, 375.1567; Found: [M+Na]+, 375.1564. Cyclohexanone 3 was dissolved in 100% dimethyl sulfoxide (DMSO), as was compound 1  is the concentration of 3.ΔSpnFtsZ (10 μM) in 50 mM PIPES pH 7.4, 50 mM KCl and 5 mM MgCl2 (PKM) buffer was incubated with different concentrations of 3  for 20 min, at 25 °C. After the addition of 1 mM GTP, the polymerization was monitored for 600 s, at 37 °C, using light scattering at 400 nm [t 400 nm  in a JASSpnFtsZ (10 μM) in PKM buffer was incubated with different concentrations of 3  for 20 min at 25 °C. The reaction mixture was allowed to polymerize by adding 1 mM GTP and incubated for 600 s, at 37 °C. The mixture was then subjected to ultra-centrifugation at 60,000 RPM for 30 min at 30 °C. The supernatant was collected and quantified using Bradford’s reagent [ reagent . The amoSpnFtsZ (10 μM) in PKM buffer was incubated without and with 10 μM 3 for 20 min, at 25 °C. The mixture was then polymerized by the addition of 1 mM GTP for 200 s, at 37 °C. Samples for EM were prepared on a fresh carbon–formvar-coated copper EM grid according to a previously described method [d method . ImagingBsFtsZ (5 μM) in PKM buffer was incubated without and with 20 μM 3 for 20 min, at 25 °C. The mixture was then polymerized by the addition of 1 mM GTP for 600 s, at 37 °C. Samples for imaging were prepared using the same procedure described above.In another experiment, SpnFtsZ in PKM buffer was incubated with or without 10 μM 3, for 20 min, at 25 °C. Upon addition of 1 mM GTP, FtsZ was polymerized for 300 s, at 37 °C. AFM samples were prepared according to a previously described method [Then, 4 μM d method . Then, 22 and 1 mM EGTA) + 10% DMSO was incubated with 3 for 10 min on ice. Then, 1 mM GTP was added to the mixture, and polymerization was monitored by light scattering in SoftMax Spectra Multi Plate reader for 30 min, at 37 °C. The excitation and emission wavelengths were set to 350 nm.Tubulin polymerization was performed as described previously . Purifie2, and 0.1 mM ZnCl2 was incubated with different concentrations  of 3 for 10 min on ice. Immediately after the addition of 250 μM para-4-nitrophenyl phosphate (PNPP), the absorbance at 410 nm was measured for 700 s in a JASCO V-730 spectrophotometer.Alkaline phosphatase assay was performed as described previously . Then, 2https://www.graphpad.com/quickcalcs/ttest1/ accessed on 28 February 2019) for the unpaired t-test. The differences were considered non-significant if the p values were more than 0.05.Statistical analyses were performed using Graph-Pad Prism online software . S. pneumoniae resistance to penicillins and macrolides is increasing, highlighting the need for new anti-pneumococcal agents [3 acts by hindering the cell division process by perturbing Z-ring dynamics inside the cell. Enol 3 was also shown to inhibit FtsZ polymerization and induce its aggregation in vitro. We ruled out the possibility that enol 3 induced non-specific inhibition of protein molecules by showing that it does not affect the activity of tubulin and alkaline phosphatase. Docking studies show that enol 3 binds near the T7 loop, which is the catalytic site of FtsZ. A possible explanation could be that 3 binding to the catalytic site of FtsZ interferes with amino acid interactions, leading to accelerated aggregate formation of FtsZ. These large aggregates may not allow further FtsZ polymerization, thereby leading to disruption of the dynamic Z-ring, as seen in the cells. Similar effects on Z-ring and FtsZ assembly were also observed in B. subtilis, indicating that enol 3 could be a broad-spectrum anti-bacterial agent useful in targeting Gram-positive bacteria.In this study, we have demonstrated the anti-bacterial potential of enol l agents . We iden3 shows strong anti-pneumococcal activity as a result of its effect upon Z-ring dynamics, prompting further pre-clinical studies to explore its potential as a treatment for bacterial infections.In conclusion, enol


Escherichia coli FtsZ filaments. We combined chemical shift prediction, homology modeling, and heteronuclear dipolar recoupling techniques to characterize the E. coli FtsZ filament interface and demonstrated that the monomers in active filaments assume a tense conformation.FtsZ filaments are the major structural component of the bacterial Z ring and are drivers of bacterial division. Crystal structures for FtsZ from some Gram-positive bacteria in the presence of GTP analogs suggest the possibility of a high-energy, “tense” conformation. It remains important to elucidate whether this tense form is the dominant form in filaments. Using dynamic nuclear polarization (DNP) solid-state nuclear magnetic resonance (NMR) and differential isotopic labeling, we directly detected residues located at the intermonomer interface of GTP-bound wild-type (WT)  Escherichia coli , a second monomer conformation has been observed when FtsZ is bound to a small-molecule inhibitor, PC190273  state (E. coli FtsZ (EcFtsZ) filaments  state . Additioilaments  and mapsilaments . The curE. coli FtsZ filaments are in the T or R state. We use magic angle spinning (MAS) nuclear magnetic resonance (NMR) under cryogenic dynamic nuclear polarization (DNP) conditions (115 K) to directly observed GTP-bound, full-length EcFtsZ filaments at atomic resolution. By focusing on the monomer-monomer interface within the filament, we were able to distinguish between T state monomers and R state monomers and determine that GTP-bound EcFtsZ filaments are composed primarily of T state monomers.However, to date, a T state has been crystallized only from SaFtsZ, and the evidence that it is a conserved, active state is minimal. Notably, the inhibitor used to stabilize SaFtsZ does not induce polymerization in EcFtsZ , and twoMAS NMR is an important structural technique that is well suited for the study of protein oligomers. MAS NMR has contributed greatly to our understanding of the structure of amyloid fibrils , membran53–6UNX] [6LL6] [While large proteins (>200 residues) remain difficult to study using traditional MAS NMR methods that rely on full spectral assignments, the availability of homology modeling, crystal structures, and modern spectroscopy techniques allows targeted biological questions to be asked. FtsZ is one such large protein. EcFtsZ has 383 total residues, consisting of a short disordered N-terminal tail (~residues 1–10), a structured domain S. aureus monomer , 34, 37,E. coli FtsZ filaments. Because FtsZ forms single-stranded filaments composed of a single monomer, the monomer-monomer interface is unique to the filament and can, in principle, be used to characterize the active monomers. In order to understand the differences in the monomer-monomer interface between the T and R states, we constructed three models of the EcFtsZ dimer. A T-state dimer model was generated by defining a dimer based on the crystal