Cordyceps militaris is a fungus that parasitizes insects. Compounds from C. militaris are valuable in medicine and functional food. There are many kinds of bacteria in the natural sclerotia of C. militaris. However, the community structure of microorganisms in samples from different places may be different, and their corresponding ecological functions require experimental verification.C. militaris from Liaoning Province, China. We isolated, identified and verified the function of culturable bacterial strains from the sclerotia.We used high-throughput sequencing technology to analyze bacterial 16S rRNA gene sequences in sclerotia of three samples of Pseudomonas, Pedobacter, Sphingobacterium, and Serratia were the dominant bacterial genera in the sclerotia. And function prediction showed that Pseudomonas and Pedobacter could be heterotrophic, Sphingobacterium could decompose urea, and Serratia could reduce nitrate. Two strains of bacteria isolated from the sclerotia of C. militaris, N-2 and N-26, were identified as Stenotrophomonas maltophilia and Pseudomonas baetica, respectively, based on culture and biochemical characteristics. When these isolated strains were co-cultured with C. militaris, the mycelium biomass and mycelium pellet diameter decreased, and the content of extracellular polysaccharide increased. Strain N-26 decreased the cordycepin content in C. militaris.C. militaris and the production of its metabolites.Bacteria in sclerotia have an important effect on the growth of  Cordyceps militaris is a member of the fungal genus Cordyceps and is used as a traditional Chinese medicine. It contains a variety of active substances including cordycepin (3′-deoxyadenosine), cordyceps polysaccharide, and cordyceps acid  isolated from C. cicadae promoted the production of N6-(2-hydroxyethyl) adenosine and decreased the production of adenosine, uridine and guanosine , Liaoning Province, China. Cultured C. militaris (L.) Link was stored in the Institute of Fungi Resources of Guizhou University .Wild C. militaris obtained from Liaoning Province was divided into three samples for analysis in this work. Sclerotia samples were prepared using the method reported in C. militaris were rinsed with sterile water to remove residual soil, soaked alternatively with 75% alcohol and 2% sodium hypochlorite three times for 20 s each time, and then rinsed with sterile water. After removal from the body surface of the insect, the sclerotia were used for analysis. Each C. militaris sclerotia sample weighed about 3.5 g. The samples were stored at −80 °C until analysis.The wild LB medium contained tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, and agar 15 g/L (pH ≈ 7.0). Potato-dextrose-agar (PDA) contained potato 200 g/L, glucose 20 g/L, and agar 15 g/L (pH ≈ 7.0). Sabouraud’s medium contained glucose 40 g/L and peptone 10 g/L (pH ≈ 7.0).C. militaris sclerotia sample (0.5 g) was ground, and its suspension was placed on Luria Bertani agar for microbial isolation at 25 °C. Then, bacteria isolated by the culture method were observed by scanning electron microscopy , and their physiological and biochemical characteristics were identified using bacterial biochemical identification strips HBIG05 and HBIG08 . Bacterial DNA was extracted according to the procedures for the Bacterial Genomic DNA Extraction Kit DP2002 . The 16S rRNA gene was amplified from all DNA extracts using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (2O (8.5 µL). The PCR conditions were: predenaturation at 95 °C for 3 min; 32 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min. PCR products were sequenced by Tsingke Biotechnology Co., Ltd. Using the sequence data, BLAST was performed against DNA sequences in GenBank, and the sequences of related species (similarity > 97%) were downloaded. Using Pseudomonas carboxydohydrogena as the outgroup, a phylogenetic tree was constructed by the neighbor-joining