CaM-dependent KCNE4 dimerization controls KCNE4 membrane targeting and modulates its interaction with Kv1.3. KCNE4, which is highly retained at the ER, contains an important ER retention motif near the tetraleucine motif. Upon escaping the ER in a CaM-dependent pattern, KCNE4 follows a COP-II-dependent forward trafficking mechanism. Therefore, CaM, an essential signaling molecule that controls the dimerization and membrane targeting of KCNE4, modulates the KCNE4-dependent regulation of Kv1.3, which in turn fine-tunes leukocyte physiology.The voltage-dependent potassium channel Kv1.3 participates in the immune response. Kv1.3 is essential in different cellular functions, such as proliferation, activation and apoptosis. Because aberrant expression of Kv1.3 is linked to autoimmune diseases, fine-tuning its function is crucial for leukocyte physiology. Regulatory KCNE subunits are expressed in the immune system, and KCNE4 specifically tightly regulates Kv1.3. KCNE4 modulates Kv1.3 currents slowing activation, accelerating inactivation and retaining the channel at the endoplasmic reticulum (ER), thereby altering its membrane localization. In addition, KCNE4 genomic variants are associated with immune pathologies. Therefore, an in-depth knowledge of KCNE4 function is extremely relevant for understanding immune system physiology. We demonstrate that KCNE4 dimerizes, which is unique among KCNE regulatory peptide family members. Furthermore, the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4 is a structural platform in which Kv1.3, Ca Furthermore, Kv channels mediate additional cellular functions, such as control of cell cycle progression, cell activation and apoptosis2. Kv1.3 is mainly expressed in the nervous and immune systems. Leukocytes present a limited repertoire of potassium channels, and Kv1.3 is essential during the immune system response4. T lymphocyte activation triggers the translocation of the channel to the immunological synapse between T lymphocytes and antigen-presenting cells, maintaining the driving force for sustained calcium signaling5.Voltage-dependent potassium (Kv) channels participate in the resting membrane potential and repolarization of excitable cells6. The KCNE family contains five single-span membrane auxiliary peptides (KCNE1-5) that modulate potassium channels7. KCNEs are present in the immune system, and their expression is regulated by several insults, highlighting the importance of this peptide family in leukocytes9. KCNE4, the largest member of the family, triggers dominant-negative effects on Kv channels. For instance, this subunit decreases Kv1.1, Kv1.3, Kv2.1 and Kv7.1 potassium current12. KCNE4 inhibits Kv7.1 current, not by altering the surface abundance of the channel but by altering the channel localization in lipid raft microdomains14. Functional interactions between KCNE4 and Kv7.1 require the presence of calmodulin (CaM). In fact, KCNE4 interacts with Ca2+/CaM through a tetraleucine domain in the juxtamembrane of the KCNE4 C-terminus15.Regulatory subunits modulate the function, trafficking and subcellular localization of Kv channels18. KCNE4, tightly regulated in leukocytes, inhibits Kv1.3 function by hindering the surface expression and lipid raft localization of the channel19. Kv1.3 and KCNE4 interact via the C-terminal domains of both proteins21. While the implicated molecular determinants of Kv1.3 are not evident, the tetraleucine domain of KCNE4 clearly participates in this interaction21. The tetraleucine motif hub forms a competing mechanism that directs the KCNE4 association with either CaM or Kv1.3, tuning the membrane targeting of the channel. The fact that several dileucine-rich domains participate in protein–protein interactions and that the tetraleucine signature of KCNE4 facilitates the association with CaM and Kv1.3 points to whether this element could be involved in further interactions regulated by CaM. In fact, KCNE4 does not inhibit Kv7.1 by impairing the association to CaM15. Furthermore, CaM interaction improves the surface targeting of Kv7.222. Therefore, the evidence suggests that the CaM interaction, and hence the molecular determinants involved, is an important intracellular signaling mechanism of ion channel function.Evidence demonstrates that alterations in Kv1.3 and KCNE4 are associated with immune system pathologiesWe show in this work that the tetraleucine motif in the juxtamembrane region of the KCNE4 C-terminus functions as a multiple platform for subunit assembly and protein interaction. In contrast with other members of the KCNE family, KCNE4 forms stable dimers. The tetraleucine domain, unique to KCNE4, mediates this dimerization. The association of KCNE4 with CaM, as well as with Kv1.3, disrupts the dimer and facilitates the cell surface expression of KCNE4. CaM fine-tunes the association capacity of KCNE4 with Kv1.3, thereby modulating channel-dependent immune responses. Our data are of physiological relevance because CaM, a very important intracellular signaling molecule, controls Kv1.3-dependent KCNE4-related immunological events.23. However, no protein data are available. We showed that KCNE4 was differentially expressed in several rat tissues and leukocytes 30. Kv1.3-loopBAD-GFP, KCNE4-loopBAD-GFP and KCNE1-loopBAD-GFP were expressed in HEK 293 cells, and GFP fluorescent immobile spots were monitored  ) or in combination with ERRM signaling (KCNE4(RM&L)). KCNE2, which neither contains tetraleucine motifs nor associates with Kv1.3, has been widely characterized20. Therefore, a KCNE2 mutant (KCNE2(L83-86)) with an embedded tetraleucine motif was also used  of Ca2+  in the C-terminal juxtamembrane region. Altering the LLDLL sequence, either full or in part, increases the surface levels of the channel45. Moreover, mutation of the carboxyl terminal dileucine sequence of the LHβ subunit increases trafficking and exocytosis46. In this context, our data indicated that disrupting or masking the tetraleucine motif hinders dimerization, thereby facilitating membrane targeting of KCNE4. Therefore, in a KCNE4 monomer, although it contains an ERRM, the strength of the retention signal may be counterbalanced by associating with CaM. This mechanism is in agreement with the Ca2+/CaM-dependent regulation of KCNE4 of Kv channels, such as Kv7.1 or Kv1.3. CaM, upon calcium stimuli, can interact with KCNE4 at the tetraleucine motif20. Therefore, CaM fine-tunes the dimerization and promotes KCNE4 anterograde trafficking through a COPII-dependent mechanism. Thus, Ca2+ signaling controls the final fate of KCNE4, which in turn modulates Kv1.3 function, which is profoundly related to the Ca2+ trigger in leukocytes. The precise balance between several interactions in a unique cluster, masking the interaction sites to prevent competing associations, fine-tunes the final function of a multitask channel such as Kv1.316. A similar cloaking mechanism is evident for E-cadherin. A dileucine motif in the juxtamembrane cytoplasmic domain is required for E-cadherin endocytosis. When mutated, this protein cannot be internalized. P-120, an E-cadherin interactor, associates and masks this motif, inhibiting endocytosis47. Furthermore, our data indicate that KCNE4 contains routing information to the plasma membrane via COPII-dependent mechanisms. KCNEs, which contain strong ERRM signals at their C-terminus, share a notable intracellular retention capacity. Surprisingly, without canonical sequences, the COPII-dependent mechanism is a major characteristic of KCNE peptides because, similar to KCNE4, KCNE1 uses this route27. In this sense, N-glycosylation facilitates KCNE anterograde trafficking through the secretory pathway, but the specific mechanisms have not been dissected48. The balance between forward trafficking and retention signaling is highly skewed toward the latter in KCNEs. Regardless of whether the assembly of KCNE and Kv channels takes place at the cell surface or through the secretory pathway, CaM controls cell surface channels by modulating KCNE4 trafficking49.Evidence links dileucine clusters with protein trafficking. For instance, the ClC-2 chloride channel, which might compensate for the malfunction of CFTR in absorptive epithelia during cystic fibrosis, contains a dileucine motif. Mutations of this signature alter the basolateral targeting of the channel20. Our data support the dimerization of KCNE4 as a regulating mechanism for such interactions and CaM displaced KCNE4 dyad formation. These data, similar to those of Kv7.1/KCNE1 and Kv7.1/KCNE3, further support that single monomeric forms of KCNE4 occupy the cleavage between subunits within the Kv tetrameric structure29. However, in contrast to Kv7.1, Kv1.3 does not need CaM to associate with KCNE415. CaM indirectly interacts with Kv1.3 in a Ca2+-dependent manner, forming a multiprotein complex with CaM kinase II (CaMKII)51. Although the regulation of Kv1.3 by Ca2+/CaM in leukocytes is under debate, CaM-dependent KCNE4 dimerization situates this important signaling molecule in the equation. Ca2+- and CaM-dependent signaling pathways are key to T cell activation. The immunological synapses enriched in lipid rafts concentrate Ca2+-dependent targets. KCNE4, retaining Kv1.3 at the ER, impairs the localization of Kv1.3 to lipid rafts. Therefore, our data are of physiological relevance because indicates that the KCNE4 tetraleucine motif is an associative platform in which different targets may compete for signaling interactions fine-tuning the cell physiology.We demonstrated that Kv1.3, KCNE4 and CaM are closely related because they interact via the tetraleucine cluster in KCNE4. A balanced competition warrants no tripartite complexes26. Mouse KCNE4 in pSGEM was obtained from M. Sanguinetti . mKCNE4 was introduced into pEYFP-N1, pECFP-N1 and pEGFP-N1 (Clontech). hKCNE1-HA and hKCNE2-HA, obtained from S. de la Luna , were introduced into pECFP-N1, pEYFP-N1 and pEGFP-N1 (Clontech). CaMYFP was obtained from A. Villarroel (Universidad País Vasco-CSIC). An endoplasmic reticulum marker (pDsRed-ER), soluble cyan fluorescent protein (pECFP-N1), green fluorescent protein (pEGFP-N1) and yellow fluorescent protein (pEYFP-N1) were obtained from Clontech. The plasma membrane marker Akt-PH-pDsRed (pDsRed-tagged pleckstrin homology (PH) domain of Akt) was a kind gift from F. Viana . The dominant-negative GTPase mutant HA-Sar1H79G was provided by R. Pepperkok 21.The rKv1.3 in pRcCMV, provided by T.C. Holmes , was subcloned into pEYFP-C1, pECFP-C1 and pEGFP-C1 (Clontech). hKv1.5 has been widely analyzed52.For counting the bleaching steps in nonmoving spots, a loopBAD-tag was added. The loop-BAD tag was introduced into GFP-Kv1.3, KCNE1-GFP and KCNE4-GFP at NruI restriction sites. The Kv2.1-loopBAD-YFP and BirA coding plasmids were previously described20. All constructs and mutants were verified by automated DNA sequencing.The KCNE4-CFP tetraleucine motif (L69–72) was mutated to alanine by single-site–directed mutagenesis using a QuikChange multisite-directed mutagenesis kit (Agilent Technologies), generating the KCNE4(L69–72A)-CFP mutant. A tetraleucine motif similar to that in KCNE4 was incorporated into KCNE2-CFP (KCNE2(L83–86)-CFP). KCNE4 (ERRM), with ERRM disruption, and KCNE4 (RM&L), containing both modified ERRM and tetraleucine motifs, have been fully characterized previouslyHEK-293 cells (Sigma-Aldrich) were cultured in DMEM (LONZA) containing 10% fetal bovine serum (FBS) supplemented with penicillin , streptomycin (100 μg/ml) (Gibco), 4.5 g/l glucose and L-glutamine (4 mM).21.For confocal imaging and coimmunoprecipitation experiments, cells were seeded (70–80% confluence) in either 6-well dishes containing poly-D-lysine-coated coverslips or 100-mm