contacts in the PDB file of the SaFtsZ T-state monomer  filament. Assuming that the two major forms of the monomer are the T and R states, we set out to identify which state is present in  ID 5MN4 ). This dN4 [5MN6 ). AdditiN6 [6UNX ) was useWhile all three models have similar interfaces, the T model interface contains nearly twice as many interface residues, defined as any residue with at least one carbon atom within 5 Å of the other monomer. Using this definition, the T model has 78 interface residues, the R1 model has 41 interface residues, and the R2 model has 38 interface residues. The majority of R1 and R2 interface residues are in what we term the hinge region of the interface . The hinWe used this information to design a set of solid-state MAS NMR experiments to probe the interface in active, GTP bound EcFtsZ filaments. Unlike X-ray crystallography, where filament structure must be inferred from crystal packing, MAS NMR allows us to study the filaments directly. However, MAS NMR is limited by low sensitivity. Long-range correlation experiments of the type used here are particularly inefficient, with as little as 2% transfer efficiency being common for 4- to 5-Å contacts  Fig. 2AFig. 2A. 15N FtsZ samples with 13C-depleted glucose (99.9% 12C) to reduce contributions from intramonomer transfers due to background natural 13C abundance  and 2-13C-glycerol + U-15N,12C mixed-label EcFtsZ (the 2-glycerol sample) were used to get broad coverage of interface residue atoms, and 13Cα-glycine- and 13Cβ-alanine-labeled EcFtsZ mixed with U-15N,12C EcFtsZ reduced spectral crowding . Additionally, the 2-glycerol and 13Cα-Gly,13Cβ-Ala samples were fractionally deuterated on the 13C-enriched monomers in order to obtain suitable transverse (T2) relaxation values for the collection of long-mixing-time spectra. For each sample, we collected a series of one-dimensional (1D) z-filtered TEDOR (ZF-TEDOR) spectra at various mixing times and observed polarization buildup in a distance-dependent manner.In order to isolate the longitudinal interface of EcFtsZ filaments, we employed a differential isotopic labeling scheme  51, 60), 6051, 6bundance . We prep13Cα-Gly,13Cβ-Ala spectra. It is noted that the while the peaks were confirmed in two data sets, the peaks were still picked judiciously by hand. Our analysis relies on the presence or absence of many such peaks, so small errors in this peak list would not affect the overall outcome of our analysis. ZF-TEDOR buildup curves were plotted for each peak by integrating a slice of the spectrum 1 ppm wide centered at the peak. Transfer efficiencies were calculated by dividing the integrated intensity of each peak by the integrated intensity of the same slice of the 1H-13C cross polarization spectrum  spectra for DNP samples used in this study. The parameters for each spectrum are as follows: for 1,3-glycerol, ns = 32 scans; for 2-glycerol, ns = 16 scans; for 13Cβ-Ala,13Cα-Gly, ns = 64 scans; for U-15N, ns = 64 scans. All samples had an MAS frequency of 14,000 Hz. All spectra were processed with 50 kHz of exponential line broadening. Download FIG S4, JPG file, 0.2 MB.Copyright © 2022 McCoy et al.2022McCoy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02358-22.5FIG S513C-glycerol + U-15N,12C sample spectra, 1,3-13C-glycerol + U-15N sample spectra, and U-15N EcFtsZ sample spectra. (B) Buildup curves from peaks in the 2-13C-glycerol sample spectra and the U-15N EcFtsZ sample spectra. Download FIG S5, JPG file, 0.3 MB.ZF-TEDOR buildup curves for selected peaks. (A) Buildup curves from peaks in the 1,3-Copyright © 2022 McCoy et al.2022McCoy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 13C chemical shift prediction using SHIFTX2  in the ZF-TEDOR 1D spectra, full characterization of the EcFtsZ interface is not possible with the data set described here. However, using the dimer models described above, we generated predicted chemical shift lists to compare to the peaks present in the spectra. We predicted the chemical shifts of the three dimer models using SHIFTX2  and extr SHIFTX2 . AdditioWe ranked each model’s prediction based on whether the predicted residue appeared at the interface, was expected to be labeled, or appeared on a lateral surface and could be explained by lateral contacts between bundled filaments. Most peaks were equally well explained by all three models, meaning that all three models had predicted chemical shifts within 0.5 ppm of the experimental peak that correspond to labeled atoms in interface residues. Predicted shifts from residues appearing at the interface and atoms that are expected to be labeled at >50% efficiency were ranked the highest, followed by labeled atoms appearing at a lateral surface, unlabeled atoms that do not appear at an interface, and, finally, peaks that cannot be explained by any predicted chemical shift.Of the 219 total peaks identified across all three samples, the T model can account for all but 24 of them . The R1 13Cα-Gly,13Cβ-Ala spectra (Cα), 72.5 ppm in the 1,3-glycerol spectra (Cβ), and potentially the peak at 22.1 ppm in the 2-glycerol spectra (Cγ2). The recurrence of the same residues across all 3 samples demonstrates that the same interface form is present across samples and these data are highly reproducible.We also identified 29 peaks that are accounted for by only the T model or only an R model . In someThe fact that the T model better accounts for the experimental data is evidence that the EcFtsZ filaments are primarily composed of T monomers. That some peaks are not well explained by the T model used in this analysis suggests that either some percentage of interfaces present in the sample contain R-state monomers—due either to filaments with mixed states or to nonpolymerized oligomers—or that the physiological interface differ from the models used for this analysis. With continued refinement of the model, and subsequent rounds of chemical shift prediction and analysis, we expect that more peaks will be well explained by the model.Next, we took the identified residues and identified their location in the interface . Glu250 The clasp region and the T7 loop are also present in the data. Ile200, Thr201, and Arg202 are in the T7 loop. Ile200 and Thr201 are expected to have contacts in the R models, but Arg202 is not, because the T7 loop is shifted away from the clasp in the R conformations. Thr65 is located in the clasp region on the N-terminal side of the interface and is predicted to have an interface contact in the R2 model and the T model. However, the chemical shift is significantly different between the two models. In the R2 model, Thr65 Cα is predicted to have a chemical shift of 61.9 ppm, whereas in the T model, it is predicted to be at 66.1 ppm, which aligns with the observed shift of 66.4 ppm. Thr215 is present in the C-terminal subdomain in the H8 helix  and 4. I13Cα-Gly,13Cβ-Ala sample spectra , whereas only one is present in either R model (Thr296 and Thr65) . In addi spectra . While t10.1128/mbio.02358-22.6FIG S613C DNP spectra of the 13Cβ-Ala13Cα-Gly EcFtsZ sample showing a cross polarization spectrum  and a representative ZF-TEDOR spectrum , annotated to show the isotopic scrambling pattern. (Bottom) 13Cβ-Ala,13Cα-Gly 13C CP spectrum (blue) and a natural-abundance 13C EcFtsZ CP DNP spectrum from the U-15N sample (orange). Download FIG S6, JPG file, 0.2 MB.