method using MEGA X software with 1,000 bootstrap replicates.Each ACTT-3′) . The reaC. militaris SYCM1910 was inoculated on the center of a PDA plate and cultured at 25 °C for 7 days. Then, bacteria were inoculated at three locations on the periphery of the colony (25 mm from the point where C. militaris had been inoculated) and culture was continued at 25 °C for 7 days.C. militaris from a 21-day-old culture on PDA plate culture was inoculated into a 250-mL triangular flask containing 100 mL Sabouraud’s medium and cultured on a magnetic stirrer (120 rpm) at 25 °C for 3 days. Then, one mL/flask of bacterial suspension  was added and culture was continued at 120 rpm and 25 °C for 7 days. The co-cultured fermentation liquid was filtered using 0.45-µm and 0.22-µm microporous membranes, and then the filtrate was analyzed for the content of cordycepin and extracellular polysaccharides (EPS).A piece of mycelium of Dry weight of mycelium pellets was determined after the fermentation broth was filtered using qualitative filter paper and the pellets were dried to constant weight at 60 °C. Then, the mycelium pellet diameter was measured using vernier calipers.18 column with mobile phase acetonitrile: water (5:95 v:v) at flow rate 1.0 mL/min, column temperature 35 °C, detection wavelength 260 nm, and with sample volume 10 µL. EPS content was determined by the anthrone sulfuric acid method  were taken, ground in liquid nitrogen, and total microbial DNA was extracted according to the instructions of the E.Z.N.A.® SOIL DNA Kit . PCR amplification used TransStart FastPFU DNA Polymerase. The reaction system contained: 5 × FastPFU buffer (4 µL), 2.5 mmol/L dNTPs (2 µL), 5 µmol/L primers 338F (5′-ACTCCTACGGGAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTA-3′) (0.8 µL each) targeting the V3–V4 region of 16S rRNA genes , repetitive sequence operational taxonomic unit (OTU) clustering was carried out with a threshold of 97% similarity, chimeras were removed in the process of clustering, and the RDP database (http://rdp.cme.msu.edu/) was used for OTU annotation.Paired-end reads obtained by MiSeq sequencing were first stitched by overlap, and sequence quality was controlled and filtered at the same time. Effective sequences were obtained by distinguishing samples according to barcode and primer sequences at both ends of the sequence, and sequence direction was corrected to obtain optimized sequences. Using UPARSE software (PRJNA722375. FAPROTAX (http://www.zoology.ubc.ca/louca/FAPROTAX/), a tool that can predict ecological functions of bacterial and archaea taxa from 16S rRNA amplicon sequencing   (MW829549).Two pure strains of bacteria, N-2 and N-26, were isolated from sclerotia of –3.8 µm)  and Gram–3.8 µm)  showed ta (Hugh)  (NCBI acPseudomonas baetica YHNG5 (P. baetica (Lopez) (NCBI accession number: MW829550).Strain N-26 is short rod-shaped (9.5–11.5 × 4.2–5 µm), Gram-negative, and its colonies are yellow, smooth, moist and opaque, with a central bulge. The semi-solid agar (dynamic test) of strain N-26 was positive, the Voges-Proskauer test was positive, and the Methyl Red test was negative. The strain was positive for ornithine decarboxylase, lysine decarboxylase and amino acid decarboxylase. The strain could not use mannitol, inositol, sorbitol, melibiose alcohol, raffinose, xylose, or maltose as carbon sources. In BLAST analysis, the 16S rRNA gene sequence of strain N-26 was 99.71% identical to that of ca YHNG5 , which lS. maltophilia N-2 had an inhibitory effect on the growth of mycelium of C. militaris. Strain N-2 released something that slowed the growth of C. militaris mycelia near the area of S. maltophilia N-2 growth   . However, addition of strain N-2 had little effect   (The dry weight of mycelium pellets decreased from 1.57 g/flask in the control to 0.21 g/flask in the presence of < 0.001) . The dia< 0.001) . The add= 0.622) . The EPS20 mg/g) .C. militaris