dishes. Metafectene PRO (Biontex) was used for transfection according to the supplier’s instructions. The amount of transfected DNA was 4 μg for a 100-mm dish and 500 ng for each well of a 6-well dish. Next, 4–6 h after transfection, the mixture was removed from the dishes and replaced with fresh culture medium. All experiments were performed 24 h after transfection30.For patch-clamp experiments, trypsinized cells from a confluent culture in a 100-mm dish were electroporated with 1 μg of DNA using a Bio-Rad Gene Pulser Xcell system with a 0.2-cm gap cuvette and a single 110-V 25-ms pulse35.For total internal reflection fluorescence (TIRF) microscopy experiments, trypsinized cells from a confluent culture in a 100-mm dish were electroporated with 25–100 ng of the desired DNA plus 100 ng of BirA DNA (encoding a biotin ligase to biotinylate the loopBAD-tagged proteins) using a Bio-Rad Gene Pulser Xcell system, as previously described. Transfected cells were plated on 35-mm glass-bottom dishes (MatTek) previously coated with collagen and EZ-Link NHS-PEG12-Biotin . The next day, TIRF experiments were performed after incubation of the cells with neutravidin (50 nM) to immobilize channels onto glass as previously described21.HEK-293 cells were washed twice in cold PBS and lysed on ice with a lysis solution  supplemented with protease inhibitors . Lysates were gently mixed for 10 min and spun . The supernatant was transferred to a new tube, and the protein contents were determined by using a Bio-Rad protein assay21.For coimmunoprecipitation, 1 mg of protein added to lysis buffer at a final concentration of 500 µl and used for immunoprecipitation  supplemented with protease inhibitors. Samples were precleared with 50 µl of protein A-Sepharose beads  for 1 h at 4 °C with gentle mixing. Next, each sample was incubated in a small chromatography column (Bio-Rad Micro Bio-Spin™ chromatography columns), which contained 2.5 µg of anti-GFP antibody previously cross-linked to protein A-Sepharose beads, for 2 h at room temperature (RT) with gentle mixing. The columns were centrifuged for 30 s at 1,000 × g. Supernatants were stored at -20 °C. The columns were washed four times with 500 µl of lysis buffer and centrifuged for 30 s at 1,000 × g. Finally, the columns were incubated with 100 µl of 0.2 M glycine (pH 2.5) and spun for 30 s at 1,000 × g for elutionIrreversible cross-linking of the antibody to the Sepharose beads was performed after incubation of the antibody with protein A-Sepharose beads for 1 h at RT. The beads were then incubated with 500 µl of 5.2 mg/ml dimethyl pimelimidate (Pierce) for 30 min at RT with gentle mixing. The columns were then washed four times with 500 µl of 1 × TBS, four times with 500 µl of 0.2 M glycine (pH 2.5) and three more times with 1 × TBS . Finally, the columns were incubated with protein lysates to perform immunoprecipitation as described above.2O and boiled for 7 min. Elutions were briefly centrifuged, and the supernatans were prepared for western blotting21.For the coimmunoprecipitation experiments in the presence of CaM, the samples were precleared for 1 h at 4 °C by mixing samples with 50 µl of protein A-Sepharose beads  previously washed with 1 × TBS and centrifuged for 30 s at 5,000 × g. Next, supernatants were incubated for 2 h at RT with 8 ng of anti-HA antibody (Proteintech) previously cross-linked to Protein A-Sepharose beads. The samples were centrifuged , and the pellet was washed 3 times with 1 × TBS buffer. Finally, proteins were eluted upon the addition of 20 µl of SDS Laemmli loading buffer (5x) and 80 µl of Hhttps://arriveguidelines.org). Rats were briefly anesthetized with isoflurane, and tissues were extracted immediately after euthanasia. Tissues were homogenized in RIPA lysis buffer  supplemented with protease inhibitors. Total lysates were spun for 10 min at 10,000 × g to remove debris. Supernatants were used to analyze protein expression by western blot.All experimental protocols were approved by the ethical committee of the Universitat de Barcelona in accordance with the European Community Council Directive 86/609 EEC. We also confirm that all experiments were carried out in compliance with the ARRIVE guidelines (Protein samples (50 μg), supernatants, immunoprecipitates and total tissue lysates were boiled in Laemmli SDS loading buffer and separated by 10–15% SDS-PAGE. Next, the proteins were transferred to PVDF membranes  and blocked in 0.2% Tween-20-PBS supplemented with 5% dry milk before immunoreaction with antibodies. Filters were immunoblotted with antibodies against Kv1.3 , HA , KCNE4 , GFP  or calmodulin . Finally, the membranes were washed with 0.05% Tween 20 PBS and incubated with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad).The dimerization of KCNE4 WT-CFP and KCNE4 (L69–72A)-CFP was analyzed in non-denaturing conditions. Briefly, total lysates were mixed with sample buffer  and loaded in a non-denaturizing SDS-PAGE (5% acrylamide/bis-acrylamide (30%-0.8% w/v), 0.29 M Tris–HCl pH 8.8, 0.1% SDS, 0.1%). Western Blot was performed as abovementioned and PVDF membranes were incubated with anti-GFP  and horseradish peroxidase-conjugated secondary antibodies (Bio-Rad).2, 1 mM EGTA and 1% Triton X-100; pH 8.5). One-half of the proteins was incubated with 5 mM DMP (dimethyl pimelimidate) for 1 h, while the other half was maintained for use as controls. The reaction was stopped by adding 1 ml of 0.5 M (pH 6.8). Protein samples (50 μg) were boiled in Laemmli SDS loading buffer and separated on 7% SDS-PAGE gels.Twenty-four hours after transfection, HEK-293 cells were washed twice in cold PBS and lysed on ice with lysis buffer  150 NaCl, 5 KCl, 10 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES at pH 7.4. Whole-cell currents were recorded using the patch-clamp technique in the whole-cell configuration with a HEKA EPC10 USB amplifier (HEKA Elektronik). PatchMaster software (HEKA) was used for data acquisition. We applied a stimulation frequency of 50 kHz and a filter at 10 kHz. The capacitance and series resistance compensation were optimized. In most experiments, we obtained 80% compensation of the effective access resistance. Micropipettes were prepared from borosilicate glass capillaries (Harvard Apparatus) using a P-97 puller (Sutter Instrument) and fire polished. The pipettes had a resistance of 2–4 MΩ when filled with a solution containing (in mM): 4 NaCl, 150 KCl, 1 MgCl2, 0.5 EGTA, 5 K2ATP, and 10 HEPES at pH 7.4. Cells were clamped at a holding potential of − 80 mV. To evoke voltage-gated currents, cells were stimulated with 200 ms square pulses to + 60 mV. Data analysis was performed using FitMaster (HEKA) and SigmaPlot 10.0 software (Systat Software). All recordings were performed at room temperature (21–23 °C).HEK-293 cells were trypsinized 24 h after transfection and plated on 35-mm glass-bottom dishes coated with Matrigelhttp://www.rcsb.org/pdb). The N-terminus, transmembrane domain and proximal C-terminus (residues 1–98), which contains the tetraleucine motif, were modeled with KCNE1 (PDB ID: 2K21). The final molecular graphic representation was created using PyMOL v1.4.1 (http://pymorl.org/) as previously described20.KCNE4 was modeled using high-resolution templates of homologs available from the Protein Data Bank  with house Mowiol mounting media. The coverslips were allowed to dry for at least one day before imaging. All procedures were performed at RT. All images were acquired with a Leica TCS SP2 AOBS microscope (Leica Microsystems) equipped with argon and helium–neon lasers. All experiments were performed with a 63 × oil-immersion objective lens NA 1.32. Colocalization analysis was performed with ImageJ software  as previously described. A pixel-by-pixel colocalization study using JACoP  was used. Mander’s overlap coefficients (MOC), which are proportional to the amount of fluorescence of the colocalizing pixels in each color channel, were obtained21. In some experiments the colocalization of KCNE4 with some subcellular markers was analyzed. HEK 293 cells were co-transfected with KCNE4 and the endoplasmic reticulum marker  and the plasma membrane marker . In addition, KCNE4 transfected cells were further processed for immunocytochemistry. Thus, after fixation, cells were further permeabilized with 0.1% Triton X-100 for 20 min. After 60 min of incubation with blocking solution , the cells were incubated with anti-GM130  and anti-TGN46 2F11  in 10% goat serum and 0.05% Triton X-100 overnight at 4 °C for cis-Golgi and trans-Golgi networks respectively. Next, the cells were incubated with Cy5-conjugated secondary antibodies for 2 h at RT and further mounted with Mowiol.For confocal image acquisition, cells were seeded on polylysine-coated coverslips and transfected 24 h later. The next day, the cells were quickly washed twice, fixed with 4% paraformaldehyde for 10 min, and washed three times for 5 min each time with PBS-KCFPafter – FCFPbefore)/FCFPbefore]*100, where FCFPafter was the fluorescence of the donor after bleaching and FCFPbefore was the fluorescence before bleaching. The loss of fluorescence as a result of scanning was corrected by measuring the CFP intensity in the unbleached part of the cell20.The fluorescence resonance energy transfer (FRET) via the acceptor photobleaching technique was measured in discrete ROIs (regions of interest). Fluorescent proteins from fixed cells were excited with the 458 nm or 514 nm beam lines using low excitation intensities. Next, 475 to 495 nm bandpass and > 530 nm longpass emission filters were applied. The YFP protein was bleached using maximum laser power. We obtained acceptor intensity bleaching of approximately 80%. Posteriorly, images of the donors and acceptors were taken. The FRET efficiency was calculated using the equation [ at 37 °C for 30 min before imaging. Neutravidin binds the cell surface biotinylated Kv1.3-, KCNE1- and KCNE4-loopBAD-GFP and, simultaneously, the biotinylated glass surface, fixing the cell surface proteins to monitor the fluorescently immobile GFP spots. The amount of DNA transfected was adjusted to achieve a low membrane density for imaging and counting. Multiple spots were imaged, but the density was low enough to minimize the probability of two channels lying within the same diffraction-limited spot. Transfected HEK-293 cells were imaged within 24 h after electroporation in HEK physiological saline buffer consisting of (in mM) 146 NaCl, 4.7 KCl, 2.5 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES at pH 7.4. Videos were processed and analyzed using Volocity software. The intensity of the 6 × 6 pixel ROIs was added and calculated for all durations of the videos. The intensity of the ROIs was plotted against time. The GFP bleaching steps were counted and statistically analyzed. GFP bleaching experiments were performed with a Nikon Eclipse Ti Perfect-Focus system equipped with a TIRF/wide-field fluorescence microscope and AOTF-controlled 405, 488, 561 nm diode lasers (100 mW each), and an Intensilight wide-field light source. A 100 × PlanApo TIRF, 1.49 NA, objective lens was used for image acquisition. Emissions were collected through a Sutter Lambda 10–3 filter wheel containing the appropriate bandpass filters. The microscope was equipped with an Andor iXonEMCCD DU-897 camera, 512 × 512. For TIRF image acquisition, an incident angle of 63.3° was used52.The single bleaching quantitative approach was first described by Isacoff and collaboratorst-test, one-way ANOVA and Tukey’s post hoc test and two-way ANOVA were used for statistical analysis (GraphPad PRISM v5.01). P < 0.05 was considered statistically significant.The results are expressed as the means ± SE. Student’s Supplementary Information.