(Top) 1D Copyright © 2022 McCoy et al.2022McCoy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 13Cα-Gly,13Cβ-Ala sample. There is a large set of overlapping peaks evident between 60 and 70 ppm. Nine peaks were identified in this region, seven of which can be attributed to individual labeled atoms based on the chemical shift predictions for the T model  cutoff of 0.5 ; Fig. 5.10.1128/mbio.02358-22.8TABLE S2Table S2, JPG file, 0.3 MB.Best-fit parameters and the fit root mean square deviation (RMSD) for each of the 19 uniquely identified peaks. Download Copyright © 2022 McCoy et al.2022McCoy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Both Ile176 and Ala72 are located in loops  where weOverall, the distances we were able to measure agree with a T-state interface. They also suggest that the interface is tighter than those observed in crystal structures. More work is needed to determine the precise contacts, which will allow more accurate mapping of the interface. Taken together, these data support a model of the EcFtsZ filament where the monomers are in the T conformation. The interface itself is tight, with contacts as short as ~4.0 Å, and the T7 loop extends into the GTP-binding pocket. The clasp region of the interface makes several close contacts across the interface, indicating that it is involved in stabilizing the filament in a straight conformation.13C and 15N nuclei at the EcFtsZ intermonomer interface. The lack of site-specific resonance assignments for the EcFtsZ monomer means that we cannot unambiguously assign peaks in the ZF-TEDOR spectra. However, the availability of crystal structures of FtsZ monomers allowed us to generate chemical shift predictions using homology models. Combining structural analysis of the models with chemical shift predictions allowed us to demonstrate that the T model best explains our data. In addition, the presence of multiple threonine resonances in our spectra indicates that the monomers are in a T state.Using DNP ZF-TEDOR experiments, we were able to directly observe contacts between d-thiogalactopyranoside (IPTG). Isotopically enriched, protonated FtsZ was expressed using a 4:1 LB:M9 transfer with precursor concentrations as follows: for U-15N FtsZ, 1 g/L 15NH4Cl + 4 g/L d-glucose; for U-15N,U-12C FtsZ, 1 g/L 15NH4Cl + 2 g/L 99.9% 12C d-glucose; for the sparsely labeled 13C FtsZ, 1 g/L 1,3-13C-glycerol + 1 g/L NH4Cl + 1 g/L sodium bicarbonate (EcFtsZ was expressed from a pET21b(+) plasmid generously provided by Anuradha Janakiraman at the City College of New York. All protein was expressed in BL21(DE3) chemically competent cells using 0.5 mM isopropyl-β-arbonate . All iso2H2O LB-adapted BL21(DE3) cells transformed with pET21b(+):EcFtsZ in a 4-mL culture of 2H2O LB for 12 h. Five hundred milliliters of 100% 2H2O M9 medium was inoculated with 1% of the overnight cultures and grown to an optical density at 600 nm (OD600) of ~0.6, which took 18 to 20 h. Expression was induced with 0.5 mM IPTG in 2H2O and halted after 4 h. For the 2H2O, 2-13C-glycerol EcFtsZ, the M9 was prepared with 2 g/L 2-13C-glycerol, 1 g/L NH4Cl, and 2 g/L 13C-sodium bicarbonate. For the 2H2O, 13Cα-glycine, 13Cβ-alanine EcFtsZ, the M9 was prepared with 4 g/L 12C-glucose (99.9%), 1 g/L NH4Cl, 1 g/L 13Cα-glycine, 1 g/L 13Cβ-alanine, 0.1 g/L l-isoleucine, and 0.2 g/L α-ketoisovalerate.Partially deuterated EcFtsZ was expressed using M9 adaptation –66. Samp–EcFtsZ was purified using ammonium sulfate precipitation and calcium sedimentation, as described elsewhere , 67. Ani10.1128/mbio.02358-22.1FIG S1FIG S1, JPG file, 0.2 MB.SDS-PAGE gel stained with silver nitrate showing the relative purity of various purification methods. Lane 1, ladder; lane 2, calcium sedimentation followed by anion-exchange chromatography; lane 3, ammonium sulfate precipitation followed by calcium sedimentation; lane 4, ammonium sulfate precipitation followed by anion-exchange chromatography. Download Copyright © 2022 McCoy et al.2022McCoy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02358-22.2FIG S22. All assays were performed at 30°C with stirring. Download FIG S2, JPG file, 0.1 MB.Ninety-degree light scattering demonstrating that the purified EcFtsZ behaves as expected. EcFtsZ polymerization was induced by 1 mM GTP (blue); 1 mM GDP (red) does not induce any increase in scattering, demonstrating that GTP is necessary for polymerization. The buffer was 50 mM MES–NaOH (pH 6.5), 1 mM EGTA, 50 mM KCl, 10 mM MgClCopyright © 2022 McCoy et al.2022McCoy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02358-22.3FIG S32; MM, 50 mM MES, pH 6.5-2.5 mM MgCl2; MEK, 50 mM MES, pH 6.5-50 mM KCl-2.5 mM EDTA; MMK2.5 + GTPγS, EcFtsZ polymerized in MMK2.5 buffer with 1 mM GTPγS instead of 1 mM GTP as a negative control. All buffers contained 50 mM MES–NaOH (pH 6.5), and all samples were polymerized with 1 mM GTP unless otherwise stated. Download FIG S3, JPG file, 0.5 MB.Representative TEM images of EcFtsZ filaments under various buffer conditions. MMK2.5, 50 mM MES, pH 6.5-50 mM KCl-2.5 mM MgClCopyright © 2022 McCoy et al.2022McCoy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2, 2 mM EDTA) diluted to 30% U-12C,U-2H glycerol, bringing the final buffer concentrations to approximately 5 mg FtsZ, 30 mM KCl, 12 mM MgCl2, 1.2 mM EDTA, 3 mM GTP, and 10 mM AMUPol . Samples were centrifuged in Bruker 1.9-mm low-temperature rotors and promptly frozen in liquid nitrogen to preserve stable filaments.All samples were polymerized in DNP buffer  Bruker Avance III-DNP system at the New York Structural Biology Center (NYSBC), which is equipped with a 395 GHz gyrotron and an HCN triple-channel probe. Additional DNP experiments were performed on a 600-MHz (14.1 T) Bruker Avance III-DNP system located at the Bruker Biospin Corporation in Billerica, MA, equipped with a 395 GHz gyrotron and an HCN triple-channel probe. All pulse sequences used in this study can be found at r/2π) of 14,000 ± 10 Hz and an estimated temperature of 115 ± 2.5 K. Standard π/2 pulse lengths of 1.5, 3.0, and 4.2 μs were used for the 1H, 13C, and 15N channels, respectively, corresponding to ω1/2π values of 169 kHz (1H), 83 kHz (13C), and 60 kHz (15N). Heteronuclear recoupling was done using ZF-TEDOR  of 14,000 ± 10 Hz and an estimated temperature of 115 ± 2.5 K. Standard π/2 pulse lengths of 1.2, 3.0, and 4.15 μs were used for the 1H, 13C, and 15N