collected in October 2019 in Liaoning Province, China; 62,929–71,212 sequences were obtained for each sample, with average length 423.45–425.61 bp. With the increase of the number of sample sequences, the Shannon-Winner index curve flattened out , Unclassified Enterobacteriaceae (14%), Pedobacter (11%), Sphingobacterium (11%), Serratia (10%), Rhodococcus (6%), and Acromobacter (6%) were dominant.Bacteria identified in the sclerotia included 21 phyla, 46 classes, 123 orders, 195 families, 321 genera, 450 species, and 549 operational taxonomic units (OTUs). At the phylum level , ProteobStenotrophomonas) is animal parasitic or symbiotic, and a human pathogen; it actively participates in the nitrogen cycle. OTU2342 (Sphingobacterium) is involved in urea decomposition. OTUs 1448, 2330 and 2314 (Pseudomonas) are chemoheterotrophic. OTUs 1539 and 1423 (Rhodococcus) degrade aromatic hydrocarbons and aliphatic non-paraffin hydrocarbons.Forty-two OTUs were common to the three samples , accountC. militaris sclerotia, S. maltophilia N-2 and P. baetica N-26. Then, we co-cultured these bacteria with C. militaris, and found that both of them increased the EPS content of C. militaris, but Pseudomonas baetica N-26 decreased the cordycepin content.In our study, we isolated two bacteria belonging to the microbiota of C. militaris sampled from Liaoning Province, China, were identified using high-throughput sequencing technology. Pseudomonas were more abundant in sclerotia of C. militaris isolated in Liaoning Province than in C. militaris isolated in Yunnan Province, while Phyllobacterium, Mesorhizobium, and Bradyrhizobium were less abundant in the former  and Liaoning Province , but their relative abundances were different, which may be related to differences of the insect host and environmental conditions   . Therefod health . We concPseudomonas has many functions, e.g., P. fluorescens secretes luciferin and inhibits the growth of Escherichia coli in insects  increased after invasion by C. sinensis, and P. fragi became the dominant bacterium and participated in the process of larval mummification  or N-26 (P. baetica), so the cordycepin content in the culture medium decreased accordingly. Several mechanisms could explain this effect. One is inhibition of the expression of cordycepin-synthesis-related genes   ; this caC. militaris and keep the insect host from decaying. Our data indicate that some symbiotic bacteria may inhibit the production of cordycepin. We speculate that other symbiotic bacteria may promote the production of cordycepin. Thus, we aim to isolate other microorganisms from the sclerotia and further explore the relationships between the isolates and C. militaris. When increasing numbers of interactions are revealed, the ecological functions of microbes in the sclerotia will become clearer.Because cordycepin has antibacterial function , we specPseudoalteromonas sp. MEBiC 03485 co-cultured with Porphyridium cruentum UTEX 161, the content of sulfated polysaccharide was increased . Numbers in parentheses are GenBank accession codes. Bar: 2% sequence divergence.Click here for additional data file.10.7717/peerj.12511/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12511/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12511/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.12511/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.12511/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.12511/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.12511/supp-8Supplemental Information 8Click here for additional data file.