Streptomyces smyrnaeus UKAQ_23, isolated from the mangrove-sediment, collected from Jubail,Saudi Arabia, exhibited substantial antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), including non-MRSA Gram-positive test bacteria. The novel isolate, under laboratory-scale conditions, produced the highest yield (561.3 ± 0.3 mg/kg fermented agar) of antimicrobial compounds in modified ISP-4 agar at pH 6.5, temperature 35 °C, inoculum 5% v/w, agar 1.5% w/v, and an incubation period of 7 days. The two major compounds, K1 and K2, were isolated from fermented medium and identified as Actinomycin X2 and Actinomycin D, respectively, based on their structural analysis. The antimicrobial screening showed that Actinomycin X2 had the highest antimicrobial activity compared to Actinomycin D, and the actinomycins-mixture  against MRSA and non-MRSA Gram-positive test bacteria, at 5 µg/disc concentrations. The MIC of Actinomycin X2 ranged from 1.56–12.5 µg/ml for non-MRSA and 3.125–12.5 µg/ml for MRSA test bacteria. An in-silico molecular docking demonstrated isoleucyl tRNA synthetase as the most-favored antimicrobial protein target for both actinomycins, X2 and D, while the penicillin-binding protein-1a, was the least-favorable target-protein. In conclusion, Streptomyces smyrnaeus UKAQ_23 emerged as a promising source of Actinomycin X2 with the potential to be scaled up for industrial production, which could benefit the pharmaceutical industry. Staphylococcus aureus (VRSA), vancomycin-resistant Enterococci (VRE), Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumonia, and Enterobacter spp., are one of the major causes of death among hospitalized patients1. The resistance to most conventional antibiotics, i.e., second-and third-generation cephalosporins, fluoroquinolones, penicillin combined with a beta-lactamase inhibitor, and carbapenems2, form this list. Owing to the presence of extremely resistant bacteria, curing antibiotic-resistant nosocomial infections presents an alarming situation for public healthcare. This necessitates the discovery of new antimicrobial agents to cope with the life-threatening infections caused by MDR pathogens4. Microbes strive against fellow microbes for their existence because of limited space and nutrients in the natural ecosystem. Therefore, microbes generally develop several strategies for surviving in their environment, one of which is the production of antimicrobial agents5. Actinomycetes, a well-known, invaluable bacterial group found in multiple habitats, is responsible for producing a wide range of industrially valuable and medically significant compounds9. Streptomyces, a commercially invaluable and medically significant actinomycetes genus, produces a wide range of biologically active compounds, i.e., antibiotics, anticancer, antiviral, herbicidal, and insecticidal agents and that has kept the focus further for continuing studies on this genus and its products12. The discovery rate of novel bioactive compounds from the soil actinomycetes has decreased over the years, while the rate of re-isolation of known bioactive compounds has increased13. Many studies, however, indicated that the mangrove soil is still the primary source for the isolation of novel bioactive actinomycetes16. To prevent re-isolation of the same species and antibiotics, the target soil sample must be very precise. A mangrove sediment area, found in Jubail, Saudi Arabia, possessed a large, unexplored region that could be an opulent source for the isolation of novel actinomycetes. Therefore, the present study aimed to isolate the novel antibiotic-producing actinomycetes from the mangrove sediment samples of Jubail, Saudi Arabia. The current research demonstrated the isolation, identification, antimicrobial screening of a novel actinomycete strain, Streptomyces smyrnaeus UKAQ_23, along with the production, fermentation-optimization, isolation, purification, structure elucidation, and antimicrobial screening of the antimicrobial compounds produced by strain UKAQ_23. An in-silico molecular docking of isolated antibiotics was performed against various antimicrobial target proteins. A detailed intermolecular interaction analysis with the binding energies and docking feasibility was undertaken.Nosocomial infections caused by multi-drug resistant (MDR) pathogens, including MRSA, vancomycin-resistant Five actinomycetes strains, namely UKAQ_04, UKAQ_05, UKAQ_07, UKAQ_09, and UKAQ_23, were isolated from the twenty-five collected mangrove sediment samples  and Streptomyces smyrnaeus . A neighbor-joining (NJ) phylogenetic tree of UKAQ_23 based on 16S rRNA gene sequencing was generated by MEGA-X  with accession number MG657032.1. A culture copy of Streptomyces smyrnaeus UKAQ_23 has been deposited at the National Centre for Microbial Resource (NCMR), Pune, MS, India, under the accession number MCC 0192.The 16S rRNA gene sequence of UKAQ_23 was compared with the available sequences in GenBank , indicating that UKAQ_23 was closely related to the genus The culture characterization of UKAQ_23 revealed that all the tested growth media had supported the growth of UKAQ_23. The color of substrate mycelium ranged from canary-yellow to yellow. The diffusible pigmentation was observed only in GM-5, GM-6, and GM-7, which were yellow, while no diffusible pigmentation was observed in GM-1 to GM-4. The color of aerial mycelium ranged from grey to yellow  and clindamycin (2 µg), but resistant to amoxycillin (10 µg), ceftriaxone (30 µg), imipenem (10 µg), and tetracycline (30 µg).The physiological characterization of UKAQ_23 revealed that the optimum growth of UKAQ_23 was observed in the presence of various carbon sources, including starch maize, starch soluble, sucrose, glycerol, D (−) mannitol, and D ( +) dextrose; nitrogen sources, including ammonium sulfate, glycine, L-asparagine monohydrate, and soybean meal; pH 6.5; temperature 30 °C, and NaCl 0.5% w/v 2SO4 0.5% w/v, NaCl 0.75% w/v, K2HPO4 0.6% w/v, CaCl2 0.15% w/v, MgSO4 0.05% w/v, CaCO3 0.015% w/v, FeSO4 0.010% w/v, ZnSO4 0.005% w/v, agar 1.5% w/v, pH 6.5, inoculum 5% v/w at temperature 35 °C, and an incubation period of 7 days  of crude antimicrobial extract from solid-state fermentation was 561.3 ± 0.3 mg/kg fermented agar in a fermentation medium (modified ISP-4 agar) consisting of starch maize 1% w/v, starch soluble 0.6% w/v, , but the interactive effects of agar concentrations and inoculum volumes were insignificant (P > 0.05). The optimal level of each variable and the effects of their interactions on antibiotic output were explored using three-dimensional (3D) response surface curves Fig. , and the, but the1 and K2, with Rf values of 0.38 and 0.28, respectively  at 239 and 440 nm for the compound K1  and 215 and 442 nm for the compound K2  were subjected to MS3. MS3 spectrum demonstrated the elimination of Me-Val residue, which is in good agreement with the proposed sequence. On the other hand, a neutral loss of 81 Da, which was interpreted as eliminating the OxoPro fragment with the molecular formula, C4H3NO, and fits into the proposed structural formula. The MS3 spectrum demonstrated that compound K1 contains Oxo-Pro moiety instead of Pro in one chain  and 6.40  together with two methyl protons at δ 2.59  and δ 2.25 , characteristic for the phenoxazinone chromophore  and 5.29 , four N-Me singlets at δ 2.94 , δ 2.93 , δ 2.89 , and δ 2.88 , as well as 10 additional methyl doublets at δ 1.30/δ 1.17 , δ 1.19/δ 1.15 , δ 0.95/δ 0.94 , δ 1.03/δ 1.00  and δ 0.79/δ 0.78 . These characteristics signals were accounted for two pentapeptide lactone rings. The 13C NMR and DEPT-135 spectra revealed the presence of 10 amino acid residues from 10 α-amino acid carbon signals at δ 50.72–70.91 and the 10 carbonyl signals at δ 165.31–173.47, indicating that K1 is a peptide  and C-13 (δ 14.48) of the phenoxazinone, 5 methylenes from which three were assigned to C-3, C-4, and C-5 of the α-ring proline, and two for the C-3 and C-5 of β-ring proline, indicating that one of the methylene carbon of this proline was modified, a 2 sp2 methines (C-7 and C-8 of phenoxazinone nucleus), 6 sp3 methines including two oxygenated (C-3 of each threonine), and four non-oxygenated , and 12 sp2 quaternary carbons including three carbonyls for C-3, C-11, and C-14 of the phenoxazinone nucleus. The ongoing data are in agreement with spectral features observed for the actinomycins. Additionally, the downfield carbonyl signal at δ 208.27 was assigned to C-4 of β-ring proline, indicating that compound K1 has dissimilar pentapeptide lactone rings, one ring contains proline, and the other contains oxoproline. After an extensive 2D-NMR  analyses, the amino acid residues in each peptide ring were identified as Thr, Val, Pro, Sar, and Me-Val in α-ring, and Thr, Val, OxoPro, Sar, and Me-Val in β-ring  of Thr with C-1 (δ 165.83) of Me-Val; H-2 (δ 2.68) and N-Me (δ 2.94) of Me-Val with C-1 (δ 165.31) of Sar; H2-2 (δ 4.75 and 3.69) and N-Me (δ 2.88) of Sar with C-1 (δ 172.57) of Pro; H-2 (δ 6.02) of Pro with C-1 (δ 173.03) of Val and H-2 (δ 3.72) and NH (δ 8.21) of Val with C-1 (δ 168.32) of Thr. The location of the α-ring pentapeptide lactone at C-14 of the phenoxazinone unit was confirmed by the long-rang correlation between H-2 (δ 4.52) of Thr and C-14 (δ 165.31) of the phenoxazinone part. Similarly, the sequence of amino acids residue in β-ring was deduced from the HMBC correlations as H-3 (δ 5.20) of Thr with C-1 (δ 166.88) of Me-Val; H-2 (δ 2.73) and N-Me (δ 2.93) of Me-Val with C-1 (δ 165.71) of Sar; H2-2 (δ 4.64 and 3.68) and N-Me (δ 2.89) of Sar with C-1 (δ 172.13) of OxoPro; H-2 (δ 6.62) of OxoPro with C-1 (δ 173.47) of Val and H-2 (δ 3.61) and NH (δ 7.68) of Val with C-1 (δ 167.99) of Thr. The location of the β-ring pentapeptide lactone at C-11 of the phenoxazinone part was confirmed by the long-rang correlations between H-2 (δ 4.58) of Thr and C-11 (δ 166.92) of the phenoxazinone chromophore. Additionally, the phenoxazinone chromophore was established from the ongoing 2D correlations: H-7 and H-8 were correlated with the carbons at δ 129.70 (C-8) and δ 125.65 (C-7) in the HSQC spectrum and were also correlated to each other in the COSY spectrum. In the HMBC spectrum, H-7 showed cross-peaks with C-5a (δ 139.93), C-9 (δ 131.59) and C-13 (δ 14.48), whereas the H-8 showed cross-peaks with C-6 (δ 127.20), C-9a (δ 128.60) and C-14 (δ 165.31). Furthermore, the methyl protons at δ 