channels, respectively, corresponding to ω1/2π values of 208 kHz (1H), 83 kHz (13C), and 60 kHz (15N). Heteronuclear recoupling was done using ZF-TEDOR using mixing times between 2.0 and 24.28 ms. 1H decoupling with an ω1,H/2π value of >80 kHz was performed using SPINAL-64 decoupling.Experiments done at Bruker BioSpin  were collected with a spinning frequency, (ωSpectra were processed using TopSpin (Bruker BioSpin). To account for low signal-to-noise ratios, peaks were picked by overlaying 1D ZF-TEDOR spectra and identifying peaks that occurred in at least 2 spectra. Data were analyzed in Python using the NMRglue library .Staphylococcus aureus crystal structure by choosing the symmetry mate that best resembled a filament for the tense (PDB ID 5MN4) and relaxed (PDB ID 5MN6) forms (6UNX) . A dimer was inferred from the 6) forms . The dims (6UNX) . The ints (6UNX) .data and data value of <0.5—i.e., the standard deviation of the residuals between the fit and the data (the RMSE) is less than half of the standard deviation of the data. If this value was over 0.5 across all fitting models, the curve was deemed too noisy for accurate fitting and was excluded. The fit error was estimated using jackknife error estimation.In order to extract internuclear distances, the buildup curves of the identified peaks were fitted using numerical simulations generated by SPINEVOLUTION  as descr51610).All spectra are available on the BMRB (ID


Antheraea assama (Muga) and Philosomia ricini (Eri) pupae, specifically Alcalase (A. assama) and Papain (P. ricini) hydrolysates obtained after 60 and 240 min, exhibited the highest ACE-inhibitory and antioxidant properties. The hydrolysates’ fractions , specifically Alc_M60min_F3 (≤3 kDa) and Pap_E240min_F3 (≤3 kDa), showed the highest antioxidant and ACE-inhibitory activities, respectively. Further RP-HPLC purified sub-fractions F4 and F6 showed the highest ACE inhibition as well as potent anti-oxinflammatory activities in lipopolysaccharide (LPS)-treated endothelial cells. Indeed, F4 and F6 ACE-inhibitory peptide fractions were effective in preventing p65 nuclear translocation after 3 h of LPS stimulation along with the inhibition of p38 MAPK phosphorylation in HUVEC cells. In addition, pretreatment with F4 and F6 ACE-inhibitory peptide fractions significantly prevented the LPS-induced upregulation of COX-2 expression and IL-1β secretion, while the expression of NRF2 (nuclear factor erythroid 2-related factor 2)-regulated enzymes such as HO-1 and NQO1 was induced by both peptide fractions. The derived peptides from edible pupae protein hydrolysates have potentialities to be explored as nutritional approaches against hypertension and related cardiovascular diseases.Food-derived bioactive peptides (BAPs) obtained from edible insect-protein hold multiple activities promising the potential to target complex pathological mechanisms responsible for chronic health conditions such as hypertension development. In this study, enzymatic protein hydrolysates from non-mulberry edible silkworm  Hyperactive or dysregulated renin-angiotensin systems and oxinflammatory processes are the primary key players, among many, participating in the pathology of hypertension ,2,3. AngBombyx mori pupae have long been commonly used as food and therapeutics in countries such as Korea, Japan, Thailand, China and India [Antheraea assama and Philosomia ricinii belonging to the Saturniidae family, are predominantly found in the state of Assam and in a few other regions of northeastern India. These silkworms are abundantly used for rearing their golden and white silk threads, locally known as Assam silk or “Muga and Eri silk” [Several food products made from insects’ flour are nowadays available commercially due to the efficient functional properties present in their proteins. However, research on the use of insect-protein-extracted peptides as potential therapeutics is relatively new and trending. Among different species of silkworms found around the world, nd India ,8. Howevri silk” . As a reri silk” . Since iri silk” , they arUntil now, no studies have been carried out on the natively available non-mulberry edible silkworm pupae obtained as indigenous reeling industrial waste to evaluate the presence of multifunctional peptides. Therefore, this is a new investigation on Muga and Eri pupae-derived whole protein extracts for the identification of ACE-inhibitory and anti-oxinflammatory peptides with the aid of six different enzyme applications. Consequently, exploring the possibility of generating bioactive peptides or hydrolysates from these unconventional edible insects could be an interesting avenue of research to facilitate the development of pharmaceutical or nutraceutical products with the potential to target hypertension and oxinflammatory phenomena, and, thus, to manage cardiovascular diseases.Philosomia ricinii  and Antheraea assama  pupae were collected from a sericulture farm located in the Sonitpur district, and some pupae were also procured in the month of March, 2017, from the local market situated in Nelie, Nagaon district, Assam, India. Samples were immediately kept at 25 °C inside zip locked plastic pouches and taken to the laboratory. Pupae were taxonomically identified and confirmed by the Zoological Survey of India in Kolkata, India. Pupae were manually cleaned to remove dirt and other impurities and then stored at −20 °C, until further experimental studies. Pupae were freeze-dried , ground and stored at 4 °C until utilized. Pupae powder was mixed with n-hexane in a ratio of 1:5 (w/v), and thus defatted twice in Soxhlet apparatus (6 h) followed by oven drying  at 50 °C.Bacillus licheniformis, ≥0.75 Anson U mL−1), Flavourzyme , Thermolysin , Papain , Pepsin , Trypsin  and ACE (angiotensin converting enzyme)  were purchased from Sigma–Aldrich Co. . Chemicals such as hippuric acid (HA), hippuryl-L-histidyl-L-leucine (HHL), 3--2, 5-diphenyl tetrazolium bromide (MTT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis 3-ethylbenzthiazoline-6- sulphonic acid (ABTS) and 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ) were also obtained from Sigma–Aldrich Co. . Specific antibodies against heme oxygenase 1 , NAD(P)H quinone dehydrogenase 1 , p65 , phospho (p)-p38  and p38  were used for immunoblotting. All other chemicals and reagents used were analytical grade.All enzymes, including Alcalase  defatted powder in water was calibrated to pH 9.5, using 1 M NaOH. The solution was then stirred for 2 h at room temperature and then centrifuged at 4 °C for 30 min at 8000× g . The collected pellet was re-extracted twice using the same procedure. Supernatants obtained from the total four extractions were adjusted to pH 4.0–5.8, using 1 M HCl to screen the isoelectric point. The pellet, having precipitated proteins at pH 4.7, was isolated from the supernatant after centrifugation