Kv7 activation by cGMP contributes to the pulmonary vasodilator action of nitric oxide, but its contribution when dilation is evoked by the atrial natriuretic peptide (ANP) sensitive guanylate cyclase, or cAMP, is unknown. Small vessel myography was used to investigate the ability of Kv7 channel blockers to interfere with pulmonary artery relaxation when cyclic nucleotide pathways were stimulated in different ways. The pan-Kv7 blockers, linopirdine and XE991, caused substantial inhibition of relaxation evoked by NO donors and ANP, as well as endothelium-dependent dilators, the guanylate cyclase stimulator, riociguat, and the phosphodiesterase-5 inhibitor, sildenafil. Maximum relaxation was reduced without a change in sensitivity. The blockers had relatively little effect on cAMP-mediated relaxation evoked by forskolin, isoprenaline or treprostinil. The Kv7.1-selective blocker, HMR1556, had no effect on cGMP or cAMP-dependent relaxation. Western blot analysis demonstrated the presence of Kv7.1 and Kv7.4 proteins, while selective activators of Kv7.1 and Kv7.4 homomeric channels, but not Kv7.5, caused pulmonary artery relaxation. It is concluded that Kv7.4 channels contribute to endothelium-dependent dilation and the effects of drugs that act by stimulating cGMP, but not cAMP, signalling.Pulmonary hypertension is treated with drugs that stimulate cGMP or cAMP signalling. Both nucleotides can activate Kv7 channels, leading to smooth muscle hyperpolarisation, reduced Ca Although these pathways in PASMCs are widely documented  are the mainstay of targeted therapies used to treat pulmonary arterial hypertension (PAH). They mimic the actions of the endothelium-derived mediators, nitric oxide (NO) and prostacyclin . Both. Both2),n , the , the 2),+ current in PASMC, hyperpolarised the cell membrane, and relaxed isolated pulmonary arteries, effects that were inhibited by Kv7 channel blockers  ethanesulphonic acid) 10, KHv/v), 0.5% sodium deoxycholate (w/v), 1% sodium dodecyl sulphate , 1 mM phenylmethylsulphonyl fluoride (PMSF) and 1× complete, Mini, EDTA-free protease inhibitor cocktail . The homogenate was centrifuged at 18,000× g for 15 min at 4 °C, the supernatant aspirated, and its protein content measured using the bicinchoninic acid (BCA) assay with Nanodrop 1000 spectrophotometer. Lysates were stored at −80 °C for later analysis.Protein was extracted from isolated pulmonary arteries, the lung and the brain. Tissue samples were homogenised in chilled RIPA lysis buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediamintetraacetic acid (EDTA), 1% NP-40 , alongside molecular weight markers . The separated proteins were transferred to a polyvinylidene-fluoride (PVDF) membrane using a Trans-Blot® Cell apparatus (Bio-Rad), washed 3 times with Tris-buffered saline containing 0.1% Tween-20 (TTBS), blocked for 1 h with TTBS containing 5% dried milk powder, washed again, and then incubated overnight at 4 °C with a primary antibody. Antibody binding was detected using a horseradish peroxidase (HRP)-linked secondary antibody with SuperSignalTM chemiluminescent substrate  and imaged with a ChemiDoc system (Bio-Rad). Primary antibodies were obtained as follows: mouse monoclonal Kv7.1 and Kv7.4 from NeuroMab , rabbit polyclonal Kv7.5 from Alomone Labs , mouse monoclonal α-tubulin from Sigma-Aldrich , anti-mouse IgG/HRP from Jackson ImmunoResearch  and anti-rabbit IgG/HRP from Dako .Aliquots of lysate containing 25 μg of protein were diluted 4:1 with 5× Laemmli buffer, and the protein was denatured by boiling for 5 min at 95 °C. Samples were then subjected to electrophoresis using a 10% SDS-PAGE gel in a Mini-PROTEINH)-anthracenone dihydrochloride), carbachol chloride, isoprenaline hydrochloride, forskolin, mefenamic acid, diclofenac sodium, levcromakalim and retigabine were sourced from Sigma-Aldrich. Linopirdine hydrochloride, HMR1556 -3,4-Dihydro-3-hydroxy-2,2-dimethyl-6--2H-1-benzopyran-4-yl]-N-methylmetanesulfonamide), treprostinil, R-L3 (5-(2-Fluorophenyl)-1,3-dihydro-3-(1H-indol-3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one), ML277 ((2R)-N-[4-(4-Methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2-piperidinecarboxamide), ICA-069673 (N-(2-Chloro-5-pyrimidinyl)-3,4-difluorobenzamide), GW0742, SC-51322 