2.59 (H3-13) correlated with C-5a (δ 139.93), C-6 (δ 127.20) and C-7 (δ 129.70) and the methyl protons at δ 2.29 (H3-12) correlated with C-3 (δ 178.53), C-4 (δ 113.00), and C-4a (δ 144.46) protons. Based on the NOESY spectrum and their similar NMR pattern, the relative configuration of compound K1 was assigned to be identical to that of Actinomycin X2  is a powerful analytical tool that has been used as a gold standard for molecular identification1 and K2 demonstrated that Actinomycin X2 had a larger zone of inhibition, i.e.,14.7 ± 2.7 mm and Actinomycin D had a smaller zone of inhibition, i.e., 9.9 ± 2.8 mm, whereas actinomycins-mixture  had a medium zone of inhibition, i.e., 13.5 ± 3.0 mm (Mean ± SD), against the Gram-positive test bacteria, at a concentration of 5 µg/disc. According to the results, Actinomycin X2 was the most potent antibiotic, while Actinomycin D was the least potent, and actinomycins-mixture showed mild potency. Levofloxacin (control antibiotic) inhibited non-MRSA bacteria with a zone of inhibition of 25.6 ± 6.91 mm and MRSA bacteria with a zone of inhibition of 28.8 ± 7.14 mm (Mean ± SD)  was omitted.The primary antimicrobial activity of the compounds KD) Figs. . Based o2 ranged from 1.56 to 12.5 μg/ml for non-MRSA test bacteria and from 3.125 to 12.5 μg/ml for MRSA test bacteria, while MIC of the isolated Actinomycin D ranged from 3.125 to 12.5 μg/ml for non-MRSA test bacteria and from 12.5 to 25 μg/ml for MRSA test bacteria. Levofloxacin (control antibiotic) inhibited the growth of all the test organisms at a concentration of 5 µg/ml  = 436.605, p = 0.000, for Actinomycin D; F  = 1371.294, p = 0.000, and for actinomycins-mixture; F  = 1730.333, p = 0.000  as determined by One-way ANOVA, i.e., for Actinomycin X2; F (9) = 397.826, p = 0.000, for Actinomycin D; F (9) = 1109.126, p = 0.000, and for actinomycin-mixture; F  = 1018.509, p = 0.000  between groups of tested bacterial strains as determined by One-way ANOVA, 1 and K2 against the DNA and eight other potential antibacterial protein targets  was observed as more potent than K1  with an appreciable difference in the binding energy . The media composition and fermentation conditions were optimized to achieve maximum antibiotic production, resulting in high yield antibiotic production in modified inorganic salt medium (modified ISP-4 agar) by solid-state fermentation. The antibiotics were extracted using a solid–liquid extraction method, and the purification was performed using various chromatographic techniques, resulting in the isolation of two purified antimicrobial compounds, K1 and K2. The structures of the compounds, K1 and K2, were elucidated employing various spectroscopic techniques, including UV, IR, mass and 1D and 2D NMR spectroscopic techniques, resulting in the identification of the compounds, K1 and K2, as Actinomycin X2 and Actinomycin D, respectively. The antimicrobial screening of isolated actinomycins, X2 and D, revealed that both actinomycins exhibited potent antimicrobial properties against the tested bacterial strains, and further research revealed that Actinomycin X2 exhibited higher antimicrobial property than Actinomycin D. As a result, we concluded that the isolated actinomycete strain, Streptomyces smyrnaeus UKAQ_23, is an opulent source for the production of Actinomycin X2 and Actinomycin D and could be used as an alternative source of said actinomycins, which could be a significant shift in the pharmaceutical industry52.Previously, we isolated Streptomyces smyrnaeus UKAQ_23 exhibited antimicrobial potential, which is consistent with a previous study in which Tatar et al. reported that Streptomyces smyrnaeus sp. nov. isolated from Izmir, Turkey demonstrated antibacterial and antifungal activities against B.cereus NRRL B-3711T, B.pumilus NRRL-BD 142, B.subtilis NRRL B-209, Candida utilis NRRL Y-900, and Aspergillus parasiticus NRRL-465. However, they were unable to isolate the antimicrobial compounds from that strain53.Our findings showed that mangrove-derived 2, and D production from mangrove-derived Streptomyces smyrnaeus UKAQ_23 are consistent with previous findings that several Streptomyces strains, e.g., Streptomyces sp. MS449, Streptomyces sp. IMB094, Streptomyces nasri YG62, Streptomyces padanus JAU4234, Streptomyces elizabethii. II, Streptomyces flavogriseus NJ-4, Streptomyces MITKK-103, Streptomyces griseoruber, Streptomyces strain M7, Streptomyces sp. HUST012,Streptomyces heliomycini, Streptomyces hydrogenans IB310 produce Actinomycin X2 and Actinomycin D57.Our findings of actinomycins, X2, and D produced by Streptomyces smyrnaeus UKAQ_23 exhibited potent antimicrobial activities against MRSA and non-MRSA Gram-positive bacterial strains, but no bioactivity was demonstrated against tested Gram-negative bacteria and fungal strains, which is consistent with previous studies; Sharma and Manhas, who demonstrated the antibacterial potential of actinomycins V, X2, and D produced by Streptomyces strain M7, and the findings revealed that actinomycins X2 and D had significant antibacterial potential against VRE, MRSA, and Bacillus subtilis with MIC values ranging from 1.95 to 8.0 μg/ml54; Wang et al. demonstrated antimicrobial activity of actinomycins X0β, X2, and D produced by Streptomyces heliomycini, and the results showed that actinomycins X0β, X2, and D had substantial antibacterial potential against S.aureus, MRSA, Bacillus subtilis, and Bacillus cereus with MIC values ranging from 0.04 to 2.48 μg/ml56.Our recent findings showed that actinomycins, X. demonstrated the antibacterial potential of four actinomycins, neo-Actinomycin A, neo-Actinomycin B, Actinomycin D, and Actinomycin X2 produced by Streptomyces sp. IMB094 and the results revealed that actinomycins D and X2 had substantial antibacterial activity against S.aureus, S.epidermidis, and E.faecalis with MIC values ranging from 0.06 to 0.5 μg/ml, while neo-Actinomycins A and B exhibited less-substantial antibacterial potential with MIC values ranging, 16–32 μg/ml by neo-Actinomycin A and > 128 μg/ml by neo-Actinomycin B, respectively. However, with MIC values ranging from 16 to 128 μg/ml, all four actinomycins had insignificant antibacterial activity against E.coli, K.pneumoniae, P.aeruginosa, S.marcescens, A.calcoaceticus, P.mirabilis, P.rettgeri, P.vulgaris, C.freundii, M.morganii, S.maltophilia, and Enterobacter spp.28.Wang et al. demonstrated that Actinomycin D produced by Streptomyces sp. HUST012 had substantial antibacterial activity against MRSA ATCC 25923, MRSE ATCC 35984, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 13883 with MIC values ranging from 0.04 to 2.24 μg/ml57, which was partially deprived of our findings, as our isolated actinomycins had shown non-substantial antibacterial activity against Gram-negative test bacteria. Kulkarni et al. demonstrated the antimicrobial potential of Actinomycin D produced by Streptomyces hydrogenans IB310. They showed that Actinomycin D had substantial antibacterial and antifungal potential against plant pathogens and suggested the potential application of Actinomycin D in agriculture to manage plants’ bacterial and fungal infections58, which was partially deprived of our findings, as our isolated actinomycins had shown non-substantial antifungal activity.Khieu et al. reported that several species of Streptomyces produce Actinomycin D analogs. However, only a few species produce in significant amount, including Streptomyces parvulus (0.152 mg/ml Actinomycin D), Streptomyces griseoruber (0.21 mg/ml Actinomycin D), Streptomyces sindenensis (0.85 mg/ml Actinomycin D), Streptomyces MITKK-103 (0.11 mg/ml Actinomycin X2), Streptomyces sp. JAU4234 (0.62 mg/ml actinomycin X2), Streptomyces nasri strain YG62 (0.15 mg/ml Actinomycin X2), and Streptomyces strain MS449 24. This study confirms our findings and suggests that the isolated strain Streptomyces smyrnaeus UKAQ 23 is suitable for industrial use as it can produce a significant amount of Actinomycin D. However, prior to its commercial employment, strain improvement is essential.Chen et alStreptomyces smyrnaeus UKAQ_23. Furthermore, to avoid the formation of fermentation products with poor antibacterial activity, pH, temperature, inoculum concentration, agar concentration, and incubation time, among other variables, must be tightly regulated throughout fermentation. This method may be used to conduct further investigation into antibiotic production. The higher yield of crude antimicrobial extract obtained using the solid-state fermentation method was consistent with previous reports63. Surprisingly, one study showed that solid-state fermentation conditions are ideal for producing antimicrobial compounds by Streptomyces youssoufiensis SF10 strain64.Our findings on antibiotic production and optimization of fermentation conditions indicated that the modified ISP-4 agar was the best fermentation medium for maximum antibiotic production at pH 6.5, temperature 35 °C, inoculum 5% v/w, agar 1.5% w/v, and incubation period of 7 days. Furthermore, the solid-state fermentation method was superior to the submerged-state fermentation method for the production of crude antibiotics, possibly due to the lack of water in solid-state fermentation. The findings obtained by optimizing the fermentation medium using RSM significantly affected antibiotic production by 1 and K2 against several drug-targets of bacterial biochemical pathways was studied for the first time by molecular docking using AutoDock Vina 1.1.266. Analysis of the docking results  and cycloheximide (50 μg/ml) to isolate actinomycetes strains. The nalidixic acid and cycloheximide were supplemented to inhibit the growth of the Gram-negative bacteria and fungi, respectively68. The inoculated plates were then incubated at 28 °C for up to 14 days. The isolated actinomycetes were sub-cultured and purified on ISP-4 slants. The purified cultures were preserved in 20% (v/v) glycerol at − 78 °C for further use.A total of twenty-five mangrove sediment samples were collected from Jubail, Saudi Arabia 8. MTSA plates consisted of starch soluble 1 g, the pancreatic digest of casein 1.5 g, papain digest of soya bean 0.5 g, sodium chloride 0.5 g, agar 1.7 g, peptone 0.4 g, tryptone 0.4 g, beef extract 0.3 g, lactose 1 g, L-cystine 0.013 g, bromothymol blue 0.002 g, agar 1.5–1.7 g, ultrapure water 100 ml, and pH 6.8 ± 0.2.Preliminary antimicrobial screening of the isolated