for 30 min and 8000× g at 4 °C. The pellets labeled as MPPC (Muga pupae protein concentrate) and EPPC (Eri pupae protein concentrate) were neutralized with 0.2 M NaOH before lyophilization and stored at −20 °C.Proteins from the defatted pupae powder of both silkworms were isolated using the alkaline extraction method, as described by Jia et al.  with modMPPC and EPPC were hydrolyzed with Alcalase , Flavourzyme , Thermolysin , Papain , Pepsin  and Trypsin  at 0.5% enzymes: substrate (E/S) ratio. The protein concentration was adjusted to 5.0% in each reaction mixture and the solutions were set to their appropriate temperature and pH point. During the reaction, pH was kept constant using either 1 M NaOH or HCl. Finally, the digestions were performed in a shaking water bath for a total of 6 h. Five hundred µL samples were taken out after 15, 30, 60, 120, 180, 240, 300 and 360 min to measure their degree of hydrolysis (DH) and ACE inhibition. At the reaction end, to inactivate the enzymes, each hydrolysate was boiled for 10 min, cooled and centrifuged at 10,000 × g for 10 min at 20 °C. Finally, the supernatants containing the peptide mix were collected and labeled accordingly. The pupae proteins treated at the same conditions for each protease with no protease addition were used as controls. The degree of hydrolysis (DH) % was calculated using the trinitro benzene sulfonic acid (TNBS) method given by Hall et al. [inhibitor and Acontrol denote the relative areas (A) of HA peaks in the presence or absence of inhibitors.ACE-inhibitory activities of hydrolysates were estimated following the protocol described by Hall et al. . The rel50  value.The analysis of hydrolysates for the DPPH radical scavenging potential was conducted as per the method illustrated by Hall et al. , in a mi50 value.The analysis of hydrolysates for the ABTS radical scavenging potential was conducted according to You and Wu , in a mi® Ultra 15 mL centrifugal filters with molecular weight (MW) cutoff membranes of 3 kDa and 10 kDa . The resultant filtrates of MW < 3 kDa, 3 < MW < 10 KDa and MW > 10 KDa were later freeze-dried to determine their biological activities.Muga and Eri pupae protein hydrolysates with the highest potential of in vitro ACE inhibition and antioxidant activities were fractionated using Amicon−1 of ultramembrane filtrates having the strongest ACE inhibition and antioxidant activities was dissolved in distilled water. Furthermore, 100 μL of each sample was loaded into RP-HPLC  connected to a PrepTM C18 column . The elution of the sample was performed in linear gradient mode at a flow rate of 1 mL min−1 from 100% eluent A (0.1% trifluoroacetic acid (TFA) in double distilled water) for 2 min; 0 to 25% of eluent B (0.1% TFA in acetonitrile) for 30 min; 25% to 100% at 45 min then back to 0% at 50 min. The eluted fractions were monitored at 214 nm absorbance followed by fraction collection according to each peak. Purification steps were replicated until sufficient samples were accumulated for the ACE-inhibition assay.In total, 100 mg mL−1 penicillin-streptomycin  and 10% fetal bovine serum . The cells were maintained at 37 °C, in a 5% CO2 atmosphere, at 95% humidity in a cell culture incubator.HUVECs were obtained from the American Type Culture Collection . The cells were cultured in endothelial growth medium  with 50 units mL5 cells mL−1) were seeded in 96-well plates. Cells were then treated with peptide fractions at concentrations of 0, 10, 20, 50, 100, 300, 500 and 1000 µg mL−1 for 24 h. At the end of the incubation period, 0.5 mg mL−1 MTT solution was added into each well and incubated for 4 h further. Later, the culture medium was removed, and dimethyl sulfoxide (150 µL well−1) was added to each well to solubilize the formazan crystals with thorough shaking for 15 min. The absorbance was read at 570 nm in a SYNERGY H1 microplate reader .The effects of peptide fractions on endothelial cell viability were determined using the MTT assay. Briefly, HUVECs (1 × 105 cells mL−1 density. HUVECs were pretreated with peptide fractions (20 µg mL−1) for 24 h and then stimulated with 1 µg mL−1 of LPS for 3 h. After LPS treatment, cells were fixed in 4% paraformaldehyde in PBS  at 4 °C for 30 min, and then permeabilized with 0.25% Triton X-100 in PBS. Blocking was performed at room temperature for 1 h using PBS containing 1% BSA . Cover slips were then incubated with the primary antibody for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 subunit , diluted 1:400 in PBS including 0.25% BSA overnight at 4 °C. The next day, the cells were incubated for 1 h with fluorochrome-conjugated secondary antibody Alexa Fluor 488 , diluted 1:200 in PBS including 0.25% BSA. After removal of the secondary antibody, nuclei were stained with 1 µg mL−1 diamidino 2-phenylindole (DAPI)  for 1 min. Using the PermaFluor mounting medium , cover slips were mounted onto glass slides. Images were acquired using a Zeiss LSM10 confocal microscope at 40× magnification and analyzed using ImageJ software  [HUVECs were cultured on cover slips at 1 × 10MD, USA) .6 cells mL−1 in a 48-well plate and pretreated with peptide fractions (20 µg mL−1) for 24 h and then treated with 1 µg mL−1 LPS for different time points. After treatment, cells were detached and washed with 1× ice-cold PBS, and cell pellets were lysed with ice-cold RIPA (radioimmunoprecipitation assay) buffer  containing 1% protease  and 1% phosphatase  inhibitor cocktails. Equivalent amounts of protein were loaded on 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and then transferred to nitrocellulose membranes. Immunoblotting was conducted with targeted primary antibodies and peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies  dil. 1:10 000. Protein bands were detected by using the Clarity Western ECL Substrate Kit  and ChemiDoc MP Imaging System hardware and software . Protein bands were normalized to the loading control β-actin . Images of bands were analyzed by using ImageJ software  [Cells were seeded at a density of 5 × 106 cells mL−1 and pretreated with peptide fractions (20 µg mL−1) for 24 h and then treated with 1 µg mL−1 LPS for 12 h. Later, the culture medium was collected and stored at ≤ −70 °C. The cells were rinsed two times with PBS and then lysed within the wells by the addition of the Cell Extraction Buffer. After centrifugation at 2000× g for 5 min, cell lysates were stored at ≤ −70 °C. IL-1β released in the media and COX-2 cellular levels were determined using specific ELISA kits , as per the manufacturer’s directions. Detection was carried out using Gen5 software .Briefly, HUVECs were cultured in a 6-well plate at a density of 5 × 10p < 0.05 significance level was used to correlate the means by employing Duncan’s test.Every experiment was carried out in at least three independent experiments, and data are indicated as mean ± standard