and iberiotoxin were obtained from Tocris Bioscience . Sildenafil citrate was supplied by Cambridge Bioscience , A23187 was from Calbiochem  and glyceryl trinitrate (GTN) from Aspire Pharma Ltd. . Depending on solubility, stock solutions were prepared as 100 or 10 mM drug in water or dimethyl sulphoxide (DMSO) and stored as frozen aliquots. DMSO was present at <0.1% in the myograph chamber.Phenylephrine hydrochloride, sodium nitroprusside (SNP), ANP (127–150 rat), XE991 -9 and the drug concentration evoking 50% of the maximum effect (EC50) were measured. All statistical analyses employed Prism software version 7.04 . The D’Agostino–Pearson omnibus and Shapiro–Wilk tests were used to assess how well data followed a normal distribution and parametric or non-parametric tests of statistical significance were applied accordingly. Two groups of data were compared using an unpaired, two-tailed t test. Comparison of three or more data sets employed one-way ANOVA with Dunnett’s multiple comparisons test. Two-way repeated measures ANOVA was employed to compare concentration–response curves when Emax or EC50 could not be resolved. Differences were considered statistically significant when p < 0.05.Data are expressed and plotted as mean ± s.e.m. of vessels from “n = 9), 61 ± 2% (n = 6) and 89 ± 3% (n = 4) of the agonist-induced tone, respectively. The maxima were essentially halved by linopirdine to 54 ± 2%  for SNP, 28 ± 2%  for GTN and to 42 ± 8%  for ANP. Inhibition occurred without a change in pEC50. The SNP pEC50 was 7.72 ± 0.07 in control conditions and 7.67 ± 0.07 in the presence of linopirdine, GTN values were 7.44 ± 0.07 (control) and 7.3 ± 0.1 (linopirdine) and ANP relaxed with pEC50 = 9.0 ± 0.1 (n = 4) before and 9.0 ± 0.2 (n = 4) after adding linopirdine. XE991 (1 μM) also suppressed the maximum relaxation evoked by SNP  and GTN  without affecting pEC50, which remained at 7.4 ± 0.2 with SNP and 7.4 ± 0.1 with GTN. In contrast to the pan-Kv7 blockers, HMR1556 (1 μM) had no significant effect on pulmonary artery relaxation evoked by these vasodilators (dilators .50 = 7.9 ± 0.2 (n = 6) in control conditions and 7.3 ± 0.2 (n = 6) in the presence of linopirdine (50 (p < 0.01) without affecting the maximum relaxation. Sildenafil produced a biphasic concentration–response relationship , but the inhibition was overcome at higher sildenafil concentrations.Riociguat relaxed pulmonary arteries almost completely with pECopirdine A. In contionship B; thus, 2+ ionophore, A23187 (n = 6) in control conditions to 69 ± 3% (n = 6) in the presence of 1μM linopirdine and 64 ± 2% (n = 6) in the presence of 1 μM XE991. These Kv7 channel blockers also caused a small but statistically significant (p < 0.05) reduction in the carbachol pEC50, which was 6.5 ± 0.1 in control conditions, 6.1 ± 0.1 after adding linopirdine and 6.1 ± 0.06 in the presence of XE991. A23187 relaxed pulmonary arteries with pEC50 = 7.7 ± 0.1 and maximum at 68 ± 6% (n = 8) of phenylephrine-induced tone. Responses to A23187 at all concentrations were almost abolished by linopirdine and XE991 (at 1 μM). In contrast to the pan-Kv7 blockers, HMR1556 did not significantly alter the carbachol or A23187 concentration–response curves.Linopirdine and XE991 also inhibited endothelium-dependent relaxation evoked either by the muscarinic receptor agonist, carbachol A, or Ca2, A23187 B. The pa50 values. Isoprenaline relaxed arteries with pEC50 = 8.1 ± 1 (n = 8) in control conditions, falling to 7.73 ± 0.09 (n = 7) in the presence of linopridine and 7.53 ± 0.07 (n = 11) in the presence of XE991 (p < 0.05). Forskolin evoked relaxation with pEC50 = 7.25 ± 0.04 (n = 6), falling to 6.98 ± 0.07 (n = 7) when exposed to linopirdine and 6.72 ± 0.07 (n = 4) in the presence of XE991 (p < 0.01). Neither linopirdine nor XE991 significantly altered the maximum relaxation evoked by isoprenaline  or forskolin .n = 7) in control