actinomycetes was conducted by the spot inoculation method using modified tryptic soy agar (MTSA) platesStaphylococcus aureus (S.aureus) ATCC 29213, MRSA-A, MRSA-B, MRSA-C, Staphylococcus saprophyticus (S.saprophyticus) ATCC 43867, Staphylococcus epidermidis (S.epidermidis) ATCC 12228, Streptococcus pyogenes (S.pyogenes)-A ATCC 19615, Streptococcus pneumoniae (S.pneumoniae) ATCC 49619, Enterococcus faecalis  ATCC 29212, Bacillus cereus (B.cereus) ATCC 10876), 7 Gram-negative bacteria, Escherichia coli (E.coli) ATCC 25922, Klebsiella pneumoniae (K.pneumoniae) ATCC 27736, Pseudomonas aerugenosa (P.aerugenosa) ATCC 9027, Proteus mirabilis (P.mirabilis) ATCC 29906, Proteus vulgaris (P.vulgaris) ATCC 6380, Salmonella typhimurium (S.typhimurium) ATCC 13311, Shigella flexneri (S.flexneri) ATCC 12022), and 2 fungal species Candida albicans  ATCC 10231 and Aspergillus niger (A.niger) ATCC 6275 were used as test organisms. The antibacterial activity was determined by incubating the inoculated plates at 35 ± 2 °C for 24 h., and antifungal activity was determined by incubating the inoculated plates at 28 ± 2 °C for up to 48 h. The antimicrobial activities of all the isolates were recorded by observing the zone of inhibition around the actinomycetes spot68. The selection of the highly potent actinomycete strain, UKAQ_23, was based on the zone of inhibition around the inoculated spot (growth).For preliminary antimicrobial screening, 10 Gram-positive bacteria, 24. The phylogenetic analysis was performed using the Maximum Likelihood approach using the Tamura-Nei model, and the phylogenetic tree was constructed using the neighbor-joining (NJ) method MEGA X69.The selected isolate UKAQ_23 was identified by 16S rRNA gene sequencingi.e., the color of substrate and aerial mycelium, spore formation, diffusible pigment on various growth media, i.e., GM-1… GM-7) at 28 °C for up to 7 days. SEM recorded the morphological characteristics54.The cultural characterization of strain UKAQ_23 was determined by recording the cultural characteristics, e.g., assimilation of different carbon sources , nitrogen sources , tolerance of NaCl concentrations (0.25–10% w/v with an increment of 0.25%), pH (5.5–10 with an increment of 0.5), temperature (25–55 °C with an increment of 5 °C), and incubation periods (1–10 days with an increment of 24 h.) on the growth of UKAQ_2375. The biochemical characterization was performed to determine UKAQ_23's ability to produce various hydrolytic enzymes. The antibiotic susceptibility pattern was determined by the disc diffusion method with amoxycillin (10 µg), ceftriaxone (30 µg), chloramphenicol (30 g), clindamycin (2 µg), imipenem (10 µg), and tetracycline (30 µg)78.The physiological characterization of strain UKAQ_23 was performed by determining the effects of various physicochemical factors, i.e., ISP-1 broth, ISP-2 broth, ISP-4 broth, and 1% (w/v) starch tryptic soy broth. The best fermentation medium (ISP-4) was selected based on its antimicrobial activity. The well diffusion method determined the antimicrobial activity of fermented broth4. The effects of different carbon and nitrogen sources on antibiotic production were determined in ISP-4 base fermentation media, i.e., FM-1… FM-24  were selected for further finer optimization based on their antimicrobial activity demonstrated against the test organism. The antimicrobial activity of each fermented broth was determined by the well-diffusion method using 80 μl of cell-free broth/well against the test organism (S. aureus ATCC 29213). The zone of inhibition was measured on an mm scale4.Initially, antibiotic production was carried out in four different fermentation media, 62. The effects of various carbon source concentrations (0.2–1% w/v with an increment of 0.2%), nitrogen source concentrations (0.25–1% w/v with an increment of 0.25%), NaCl concentrations (0.5–2% w/v with an increment of 0.25%), K2HPO4 concentrations (0.3–1.5% w/v with an increment of 0.3%), CaCl2 concentrations (0.05- 0.40% w/v with an increment of 0.05%), MgSO4 concentrations (0.05–0.40% w/v with an increment of 0.05%), CaCO3 concentrations (0.005–0.030% w/v with an increment of 0.005%), FeSO4 concentrations (0.005–0.030% w/v with an increment of 0.005%), ZnSO4 concentrations (0.005- 0.030% w/v with an increment of 0.005%), pH (6–7.5 with an increment of 0.5), temperature (25–40 °C with an increment of 5 °C), inoculum concentrations , agar concentrations (1–2% w/v with an increment of 0.25%), and incubation periods (1–10 days with an increment of 24 h.) were determined.Initially, the production of antibiotic was carried out in submerged-state fermentation, but owing to large fluctuation in the production of antibiotic, the optimization was shifted to solid-state fermentationi.e., pH 6.5, temperature 30 °C, carbon source 1% w/v, nitrogen source 0.5% w/v, NaCl 0.50% w/v, inoculum 5% v/w, agar 1.5% w/v, and incubation period of 7 days. After completing each set of fermentation, the yield of antimicrobial extract was measured in mg/kg of fermented agar74.The effects of media constituents and fermentation conditions on antibiotic production were determined by varying the one factor at a time and base fermentation conditions were kept constant, 79. The predicted model was validated and found to be statistically suitable for use. A total of 27 experiments were carried out to optimize the main factors for a 3-levels-4-factors BBD with three replicates at the center. After evaluating the responses for each trial, each response was fitted to an individual second-order polynomial model. For media optimization, a total of twenty-seven BBD experiments were run in one block. The minimum and maximum ranges of variables investigated in terms of their actual and coded values are mentioned  were chosen for further antibiotic-production-optimization by RSM using the Box-Behnken design (BBD)61  and acetone ((CH3)2CO) in a ratio of 60:40. The TLC was run on pre-coated silica gel  sheets2. The plates were visualized under UV light. The bioautography was performed on a developed TLC plate80. S.aureus ATCC 29213 was employed as a test organism.A thin layer chromatography (TLC) was employed to assess the purity and number of compounds present in the crude antimicrobial extract using a solvent system composed of dichloromethane , size-exclusion chromatography , and silica gel  column chromatography1 and K2. The homogeneity of purified compounds was assessed by HPLC .A solvent system composed of dichloromethane and acetone (60:40) was employed in the preparative TLC and silica gel column chromatography, while pure methanol was employed in the size-exclusion chromatography. The purification steps culminated into two pure antimicrobial compounds named K1 and K2, were carried out by recording the color, appearance, solubility, melting point (°C), UV–Visible  absorbance, FT-IR  absorbance, monoisotopic masses, ( +)-HR-ESI-MS, LC–MS, LC–MS-MS, 1D , and 2D  NMR spectroscopy85.The physicochemical characterization and structure elucidation of isolated antimicrobial compounds, Ke.g., water, ethanol, methanol, acetone, acetonitrile, dimethylformamide, dimethyl sulphoxide, chloroform, ethyl acetate, dichloromethane, and n-hexane at a concentration of 10 mg/ml. The melting points of the compounds were determined by the capillary method with the Dynalon DMP100 Digital Melting Point Device . The compounds were dissolved in methanol at a concentration of 1 mg/ml, and UV–Visible spectra were recorded with the Ultraspec 8000 spectrophotometer . FT-IR spectra were recorded with the Nicolet iS20 FT-IR spectrometer . The MSn analysis was performed on LCMS-IT-TOF . The instruments parameters were as follows: Positive-ion mode, calibration with the Tune Solution, mass accuracy was better than 5 ppm, scan range: 50–2000 m/z, drying gas: nitrogen (flow rate: 1.5 l/min), temperature: 200 °C, solvents: isocratic elution 50% B in A , flow rate: 0.1 µl/min, injection volume: 1 µl.LC-MS analysis was performed on Shimadzu LCMS-9030 quadrupole time-of-flight (Q-TOF). Separation was carried out on an Aeris Peptide XB-C18  column. Eluent A: 0.1% formic acid in H2O, eluent B: 0.1% formic acid in MeCN. The gradient conditions (B%) were from 5 to 85% B in 15 min. Flow rate: 0.1 ml/min, injection volume: 1 µl. The NMR spectra were recorded on the Bruker 700 MHz AVANACIII NMR spectrometer equipped with Bruker CPTCI multinuclear probe. Topspin 4.0.4 software  was used for both the data collection and spectral processing. The obtained data were compared with the standard actinomycins, X2 and D, and previously reported NMR and MS data of the actinomycins.The solubility of compounds was determined by dissolving the compounds in various solvents, 1, K2, and K1 + K2)78. The isolated antimicrobial compounds were dissolved in methanol, and then diluted samples were dispensed on sterile paper discs (6 mm size). Each disk consisted of 5 µg of the antimicrobial compound. This experiment investigated the antimicrobial efficacy of isolated antimicrobial compounds on the tested microorganisms. A disk containing 20 μl of methanol was used as a negative control, while Levofloxacin (5 µg/disc) was used as positive control antibiotic. Each test was performed in triplicate. The diameters of the inhibitory zones were measured on an mm scale. The results were recorded in Mean ± Standard Deviation (SD).The standard disc diffusion method determined the primary antimicrobial activity of the isolated antimicrobial compounds (K1 and K2) was determined by performing the MIC and MBC. MIC was determined by the resazurin-based micro broth dilution method, while MBC was determined by standard spot inoculation method86. The antimicrobial compounds were dissolved in methanol at a concentration of 200 µg/ml, and then, various concentrations (0.098–50 µg/ml) were prepared in Mueller–Hinton broth (MHB) by following the two-fold serial dilution method in microtiter plates (columns 2–11). Levofloxacin (5 µg/ml) was used as a control antibiotic (column 1). A 100 µl suspension of each test organism (0.5 McFarland) was dispensed in its respective well in columns 1-11. Each well in columns 1-11 contained an equal volume of the test antibiotics and suspensions of the test bacteria. Following the addition of the bacterial suspensions, the plates were incubated at 35 °C for 18-24 h. After incubation, 30 μl of sterile resazurin dye (0.015% w/v) was dispensed into