deviation. Results were statistically analyzed, using SPSS 19.0 version . All the data collected were used for a one-way analysis of variance (ANOVA). Bombyx mori pupae due to its broad specificity for cleavage sites [In this study, we optimized the proteolytic digestions of MPPC and EPPC in the presence of six different proteases along with hydrolysis time (0 to 360 min) to evaluate the possibility of releasing ACE-inhibitory hydrolysates with the highest activity. The effect of the relationship between applied enzymes at the same concentration and the incubation time on the DH of each protein concentrate (MPPC and EPPC) is plotted in ge sites ,13,17. TAlcalase (69.49 ± 1.04%) > Thermolysin (37.77 ± 1.33%) > Pepsin (33.22 ± 1.45%) > Flavourzyme (25.16 ± 0.32%) > Papain (23.37 ± 1.54%) > Trypsin (9.38 ± 1.08%).For EPPC, DH variation ranged from 5.59 to 65.73%. Thus, the relative highest DH of Eri pupae protein obtained by each enzyme after 360 min was the following B:Alcalase (65.73 ± 0.98%) > Flavourzyme (59.24 ± 1.25%) > Thermolysin (38.77 ± 1.33%) > Pepsin (26.05 ± 0.45%) > Papain (24.38 ± 1.54%) > Trypsin (11.57 ± 1.08%).G. sigillatus and T. molitor (28.1 to 33.8% at 3.0% (E/S)) and Purschke et al. [Locusta migratoria . The results were consistent with data  studied by Hall et al. [w/v) concentration. Such variation might be influenced by prior heat treatment at 90 °C that can affect DH. Compared to DH results achieved for A. diaperinus (20.5% (0.5% E/S), 20.3% (1.5% E/S) and 25.0–28.4% (3.0% E/S); 4 h) using Alcalase as presented by Sousa et al. [We also observed that only the control solution based on Thermolysin parameters achieved a hydrolysis rate of 7.2% for MPPC and 8.0% for EPPC after 360 min of incubation (not shown). This indicates that heating at 70 °C, the optimum temperature for Thermolysin activity, might have played a significant role in enhancing protein cleavage. The DH value seen in this study for Alcalase hydrolysate (Muga and Eri) after 2 h was potentially greater than the published data shown by Dion Poulin et al.  for G. se et al.  for Locul et al. ,13 aftera et al. . These dp > 0.05); 180 min (72.89 ± 1.36%) and 240 min (77.35 ± 1.57%) for Pepsin as shown in p > 0.05); 180 min (81 ± 2.36%) and 240 min (83.21 ± 2.51%) for Papain (p > 0.05); 60 min (75.31 ± 2.57%), 120 min (78.80 ± 1.79%) and 180 min (82.07 ± 2.56%) for Pepsin. It was noted that with the extended hydrolysis time, the ACE-inhibition activities exhibited by Alcalase-, Papain- and Pepsin-treated hydrolysates were mostly unstable for both types  against ACE. The IC50 values of the selected hydrolysates ranged between 0.12 ± 0.01 and 1.71 ± 0.29 mg mL−1, respectively  were slightly lower than the hydrolysates generated from Tenebrio molitor larva, Spodoptera littoralis and mulberry silkworm (B. mori) [50 values found in our study were higher than those of the ultrasonic-pretreated Alcalase-derived hydrolysates of silkworm pupae protein (91.3 µg mL−1) [Therefore, the hydrolysates that exhibited potent ACE-inhibitory activity >70% were finally selected and evaluated for their minimum inhibitory concentration  ,23,24, dµg mL−1) . Such a Since the proteolytic hydrolysis of total protein extracts can give rise to a number of active peptides in a hydrolysate, it seems reasonable to presume that the hydrolysates generated in our study might display versatile behaviors. Therefore, the antioxidant properties of eight selected hydrolysates shown in  were als50 values for DPPH radical scavenging activity ranged from 0.48 ± 0.27 to 3.88 ± 0.91 mg mL−1, where hydrolysate Pap_E240min was the most effective at scavenging DPPH, and Pep_E60min (Pepsin-treated Eri pupae protein hydrolysate at 60 min) was the least. In contrast to DPPH results, Pep_E60min hydrolysate exhibited the strongest ABTS radical scavenging activity with no significant difference from Pap_E240min hydrolysate (p > 0.05), while the lowest value was shown by Pep_M180min (Pepsin-treated Muga pupae protein hydrolysate at 180 min). The EC50 values attained by the ABTS method were higher than those found by the DPPH method, which signifies that DPPH was more sensitive towards the scavenging ability of protein hydrolysates at lower sample concentrations.The electron-donating ability in the tested hydrolysates was investigated using the DPPH and ABTS radical scavenging assays: the primary mechanism by which antioxidants can inhibit oxidative propagation. Tested hydrolysates demonstrated that their EC50 value obtained for the raw (5.3 µg/mL), boiled form of T. molitor (28.9 µg/mL), baked G. sigillatus and S. gregaria (28.5 µg/mL) [Zophobas morio (4.6 µg/mL) against ABTS, and Amphiacusta annulipes (19.1 µg/mL) and silkworm protein (57.91 µg/mL) hydrolysate against DPPH achieved after in vitro gastrointestinal digestion [Based on our data, it can be confirmed that the selected hydrolysates also efficiently displayed antioxidant capacities. However, the strongest antiradical property was lower than the IC5 µg/mL) . These vigestion ,27.50, 0.49 ± 0.25 µg mL−1) and Pap_E240min  hydrolysates had significantly higher binding abilities to ACE (p < 0.05)  and ABTS  free radical scavenging ability. Otherwise, fraction three of the Pap_E240min hydrolysate held a higher DPPH  and ABTS scavenging ability . Altogether after filtration, the fractions < 3 kDa showed significantly higher ACE-inhibitory and antioxidative potentials. The highest antioxidant potential of <3 kDa peptide fraction (from Eri pupae) had a lower EC50 value than the studied fractions obtained from T. molitor (24.31 µg/mL) [When the ACE-inhibitory and antioxidative properties of eight selected hydrolysates were analyzed, the hydrolysates Alc_M60min and Pap_E240min presented the most efficient bioactivities relative to the others. From a pharmacology point of view, molecules with lower molecular weight generally have greater pharmaceutical properties and nutraceutical uses . Therefo < 0.05) . However < 0.05)  dependin−1) and F6 (0.03 µg mL−1) acquired from Alc_M60min_F3 and Pap_E240min_F3, respectively, were preferred. Specified fractions were accumulated, lyophilized and then used in in vitro experiments to evaluate their activities.After fractionation through ultrafiltration membranes, the peptides present in the Alc_M60min and Pap_E240min hydrolysates were segregated into specified sizes and with discrete hydrophilic and hydrophobic proportions. To achieve ACE-inhibitory peptides, the most potent ultrafiltrate fractions, Alc_M60min_F3 A and PapInflammation is one of the key participants in the progression of hypertension and its downstream effects. Furthermore, inflammatory processes in hypertension are strongly associated with oxidative stress events, generating a vicious circle of oxinflammation ,2,3,4. T−1 to 1000 µg mL−1. As indicated in −1, reduced cellular viability below 70%. Thus, for