conditions, 48 ± 8%  in the presence of linopirdine and 36 ± 5%  in the presence of XE991. This occurred without a change in the treprostinil pEC50, which was 7.79 ± 0.04 in control conditions, 7.6 ± 0.03 in the presence of linopirdine and 7.7 ± 0.09 in the presence of XE991. Treprostinil is known to bind to and activate peroxisome proliferator-activated receptors (PPARs) independently of cAMP [50 = 5.78 ± 0.06 (n = 8) in control conditions and 5.3 ± 0.07 (n = 8) in the presence of XE991. A maximum relaxation of 102 ± 2% (n = 8) was achieved both before and after applying XE991.Linopirdine (1 μM) and XE991 (1 μM), but not HMR1556 (1 μM), inhibited pulmonary artery relaxation evoked by treprostinil A. This i of cAMP . As the  of cAMP , we inven = 8, p < 0.0001). It was reduced further to 16 ± 2%  when XE991 (1 μM) was added. The treprostinil pEC50 was reduced to 7.3 ± 0.2  by L-NAME, but was not further affected by XE991 . Treprostinil lacks receptor selectivity. While its activation of IP, DP1 and EP2 receptors leads to stimulation of AC and cAMP synthesis, it can also bind to prostanoid receptors that evoke rat pulmonary artery contraction and counteract relaxation [3 receptor (Ki > 2μM), thought to be the main mediator of contraction, it binds the EP1 receptor (Ki = 285 nM) with only 10-fold lower affinity than IP [1 and EP3 receptors, SC-51322 (10 μM) enhanced the treprostinil-induced relaxation  measured in the presence of L-NAME  inhibition of phenylephrine-induced tone with pEC50 = 6.62 ± 0.04 (EC50 = 240 nM). ML277 relaxed phenylephrine-contracted arteries up to a maximum of 84 ± 6% (n = 8) with pEC50 = 6.74 ± 0.02 (EC50 = 182 nM). The effect of K+ channel activation on membrane potential, and hence vascular tone, can be prevented by depleting the transmembrane K+ gradient. This was performed by raising the extracellular K+ concentration to 80 mM, which causes depolarisation and evokes contraction. R-L3 and ML277 were less effective at relaxing arteries contracted with 80 mM K+, compared with phenylephrine, but their effects were not abolished  and 99 ± 5% maximum . ICA-069673 was originally identified as a potent Kv7.2/Kv7.3 activator that is inactive at Kv7.1 channels [50 = 5.21 ± 0.06  and 104 ± 1% maximum , an activator of the Kv7.1 isoform ,31, relachannels , but at channels . ICA-069ximum  and 6.46 ± 0.08 , respectively, without changing the maximum relaxation. In contrast, HMR1556 had no effect on the ICA-069673 concentration–response curve  shifted the RL-3 B and ML2se curve E. It alsse curve F, or theomakalim G. As BKCarteries ,37, we t blocker , to inteWestern blot analysis confirmed the expression of Kv7.1 and Kv7.4 proteins in pulmonary arteries, but not Kv7.5. The expression of Kv7 subunits was investigated initially in whole lung A by usinThe results of this study suggest that Kv7 channels contribute substantially to pulmonary vasodilation mediated by the cGMP-signalling pathway but have a lesser role in cAMP signalling. Pharmacological and expression profiling further suggests that Kv7.4 is the isoform most likely to act as the functional target of cGMP in rat pulmonary arteries.The broad-spectrum Kv7 channel blockers, linopirdine and XE991, were recently shown to impair pulmonary artery relaxation induced by NO donors and riociguat . Here, s2+ influx and muscle relaxation. The pulmonary artery resembles the aorta (at least in rat), where relaxations induced by activating the pGC with ANP or the sGC with NO donors were both impaired by linopirdine, but it differs from the renal artery where only the pGC was linked to Kv7 channels [The pattern of inhibition depended on the method of raising cGMP. The Kv7 blockers suppressed maximum responses to SNP, GTN, ANP and endothelium-derived NO, suggesting that a key pathway required to generate full relaxation had been removed. The results support the finding of Mondéjar-Parreño and colleagues  that