each well of columns 1-11. The plates were kept at room temperature for 5 h. After 5 h, the results of MIC were recorded. The prepared concentrations of the antimicrobial compounds were evaluated for their antimicrobial efficacy against selected test organisms. The lowest concentration of the tested antimicrobial compound showed no color change from blue to pink was considered MIC. The lowest concentration of the tested antimicrobial compound showed no isolated colony on the inoculated plate considered MBC. The results were expressed in µg/ml.The secondary antimicrobial activity of isolated antimicrobial compounds . The post hoc test (Tukey method) was performed to determine the significance of interactions among the means of groups, where p = 0.05 was considered as statistically significant. The statistical analyses were performed with SPSS software, version 20.0 87.The statistical software package Minitab 19.2020.1 was used to analyze the experimental design applied in RSM during optimization of fermentation conditions for maximum yield of antibiotic. A One-Way ANOVA statistical test statistically analyzed the results of primary antimicrobial activity of the isolated antimicrobial compounds Khttp://www.rcsb.org/pdb/home/home.do) with PDB IDs 3TTZ, 2VEG, 3SRW, 2VF5, 1JZQ, 1JIJ, 3UDI and 2X5O. Also, Actinomycin D co-crystallized with DNA fragment (PDB ID: 1MNV) was also included in the study. Biovia Discovery Studio Visualizer 2020 and MGLTools 1.5.6 were used for the preparation of receptors88.X-ray crystal structures of several protein targets known to be associated with antimicrobial biological activity elicitation and employed in antimicrobial drug discovery and development, e.g., DNA gyrase, dihydropteroate synthase, dihydrofolate reductase, glucosamine-fructose-6-phosphate aminotransferase, isoleucyl-tRNA synthetase, tyrosyl-tRNA synthetase, penicillin-binding protein-1a, and UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank  and non-bond interactions profile90. Molecular interactions analyses were performed in Biovia Discovery Studio Visualizer 202091, and PyMoL 1.7.492 programs.All the co-crystalized ligands, water molecules, and cofactors were deleted, and Gasteiger charges were added to each receptor individually. Two-dimensional chemical structures of the compounds KA patent has been filed on January 22, 2021, at the office of Intellectual Property of India. The patent filing reference number is 202111003185. The final decision is awaited.Supplementary Information.


Purpose: To investigate the stability of intraocular lens (IOLs) with different haptics by swept-source anterior-segment optical coherence tomography (AS-OCT).Methods: Sixty-eight eyes from 55 patients received the implantation of Rayner 920H (Closed C-loop Group), Zeiss 509M (Plate Group) or Lenstec SOFTEC HD (C-loop Group) IOLs. The tilt and decentration of IOLs were evaluated using AS-OCT at least 1 month postoperatively.Results: Mean decentration and tilt of IOLs were 0.18 ± 0.12 mm (range 0.02 to 0.59 mm) and 5.63 ± 1.65° (range 2.2 to 9.6°) respectively. Decentration was significantly smaller in the plate haptic group (0.12 ± 0.06 mm) as compared to the C-loop group . The tilt of IOL was also significantly smaller in the plate haptic group (4.96 ± 0.89°) as compared to the C-loop group . There was marginal difference between the Closed C-loop group (5.52 ± 1.74°) and C-loop group .Conclusions: The Plate-haptic IOLs should have better stability for the decentration and tilt than the C-loop design IOLs. Cataract is the leading cause of reversible blindness in the world , 2. PhacThe maintenance of IOL stability relies on the support of the haptics in the capsular bag . PreviouIOLs with Plate-haptics, C-loop or Closed C-loop are widely used in clinical practice. It is not clear whether these IOLs with different haptics would have different performance in stability. The CASIA2  is a new generation anterior segment optical coherence tomography (AS-OCT) with a 1,310 nm swept-source laser . The lenThis study was a retrospective study. It has been approved by the Human Medical Ethics Committee of the Joint Shantou International Eye Center (JSIEC) of Shantou University and the Chinese University of Hong Kong, which was in accordance with the tenets of the Declaration of Helsinki.Sixty-eight eyes from 55 Chinese adults who had underwent cataract surgeries were involved. All patients were followed up at JSIEC from May 2019 to January 2020. All subjects received complete ophthalmic examinations, including visual acuity, intraocular pressure (IOP), dilated fundus stereoscopic examination. The exclusion criteria were as follows: (1) age <18; (2) follow-up time <1 month; (3) history of eye surgeries other than cataract surgery; (4) history of complications during or after cataract surgery; (5) severe anterior capsule contraction; (6) angle closure glaucoma; (7) high myopia or axial length (AL) ≥ 26 mm.All 68 eyes from 55 Chinese adult study subjects received routine phacoemulsification and IOL implantation in the capsular bag. Closed C-loop IOLs (Rayner 920H) were implanted in 25 eyes of 22 subjects. C-loop IOLs (Lenstec Softec HD) were implanted in 24 eyes of 19 subjects. Plate-haptics IOLs (Zeiss 509M) were implanted in 19 eyes of 14 subjects. The information of IOLs used in the current study was shown in All studied eyes were dilated using a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride  at 30 min before CASIA2 examination. All examinations were performed by a single experienced operator. “Post-op Cataract” was selected in Exam Protocol Selection. Trace Line mode was used with “Semi-Auto”. Eight anterior segment images were obtained from 8 scans . Three-dimensional anterior segment was reconstructed. The positions of IOLs were assessed automatically by the lens analysis mode in the device-specific software. IOL decentration was defined as a distance between vertex normal of the cornea and the center of equator circle of IOLs, while IOL tilt was defined as a tilt between vertex normal of the cornea and the optic axis of IOLs . Moreovepost-hoc test. χ2 test was used for the comparisons on gender and laterality. P < 0.05 was considered as statistical significant.Statistical analysis was performed with commercially available software . Data distribution for normality was checked using Shapiro–Wilk's test. According to the data normality of the groups, one-way analysis of variance (ANOVA) or Kruskal-Wallis H test was used to compare the age, AL, IOL decentration and tilt among different groups. Multiple testing correction between groups was adjusted by the Sixty-eight eyes from 55 study subjects were included in the analysis. There were 25 eyes in the Closed C-loop Group, 24 eyes in the C-loop Group and the 19 eyes in the Plate Group. The demographics of the study subjects were shown in P = 0.02). The IOL decentrations of the three studied groups were shown in P = 0.02). There was no statistically significant difference between the Plate and Closed C-loop groups in decentration, and no significant difference in decentration was found between the Closed C-loop and C-loop groups. Decentration for IOLs was toward each quadrant without obvious tendency (mean: 155.50° for right eyes and 87.50° for left eyes). For the right eyes, mean IOL decentration azimuth of the Closed C-loop, C-loop and Plate groups was 159.08 ± 103.33° (range 17 to 324°), 155.92 ± 73.56° (range 10 to 253°) and 135.33 ± 83.94° (range 16 to 275°) respectively. For the left eyes, mean IOL decentration azimuth of the Closed C-loop, C-loop and Plate groups was 186.33 ± 122.14° (range 21 to 343°), 226.08 ± 105.65° (range 61 to 341°) and 249.40 ± 102.89° (range 4 to 329°) respectively. This tendency was not affected by the haptic design .Mean decentration for all types of IOLs was 0.18 ± 0.12 mm (range 0.02 to 0.59 mm). There were significant differences among the three studied groups in decentration . IOL tilts of the three studied groups were shown in P = 0.01). Ther was marginal difference in IOL tilt between the Closed C-loop Group (5.52 ± 1.74°) and C-loop Group , and no significant difference was found between the Plate Group and Closed C-loop Group in IOL tilt. Mean IOL tilt direction was toward the inferotemporal direction (204.74° for right eyes and 290.85° for left eyes), and both eyes presented a mirror symmetry relationship. For the right eyes, mean IOL tilt azimuth of the Closed C-loop, C-loop and Plate groups was 209.92 ± 20.68° (range 182 to 258°), 195.83 ± 31.13° (range 119 to 247°) and 209.11 ± 21.26° (range 186 to 243°) respectively. For the left eyes, mean IOL tilt azimuth of the Closed C-loop, C-loop and Plate groups was 325.50 ± 28.33° (range 244 to 352°), 221.58 ± 158.12° (range 15 to 305°) and 332.40 ± 11.30° (range 312 to 347°) respectively. This tendency was not affected by the haptic design .Mean tilt for all IOLs was 5.63 ± 1.65° (range 2.20 to 9.60°). There were significant differences among the three studied groups in IOL tilt , Shantou, China. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.GW contributed to administrative, research conception, technical, and material supports. ZX contributed to research conception, data collection, analysis, and writing of the manuscript. MZ and ZZ contributed to data collection. All authors saw and approved the final version.This study was supported by Guangdong Medical Research Foundation (Grant No. A2016514), Shantou Municipal Science and Technology Project (Grant No. 190917155269927), 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant (Project Number: 2020LKSFG06B), and Grant for Key Disciplinary Project of Clinical Medicine under the Guangdong High-level University Development Program, China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pseudopterogorgia americana (PPA) were investigated. PPA increased the expression levels of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), but not inducible nitric oxide synthase and nitric oxide, in macrophages. A mechanistic study revealed that PPA activated macrophages through the toll-like receptor-4 and induced the generation of reactive oxygen species (ROS), increased the phosphorylation levels of protein kinase C (PKC)-α, PKC-δ and mitogen-activated protein kinases (MAPK), and activated NF-κB. The inhibition of ROS and knockdown of PKC-α reduced PPA-mediated TNF-α and IL-6 expression; however, the knockdown of PKC-δ significantly increased PPA-mediated TNF-α expression. In addition, the inhibition of c-Jun N-terminal kinase-1/2 and NF-κB reduced PPA-mediated TNF-α, IL-6 and COX-2 expression. Furthermore, the inhibition of ROS, MAPK and PKC-α/δ reduced PPA-mediated NF-κB activation, indicating that ROS, MAPK and PKC-α/δ function as upstream signals of NF-κB. Finally, PPA treatment decreased the phagocytosis activity of macrophages and reduced cytokine expression in bacteria-infected macrophages. Taken together, our current findings suggest that PPA can potentially play a role in the development of immune modulators in the future.Polysaccharides from marine organisms produce an important regulatory effect on the mammalian immune system. In this study, the immunomodulatory properties of a polysaccharide that was isolated from the coral  The ocean is rich in biodiversity and this makes it a very important source for biomedical researchers to develop active substances. The interest is due, first, to the ocean’s high content of biologically active substances, which are effective in the treatment and prevention of multiple diseases. In addition, they are used in the food, cosmetic and other industries, and thus biologically active substances must be extracted from these organisms . Thanks Codium fragile (green sea fingers) enhance the cytotoxicity of natural killer cells toward HeLa cells by increasing the release of cytokines and cytotoxic granules [Chlamys farreri  and sea cucumber viscera promote phagocytosis and increase cytokine production in macrophages [Cuminum cyminum activate RAW264.7 cells and NK-92 cells, causing them to produce cytokines [Marine-derived polysaccharides are an important resource for the development of immunomodulators. Sulfated polysaccharides from sea cucumbers, fucoidan from brown algae and iota-carrageenan from red algae showed inhibitory activity against SARS-CoV-2 to combat COVID-19 ,6,7. Sulgranules . Polysacrophages . The watytokines . Marine-ytokines , antitumytokines  and prebytokines . In addiytokines , renal dytokines , cardiovytokines  and neurytokines . Howeverytokines . The cheytokines .Pseudopterogorgia americana, which is widespread in the waters of the Caribbean Sea, and its physicochemical and biological properties were studied [−1 in the IR spectrum. In addition, the residues of D-arabinose, D-fucose, D- and L-galactose, and D-glucose in molar ratios of 35.6:24.5:33.3:6.6 were identified in a hydrolysate of PPA. The chemical structure of PPA was characterized by methylation studies, partial acid hydrolysis, periodate oxidation and Smith degradation. A study of oligosaccharides obtained by partial acid hydrolysis of PPA showed that the polysaccharide is branched; linear sections of the carbohydrate chain with 1,2-bonds between the galactose residues and 1,4-bonds between the glucose residues are present. Residues of D-arabinose and D-fucose occupy the terminal position and are incorporated in the carbohydrate chain by 1,3- and 1,2-bonds, respectively. Sulfate groups are located at C-2 and C-3 of D-glucose residues and at C-3 of L-fucose residues [We previously isolated mucus glycoprotein from the soft coral  studied ,23. The Pseudopterogorgia americana (PPA) were investigated. The cytokine induction activity of PPA was investigated and the signalling pathways that regulate PPA-mediated cytokine production were also dissected. The effect of PPA on the phagocytic activity of macrophages in response to bacterial infection was also studied.Macrophages are one of the important antigen-presenting cells that play important roles in innate immunity and regulate adaptive immunity. Cytokines secreted from activated macrophages cause inflammatory responses and are essential for host defences against invading pathogens . HoweverAntibodies against proIL-1β, iNOS, COX-2, NF-κB SN50 cell permeable inhibitory peptide (sc-3060) and pyrrolidine dithiocarbamic acid ammonium salt (PDTC) were obtained from Santa Cruz Biotechnology . Antibodies against actin, LPS, pharmaceutical inhibitors, N-acetyl cysteine (NAC), polymyxin B (PMB) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich . PD98059, SB203580, SP600125 and antibodies against phospho-proteins were obtained from Cell Signaling Technology . The NF-κB reporter reagent (QUANTI-Blue) and ATP were obtained from InvivoGen . ELISA kits were obtained from Affymetrix eBioscience . The intracellular ROS indicator was obtained from Molecular Probes . The florescent E. coli particles and Pierce Limulus Amebocyte Lysate (LAL) Chromogenic Endotoxin Quantitation Kit were obtained from Thermo Fisher Scientific . M-CSF was obtained from Peprotech . Lipid IVa was obtained from MyBioSource .J774A.1 macrophages, RAW264.7 macrophages and THP-1 monocytes were obtained from the American Type Culture Collection . THP-1 monocytes were incubated for 48 h with 50 nM PMA and differentiated THP-1 macrophages were obtained by washing out the nonadherent cells. Stable PKC-α and PKC-δ knockdown RAW264.7 and mock cells were obtained from stable transfection with shRNA plasmids that target PKC-α , PKC-δ  and a scrambled sequence , respectively, which were obtained from OriGene Technologies, Inc. . The RAW-Blue cell line is a RAW264.7 cell-based NF-κB reporter cell line that was obtained from InvivoGen . Primary wild-type and TLR4-knockout macrophages were obtained from the bone marrow of wild-type and toll-like receptor 4 (TLR4)-knockout mice, respectively. Briefly, bone marrow cells were isolated from C57BL/6 mice femur and tibia and incubated for 7 days in a culture medium containing M-CSF. The isolation of bone marrow from mice was performed with the approval of the Institutional Animal Care and Use Committee of the National Ilan University .Pseudopterogorgia americana was ground into small pieces, homogenized with a 0.9% solution of sodium chloride in a ratio of 1:0.5 at 4–6 °C for 15 min, and added to a 0.9% solution of sodium chloride to 5 L for extraction  with stirring for 12–14 h at 4–6 °C. Then, the mixture was centrifuged for 30 min at 4000 rpm, and the insoluble material was discarded. A four-fold volume of 96% ethanol  cooled to 4 °C was added to the supernatant to precipitate the high molecular weight fraction. For more complete precipitation, the mixture was incubated in a refrigerator for 12–14 h at 4 °C. As a result, a high molecular weight fraction in the form of flocculent flakes floating on the surface of the solvent was obtained. After filtration through a nylon membrane, the precipitate was wrung out, washed three to four times with anhydrous 96% ethyl alcohol that had been cooled to 4 °C, dried in air at room temperature (19–20 °C) and dissolved in distilled water until complete dissolution (requiring approximately 3 L of distilled water) with magnetic stirring. The resulting viscous solution was dialyzed against distilled water for 70–72 h with a double change of water daily. The dialyzed product was freeze-dried, and a snow-white powder with a yield of 19.0 g  was obtained. The homogeneity of the PPA was confirmed according to gel filtration. The gel filtration chromatography showed that the PPA formed a single symmetrical peak, indicating homogeneity of preparation. The glucose content in the PPA is around 55.3%, and determined by the phenol-sulfuric method, using glucose as standard. The protein and nucleic acid content in the PPA was less than 2% and 0.8% respectively [PPA was isolated, and its structure was established as described in previous reports ,23. Brie2O (5 µL/mL) for 24 h, followed by changing to a fresh medium. The cells were infected with Shigella sonnei at a multiplicity of infection (MOI) of 50 or Escherichia coli at an MOI of 30 for 5 h. The levels of TNF-α and IL-6 in the supernatants were analysed using an ELISA.For IL-1β precursor (proIL-1β) production, J774A.1 macrophages were incubated with PPA or LPS (1 µg/mL) for 6 h with or without PMB (10 µg/mL). The level of proIL-1β in the cell lysates was analysed using Western blotting. For IL-1β production, J774A.1 macrophages were incubated with PPA or LPS (1 µg/mL) for 5 h, followed by stimulation with ATP (5 mM) for 0.5 h. The levels of IL-1β in the supernatants were analysed using an ELISA. For COX-2, IL-6, TNF-α, iNOS and NO production, RAW264.7 macrophages or THP-1 macrophages were incubated with PPA for 24 h. The concentration of cytokines and NO in the supernatants was analysed using ELISAs and the Griess reaction, respectively. The levels of COX-2, iNOS and actin in the cell lysates were analysed using Western blotting. For the phosphorylation levels of MAPKs, PKC-α/δ and IκB-α, RAW264.7 macrophages were incubated with 10 µg/mL PPA for 0–60 min, and protein phosphorylation levels were analysed using Western blotting. For the effect of MAPK inhibitors on the phosphorylation levels of MAPK in PPA-stimulated cells, the cells were treated with 10 µg/mL PPA for 20 min. All the inhibitors were added to culture medium 0.5 h before PPA or LPS treatment. For bacteria-induced cytokine production, J774A.1 macrophages were incubated with 10 µg/mL PPA, 0.1 µg/mL LPS or sterile HBriefly, we added 50 µL of each sample or standard to the antibody-coated 96-well plate, followed by adding 50 µL of biotin conjugate (detection antibody) to the wells. The plate was covered with an adhesive film and incubated at room temperature for 2 h on a microplate shaker. After washing the plate three times with washing buffer (PBS with 0.05% Twee 20), 100 µL of diluted Streptavidin-HRP was added to all wells and incubated at room temperature for 1 h on a microplate shaker. After washing, 100 µL of TMB substrate solution was added to all wells and the plate was incubated for 30 min in the dark. A total of 100 µL of stop solution (1 M phosphoric acid) was added to all wells when the highest standard developed a dark blue colour. The absorbance of the wells was measured using an ELISA microplate reader with 450 nm as the primary wavelength and 620 nm as the reference wavelength.Briefly, we added 50 µL of each sample or standard to the 96-well plate, followed by adding 50 µL of sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) to the wells. The plate was incubated at room temperature for 10 min in the dark, followed by adding 50 µL of NED solution (0.1% N-1-napthylethylenediamine dihydrochloride in water) to all wells and being incubated at