the subsequent experiments, 20 µg mL−1 of each peptide fraction was chosen to evaluate the anti-oxinflammatory activities in endothelial cells.To characterize the effects of peptide fractions on cell viability, HUVECs were treated for 24 h with different doses of F4 and F6, ranging from 10 µg mL−1 LPS stimulation for 3 h promoted significant p65 nuclear translocation, whereas pretreatment with F6 for 24 h was significantly effective in preventing this effect. However, F4 did not significantly decrease LPS-induced NF-κB activation, although a slightly decreasing trend was evident. Our results suggest that the hydrolysate fractions can promote anti-inflammatory effects due to their ability to regulate NF-κB. In fact, we observed that treatment with the hydrolysate fractions could suppress the LPS-induced phosphorylation of p38 MAPK . As shown in p38 MAPK B, a critTo further confirm whether the silkworm F4 and F6 peptide fractions could suppress inflammation, we evaluated the levels of cyclooxygenase-2 (COX-2), which is an enzyme responsible for the production of the proinflammatory mediators such as prostaglandins . As showSince IL-1β is a proinflammatory cytokine involved in hypertension and cardiovascular disease , we alsoDue to the antioxidant activity shown by the <3KDa fractions of both F4 and F6, as a next step in the evaluation of their potential ability to affect oxinflammatory processes, we analyzed levels of the antioxidant enzymes heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) in LPS-treated endothelial cells. LPS treatment alone did not affect HO-1 expression levels, but slightly increased NQO1 expression compared to control cells A,B. HoweThis study illustrates the efficient retrieval of underexploited protein from non-mulberry silkworm waste generated after the reeling process, along with the production of its hydrolysates, which displayed interesting bioactivities. We have demonstrated that enzyme-specific hydrolysis is a very important step in validating the production of multifunctional peptides that could have potential benefits for human health. In fact, our results indicate a possible correlation between the ACE-inhibitory and antioxidant properties of <3KDa peptide fractions, observed in vitro, and their potential anti-oxinflammatory activities, validated in LPS-treated endothelial cells. Future research is required to isolate and characterize potent peptides for each bioactivity and to evaluate the inhibition modes of ACE, the mechanisms involved in the anti-oxinflammatory activity and the efficacy and bioavailability of these peptides in vivo. Overall, our findings provide initial data to support the therapeutic potential of using non-mulberry silkworm pupae proteins as a source to generate multifunctional hydrolysates, which can be incorporated into pharmaceutical products such as functional foods and considered as natural alternatives to treat hypertension and related cardiovascular diseases.


Ircinianin lactones B and C (7 and 8) represent new ircinianin terpenoids with a modified oxidation pattern. Despite their labile nature, the structures could be established using a combination of spectroscopic data, including HRESIMS and 1D/2D NMR techniques, as well as computational chemistry and quantum-mechanical calculations. In a broad screening approach for biological activity, the class-defining compound ircinianin (1) showed moderate antiprotozoal activity against Plasmodium falciparum (IC50 25.4 μM) and Leishmania donovani (IC50 16.6 μM).Two new ircinianin-type sesterterpenoids, ircinianin lactone B and ircinianin lactone C ( Ircinia is well known for producing sesterterpenes (C25-terpenes) c = 0.004, MeOH) +  ; IR : Yellow amorphous powder; HPLC-UV profile: see m/z 413.2325 [M + H]+ ; 13C NMR data, see Oxoircinianin (4): White amorphous solid; HPLC-UV profile: see m/z 470.2540 [M + H]+ ; 1H and 13C NMR data, see Ircinianin lactam A (5): Yellow oil; HPLC-UV profile: see m/z 486.2486 [M + H]+ ; 1H and 13C NMR data, see Oxoircinianin lactam A (6): Yellow amorphous powder; HPLC-UV profile: see m/z 413.2327 [M + H]+ ; 1H and 13C NMR data, see Ircinianin lactone A (7): Yellow amorphous powder; HPLC-UV profile: see m/z 443.2431 [M + H]+ : Yellow amorphous powder; HPLC-UV profile: see m/z 429.2281 [M + H]+ , were adapted from the crystal structure of -5′-[(E)-5-(furan-3-yl)-2-methyl-pent-1-en-1-yl]-3-hydroxy-3′,4,7′-tri-methyl-1′,2′,3′,3a′,5′,7a′-hexa-hydro-5H-spiro--5-one, as published in [R- and the S-enantiomer of 8, 30 distinct conformations were retrieved. Subsequently, these conformations were submitted to a free geometry optimization using B3-LYP/SV(P) [Both, the ished in . MOE  ,58,59 emYP/SV(P)  in TURBOYP/SV(P) . The dieYP/SV(P)  within T5 colony-forming units (CFU)/mL. After an overnight incubation at 37 °C, the MIC was read as the lowest compound concentration preventing visible bacterial growth. The strain panel included representative species of nosocomial pathogens, known as “ESKAPE” bacteria. Specifically, the following strains were used: Enterococcus faecium BM 4147-1, Staphylococcus aureus ATCC 29213, Klebsiella pneumoniae ATCC 12657, Acinetobacter baumannii 09987, Pseudomonas aeruginosa ATCC 27853, and Enterobacter aerogenes ATCC 13048. Bacillus subtilis 168, Escherichia coli ATCC 25922, and Mycobacterium smegmatis mc2 155 ATCC 700084 were used as further reference strains.The minimal inhibitory concentration (MIC) was determined as described previously  in catio1 was tested against the human cytomegalovirus (HCMV) and the severe acute respiratory syndrome coronavirus 2 (SARS CoV2), essentially as reported before [The antiviral activity of d before ,66.Activity against HCMV: Human foreskin fibroblasts (HFF) (<25 passages) cultured in DMEM cell culture medium supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin were seeded to a final cell concentration of 1 × 104 cells per well. After 24 h of incubation at 37 °C, with 5% CO2 and 95% relative humidity and medium exchange, 1 was added at the concentrations of 1 and 10 µM. Infection was performed directly afterwards using HCMV TB40EdelUL16EGFP with MOIs of 0.3 and 1. 120 h post infection, the cells were fixed and permeabilized with 80% acetone in H2O at room temperature for 5 min. Intracellular staining for immediate early HCMV proteins was conducted by a 90 min incubation of 1:1000 IE1/2 HCMV antibody in PBS at 37 °C, a subsequent 45 min incubation of 1:2000 ALEXAFluor514 in PBS at 37 °C and a nuclear counterstaining with DAPI 1:20,000 in PBS for 8 min at room temperature. Each step was followed by a washing step, three times with PBS. Images were taken using the Cytation3 multiplate reader.Activity against SARS-CoV2: CaCo2 cultured in DMEM cell culture medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% non-essential amino acids were seeded to a final cell concentration of 1 × 104 cells per well. After 24 h of incubation at 37 °C, with 5% CO2 and 95% relative humidity and medium exchange, 1 was added at the concentrations of 1 and 10 µM. Infection was performed directly afterwards using SARS-CoV2 mNeonGreen at 1:2000 dilution. 