NO channels . Multiplchannels , but the50 of 2-4 nM [The clear inhibition of sildenafil-induced relaxation by Kv7 blockers at sub-micromolar sildenafil concentrations was overcome at higher sildenafil concentrations. This may reflect a lack of sildenafil selectivity at higher concentrations, suggested by its biphasic concentration–response relationship. Deviation from the classical sigmoidal shape has been noted before for sildenafil . Relaxatf 2-4 nM , while rf 2-4 nM . Thus, t50 rather than reducing the maximum relaxation. This differential effect of the blockers on relaxation induced by riociguat versus NO donors is also apparent in the concentration–response curves reported by Mondéjar-Parreño et al. [50 ~20 nM [While Kv7 blockers inhibited relaxation to riociguat, they increased its ECo et al.  but was o et al. . Its abio et al.  with IC50 ~20 nM . At a 500 ~20 nM . RiociguLinopirdine and XE991 reduced the potencies of isoprenaline and forskolin to relax pulmonary arteries without impairing the maximum relaxation. Such inhibition is consistent with the known ability of cAMP to enhance Kv7 channel activity, but the effect was small in comparison to the rat renal artery, where linopirdine and Kv7.4 gene silencing caused a ≥10-fold increase in the concentration dependence of isoprenaline and forskolin evoked relaxation . Downreg1 and/or EP3 receptors. Despite a low affinity of treprostinil for EP3 receptors [1 receptors. They do not contribute to contraction in the human pulmonary artery [At first glance, the large effect of Kv7 channel blockers on treprostinil relaxation conflicts with their effects on isoprenaline and forskolin and is more similar to the inhibition of cGMP-meditated relaxation. IP receptors are present on endothelial cells, and their activation stimulates NO synthesis . Thus, eceptors , they apeceptors . Treprosy artery ,52, but 50 values < 250 nM, and the effects were antagonised by HMR1556 at 1μM. Importantly, HMR1556 did not interfere with relaxation evoked by the Kv7.4 channel activator ICA-069673 [ATP channel activator, levcromakalim, indicating specificity for the Kv7.1 activators. Consistent with K+ channel activation as a mechanism of relaxation, R-L3 and ML277 were both less effective when tested on pulmonary arteries contracted with 80 mM K+ instead of phenylephrine. Nevertheless, substantial relaxation remained in those conditions; thus, part of the actions of both drugs must be unrelated to Kv7.1 channels. The selectivity of Kv7.1 modulators remains to be widely tested, but the relaxation of rat [Rat pulmonary arteries expressed Kv7.1 protein and responded with concentration-dependent relaxation to three activators of Kv7.1 . ML277 and R-L3 both relaxed the rat pulmonary artery with ECA-069673 , the Kv7A-069673  or the Kn of rat  mesentern of rat . DespiteKv7.5 protein could not be detected either in whole lung or isolated pulmonary arteries, despite the Kv7.5 mRNA transcript being present in rat PASMCs ,55. This+, consistent with K-channel activation as the main cause of relaxation. The diclofenac EC50 for relaxation (~200 μM) is close to its EC50 for activating Kv7.4 . Diclof (102 μM  and to ichannels . Althoug2+-activated K (BKCa) channels in PASMCs [Ca channels. It can inhibit other Ca2+-activated channels, as well as Kv1.2 and Kv1.3 channels [Ca channel blocker, iberiotoxin [+ current or artery relaxation evoked by NO donors [Ca channels are therefore not involved in cGMP- or cAMP-dependent pulmonary vasodilation.Both cAMP and cGMP have been proposed to relax pulmonary arteries by activating large-conductance Can PASMCs ,37,59, an PASMCs ,61. The n PASMCs ,59, whicchannels . Moreovechannels ,63. The riotoxin , was recO donors . We addi2+ influx, which promotes vasodilation [Kv7.1 and Kv7.4 subunits are both downregulated in the lungs of patients  and animdilation ,7 and prdilation ; thus, tdilation ,57,67 andilation ,67. Kv7.The