room temperature for an additional 10 min in the dark. The absorbance of the wells was measured using an ELISA microplate reader at 540 nm.2O (vehicle) (5 µL/mL) for 24 h. Twenty microliters of conditioned medium were mixed with 180 μL of SEAP detection medium QUANTI-Blue for 30 min in a 96-well plate. The NF-κB activity was analysed by measuring the SEAP levels using an ELISA microplate reader at 620 nm.RAW-Blue cells, an NF-κB reporter cell line, are derived from RAW264.7 macrophages, which stably express a secreted embryonic alkaline phosphatase (SEAP) gene that is inducible by an NF-κB transcription factor. RAW-Blue cells were treated with PDTC, sc-3060 or DMSO (vehicle) (1 µL/mL) for 0.5 h and then stimulated with PPA or sterile H2O (vehicle) (5 µL/mL) for 0.5 h and then with 2 µM intracellular indicator  for 0.5 h. Cells were stimulated with 10 μg/mL PPA or 1 μg/mL LPS for 0–60 min. The ROS production was analysed by measuring the fluorescent signal using a fluorescent photometer.RAW264.7 macrophages were treated with NAC or sterile H2O (vehicle) (5 µL/mL) for 24 h. Then, the cells were infected with S. sonnei at an MOI of 50 or E. coli at an MOI of 30 for 15 min, and the extracellular bacteria were washed out by PBS. To make sure that there was no extracellular bacterial growth, cells were incubated for 1 h at 4 °C with PBS containing 10 µg/mL gentamicin, an antibiotic that has no effect on intracellular bacterial growth. The intracellular bacterial growth was analysed by counting the colony-forming units (CFUs). For the analysis of the phagocytosis of fluorescent beads with the J774A.1 macrophages, cells were treated with 10 µg/mL PPA, 0.1 µg/mL LPS or sterile H2O (vehicle) (5 µL/mL) for 24 h. Then, the cells were treated for 1 h with 2 µg/mL pHrodo Green E. coli BioParticles Conjugate. The fluorescent signal was analysed using flow cytometry.For the analysis of bacterial phagocytosis with the J774A.1 macrophages, cells were treated with 10 µg/mL PPA, 0.1 µg/mL LPS or sterile HTotal RNA was isolated from PKC-α knockdown, PKC-δ knockdown or control cells, and the cDNA were synthesized using reverse transcription. Quantitative PCR was performed with a StepOne real-time PCR system purchased from Applied Biosystems . The mRNA levels were normalized to GAPDH. The primers were purchased from Genomics , and the sequences are as follows: PKC-α, forward: 5′-ttcccaatcatcatagcaca-3′; PKC-α, reverse: 5′-gagatagttatcaaccgagcag-3′; PKC-δ, forward: 5′-gttcatcgccaccttctttg-3′; PKC-δ, reverse: 5′-atttcttatggatggcagcg-3′. GAPDH, forward: 5′-gaagggtggagccaaaagg-3′; GAPDH, reverse: 5′-gatggcatggactgtggtca-3′.t-tests and ANOVA with Dunnett’s multiple comparisons test were used for the statistical analysis of two groups and three or more groups, respectively. Error bars represent standard deviations of three separate experiments. *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001, respectively.Two-tailed p < 0.001) (p < 0.001) (p < 0.001 at 6–24 h) (p < 0.001) (p < 0.001)  (p < 0.001), confirming the macrophage-activation property of PPA (p < 0.001) (p < 0.001) (p < 0.001); however, the IL-1β expression that was increased by PPA and ATP was less than that of LPS and ATP (p < 0.001 at 3 and 10 µg/mL)  A. We tes< 0.001) A. PPA is< 0.001) B and IL- 6–24 h) C. PPA do< 0.001) D, IL-6 (< 0.001) E and COX0 µg/mL) F. Furthey of PPA G. Notabl< 0.001) H and nit< 0.001) I in RAW20 µg/mL) J. These p < 0.001) (p < 0.001)  A. To fur< 0.001) B,C. Thesp < 0.001)  (p < 0.001)  A. In addt 10 mM) B and IL-< 0.001) C in PPA-< 0.001) D. Based p < 0.001) and PKC-δ  (p < 0.001) and PKC-δ (p < 0.001) using shRNA to elucidate their roles in TNF-α and IL-6 expression in PPA-treated cells (p < 0.001) (p < 0.001) (p < 0.001)  A. We knoed cells B. Signif< 0.001) C and IL-< 0.001) D were de< 0.001) C. Howeve< 0.001) D.p < 0.001 at 20–60 min), JNK1/2  and p38  (p < 0.05 at 5 μM), JNK1/2 inhibitor SP600125 (p < 0.001) and p38 inhibitor SB203580 (p < 0.001), respectively , SP600125  and SB203580  (p < 0.001)  A, and thectively B. In addat 2 μM) C, but PP< 0.001) D. Thus, < 0.001) E, indicap < 0.001), a decrease after 20 min, and an increase again at 60 min (p < 0.001) (p < 0.001) (p < 0.001) (p < 0.001) and sc-3060 . PDTC also significantly reduced COX-2 expression in 10 μg/mL of PPA-activated macrophages (p < 0.001)  A. In add< 0.001) B; howeve< 0.001) B. In add< 0.001) C and IL-< 0.001) D express< 0.001) E. Taken p < 0.001) (p < 0.001) (p < 0.001) and PKC-δ inhibitor rottlerin (p < 0.001) significantly suppressed NF-κB activation  A. In add< 0.001) B. These tivation C.S. sonnei for 15 min. The number of engulfed S. sonnei in macrophages was analysed using the CFU assay. We observed a greater number of engulfed S. sonnei in PBS-treated cells  than in PPA-treated cells (8900 ± 895 CFUs) (p < 0.001) or LPS-treated cells (9550 ± 358 CFUs) (p < 0.001)  (p < 0.001) or LPS-treated cells  (p < 0.001) was lower than that in PBS-treated cells   or 0.1 µg/mL of LPS-treated cells (p < 0.001) was lower than that in PBS-treated cells  and IL-6 (p < 0.001) expression  A. Under 06 CFUs) B. The phed cells C. These pression D.It is necessary to check for possible LPS contamination in the samples when investigating their immune modulatory properties, as macrophages are highly sensitive to LPS . AlthougThe activation of TLR4 by LPS induces MyD88-dependent and MyD88-independent pathways, the former controlling the expression of proinflammatory cytokine and the latter controlling Type I interferons and interferon-inducible genes . In the Klebsiella pneumonia induced ROS production and MAPK phosphorylation through TLR4 in J774A.1 macrophages; however, the inhibition of ROS by NAC did not affect the phosphorylation levels of MAPK [It has been demonstrated that ROS acted upstream of MAPK in LPS-activated J774A.1 macrophages . Interes of MAPK . In addi of MAPK . These r of MAPK . It is i of MAPK . In thisAntrodia cinnamomea [Auricularia auricula-judae [Klebsiella pneumonia activate macrophages through TLR4 [Antrodia cinnamomea [Auricularia auricula-judae [Ganoderma lucidum [Polysaccharides have attracted significant attention from scientists due to their immunomodulatory properties. We isolated galactomannan with an octasaccharide-repeating unit from the Taiwanese medicinal fungus nnamomea  and glucla-judae , and thela-judae . Toll-liugh TLR4 ,39. Thisnnamomea , Auriculla-judae , and Gan lucidum  activateG. lucidum inhibit phagocytic activity and reduce the expression of IL-1β and IL-6 in LPS- or amyloid-beta-activated microglia [Phagocytosis is an important innate immune defence process by which immune cells use their plasma membrane to engulf pathogens or cell debris. The first step in phagocytosis is the recognition of microbial pathogens by the cell surface receptors that bind pathogen-associated molecular patterns or opsonins . The welicroglia . Fucoidaicroglia .Auricularia auricula-judae, induced LPS tolerance, but enhanced the phagocytosis of macrophages [S. sonnei and E. coli by macrophages; however, further studies are needed to determine whether PPA inhibits phagocytosis by reducing the expression of pattern-recognition receptors or the key proteins related to phagocytosis.In general, immunomodulatory substances, which induce the secretion of proinflammatory cytokines, enhance phagocytosis. It has been demonstrated that phagocytosis activity was reduced in LPS-tolerant macrophages, but LPS-tolerant macrophages exhibited enhanced hydrogen peroxide production and antigen presentation molecule expression. These results suggested that LPS tolerance drives macrophages from a proinflammatory mediator-producing stage toward an enhanced antimicrobial activity state . Howeverrophages , which iS. sonnei- or E. coli-infected macrophages. LPS tolerance can be induced by the down-regulation of the important TLR4-mediated signalling pathways, including MAPK phosphorylation and NF-κB activation [In this study, we found that a PPA treatment for 24 h induced an LPS tolerance-like phenomenon in macrophages, as PPA reduced TNF-α and IL-6 expression in tivation . We suggtivation . NotablyPseudopterogorgia americana, suggesting the potential of PPA to be an immunomodulatory in the future.Although PPA did not induce iNOS expression or NO production and produced lower levels of IL-1β than LPS in the macrophages, we did not test the effects of PPA on the activation of other immune cells, for example, neutrophils and mast cells. In addition, the main limitation of this study is that the structure–activity relationship of PPA has not yet been systematically elucidated. The sulfate groups of PPA may play important roles in its immune modulatory activities; however, this needs further investigation. Another limitation is the lack of an animal model to assess the immunomodulatory activity of PPA in vivo. Investigations of the effect of the administration of PPA to mice on cytokine expression and the immune response upon bacterial infection are important. Based on the current findings, we cannot conclude that PPA did not cause severe inflammation similarly to LPS. Taken together, this study provides scientific evidence for the immunomodulatory properties of polysaccharides from the coral Pseudopterogorgia americana (PPA) activate macrophages through TLR4. PPA induces pro-inflammatory mediator expression in macrophages through ROS-, MAPK-, PKC-α/δ- and NF-κB-dependent pathways (The polysaccharides from the coral pathways . PPA pre


Correction to: BMC Neurol 19, 149 (2019)https://doi.org/10.1186/s12883-019-1379-2η2 value of 0.46. Using a partial η2 value of 0.46, which corresponds to an effect size f of 0.92, and five repeated measurements in 20 participants would yield a power of 0.999. The original sentence “Based on the data presented in the aforementioned study, a calculation (G*Power 3) revealed a power of 83% to detect significant differences when using SARA as the primary outcome measure ” is therefore replaced as follows: “Based on the reported partial η2 value of 0.46 in the aforementioned study, which corresponds to an effect size f of 0.92, a sample size of 20 participants who each have five measurements would yield an estimated power of 0.999 (G*Power 3.1) to detect significant differences when using SARA as the primary outcome measure (α = 0.05).”Following publication of the original article , the autThe original article has been corrected.