48 h post infection, the supernatant was taken to reinfect another plate of Caco2 for evaluation of viral supernatant as a sign for active replication of the virus. The initially infected cells were fixed with 2% paraformaldehyde in PBS and stained with Hoechst 33342 at 37 °C for 10 min. Afterwards, the cells were washed with PBS 3 times. 48 h post infection, the reinfected cells were fixed and stained as described above. Images were taken using the Cytation3 multiplate reader.L. sigmodontis adult female worms were isolated from chronically infected cotton rats (UKB). Three L. sigmodontis female adult worms per condition were cultured in supplemented Minimal Essential Medium (MEM) on a LLCMK2 cell layer, as previously described [1 was tested at concentrations of 0.1, 1 and 10 µM. Filarial viability was assessed by determining the mean worm motility scores on a scale of 0 (immotile) to 2 (maximum motility) before drug addition and at 1 h, 1 day, 2 day, 4 day, 6 day, 8 day, 10 day, 12 day and 14 day post drug addition. The drug-containing media was renewed every other day. Depletion of Wolbachia endosymbionts from female adult worms was analyzed after two weeks of in vitro culture and determined as previously described [Wolbachia ftsZ.escribed . The posescribed  using prActivity against Trypanosoma brucei rhodesiense STIB900. The stock was isolated in 1982 from a human patient in Tanzania, and after several mouse passages, cloned and adapted to axenic culture conditions [3 bloodstream forms of T. b. rhodesiense STIB 900 in 50 µL were added to each well and the plate was incubated at 37 °C under a 5% CO2 atmosphere for 70 h. 10 µL of a resazurin solution  was then added to each well and incubation continued for a further 2–4 h [50 values by linear regression [50 0.013 ± 0.006 μM).nditions . Minimumgression  and 4-paActivity against T. cruzi. Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtiter plates at 2000 cells/well in 100 μL RPMI 1640 medium with 10% FBS and 2 mM L-glutamine. After 24 h, the media in each well was removed and replaced with 100 μL of fresh media containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 containing the β-galactosidase (lacZ) gene [1 from 100 to 0.002 μg/mL. After 96 h of incubation, the plates were inspected under an inverted microscope to assess growth and sterility of the controls. Then, the substrate CPRG/Nonidet (50 μL) was added to all wells. A color reaction developed within 2–6 h and could be read photometrically at 540 nm. Data were analyzed with the graphic program Softmax Pro (Molecular Devices), which calculated IC50 values by linear regression [50 2.31 ± 1.15 μM).cZ) gene . After 4gression  and 4-paActivity against L. donovani axenic amastigotes. Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown in axenic culture at 37 °C in SM medium [2 in air. 100 µL of culture medium with 105 amastigotes from axenic culture with or without a serial drug dilution were seeded in 96-well microtiter plates. Serial drug dilutions ranged from 100 to 0.002 μg/mL. After 70 h of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10 μL of resazurin (12.5 mg resazurin dissolved in 100 mL distilled water) were then added to each well and the plates were incubated for another 2 h. Then, the plates were read with a Spectramax Gemini XS microplate fluorometer  using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. From the sigmoidal inhibition curves the IC50 values were calculated by linear regression [50 0.65 ± 0.27 μM)M medium  at pH 5.gression  and 4-paActivity against P. falciparum. In vitro activity against erythrocytic stages of P. falciparum was determined using a 3H-hypoxanthine incorporation assay [3 (2.1 g/L), neomycin (100 U/mL), AlbumaxR (5 g/L) and washed human red cells A+ at 2.5% haematocrit (0.3% parasitaemia). Serial drug dilutions ranged from 100 to 0.002 μg/mL. The 96-well plates were incubated in a humidified atmosphere at 37 °C; 4% CO2, 3% O2, 93% N2. After 48 h 50 μL of 3H-hypoxanthine (=0.5 μCi) was added to each well of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Betaplate™ cell harvester  and the red blood cells were transferred onto a glass fiber filter then washed with distilled water. The dried filters were inserted into a plastic foil with 10 mL of scintillation fluid and counted in a Betaplate™ liquid scintillation counter . IC50 values were calculated from sigmoidal inhibition curves by linear regression [50 0.006 ± 0.002 μM).on assay ,75 againon assay . Compoungression  using MiOne dose NCI-60 panel: 1 was selected for the anticancer drug screening service as a part of the Developmental Therapeutics Program at the National Cancer Institute (NCI). In vitro tumor growth inhibitory effects were explored using a standard protocol with a single high dose test against a panel comprising 60 human cancer cell lines [1 was conducted.ll lines ,78. It iIn vitro cytotoxicity assay with HeLa cells: The cytotoxicity test against the HeLa human cervical carcinoma cell line was performed as described previously [4 cells per well. After 24 h of incubation at 37 °C, with 5% CO2 and 95% relative humidity, resazurin was added at a final concentration of 200 μM, and cells were again incubated overnight. Cell viability was assessed by determining the reduction of resazurin to the fluorescent resorufin. Fluorescence was measured in a TECAN Infinite M200 reader at an excitation wavelength of 560 nm and an emission wavelength of 600 nm in relation to an untreated control.eviously  in RPMI In vitro cytotoxicity assay with L-6 cells. Assays were performed in 96-well microtiter plates, each well containing 100 μL of RPMI 1640 medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum, and 4000 L-6 cells  [50 values were calculated by linear regression [50 0.011 ± 0.005 μM).oblasts) ,80. Serigression  and 4-paCiona savignyi) and the blue mussel . Methods followed those published by Grant et al. [Isochorysis galbana until ready to spawn. Larval spawning and rearing procedures followed previously described methods for C. savignyi [M. gallorprovincialis [1 ranging from 0.1–100 µg/mL. Controls were included, and three replicates were performed in all cases. After 5 days of incubation at 18 ± 1 °C, the number of successfully settled and metamorphosed individuals were counted in each well. Sigmoidal dose-response relationships were explored using R Statistical Software [Antifouling activity was assessed via inhibition of settlement and metamorphosis of larvae of the Pacific transparent sea squirt (t et al. . Adults savignyi  and M. gincialis . CompeteSoftware  to deter