main limitation is the reliance on drugs to interfere with cellular pathways, especially where selectivity has not been widely tested. Although gene silencing could provide more specific knockout of channel subunits from the artery, in our hands it does not suppress expression reproducibly or reliably. To minimize the problem, drug concentrations were selected to avoid known off-target interactions. While no Kv7 modulator is specific enough to rely on by itself, conclusions were drawn from the pattern of effects of multiple drugs with similar or opposing actions on different Kv7 channels. While the evidence shows that cAMP does not act through Kv7 channels in pulmonary artery, its effects on the channels have not been investigated directly using electrophysiological techniques.Ca channels, in mediating cGMP-dependent pulmonary vasodilation. Whether resulting from endothelium-released NO, ANP, NO donors, guanylate cyclase stimulators, or PDE5 inhibition, relaxation was inhibited by Kv7 channel blockers. Any contribution of Kv7 channels to cAMP-dependent pulmonary vasodilation was, however, small, with other mechanisms dominating. Kv7.4 is the best candidate for mediating cGMP relaxation, because: (1) blocking Kv7.1 channels did not affect relaxation; (2) Kv7.5 channels are poorly expressed in PASMC; (3) drugs that directly activate Kv7.4, but not Kv7.5, produced robust pulmonary artery relaxation.The results of this study strongly support a role for Kv7 channels, but not BKThe effects of therapeutic drugs that depend on cGMP for their action may be limited by downregulation of Kv7.4 channels in PAH. As Kv7 channel activators can reduce PAH, they offer an additional target for drug development.


Limited data from clinical trials in multiple sclerosis (MS) reported that minocycline, a widely used antibiotic belonging to the family of tetracyclines (TCs), exerts a beneficial short-lived clinical effect A similar anti-inflammatory effect of minocycline attributed to a deviation from Th1 to Th2 immune response has been reported in experimental models of MS. Whether such an immunomodulatory mechanism is operated in the human disease remains largely unknown.in vitro immunomodulatory effect of tetracyclines, and in particular minocycline and doxycycline, in naïve and treated patients with MS.To assess the Peripheral blood mononuclear cells from 45 individuals  were cultured with minocycline or doxycycline and conventional stimulants (PMA/Ionomycin or IL-12/IL-18). IFN-γ and IL-17 producing T-, NK- and NKT cells were assessed by flow cytometry. The effect of TCs on cell viability and apoptosis was further assessed by flow cytometry with Annexin V staining.+ T lymphocytes from MS patients (naïve or treated) stimulated with IL-12/IL-18 but did not decrease IFN-γ producing CD8+ T cells from naive MS or treated RRMS patients. They also decreased IL-17+ T and NKT cells following PMA and Ionomycin-stimulation. Tetracyclines did not affect the viability of cell subsets.Both tetracyclines significantly decreased, in a dose dependent manner, IFN-γ production in NKT and CD4in vitro suppress IFN-γ and IL-17- producing cells from MS patients, and this may explain their potential therapeutic effect in vivo.Tetracyclines can  Multiple Sclerosis (MS) is a chronic autoimmune inflammatory demyelinating disease of the central nervous system (CNS) . Based o+ Th1 cells produce inflammatory mediators, such as interferon-γ (IFN-γ) which lead to autoantigen-specific inflammatory attack resulting in myelin degeneration  , 21, sucin vitro effect of both minocycline and doxycycline on IFN-γ and IL-17 production by peripheral cell populations in patients with MS (newly-diagnosed naive and treated RRMS).The precise cellular source and mechanism of cytokine modulation by TCs in MS patients, remains largely unknown and studies conducted so far limited their assessment in particular cell-subsets, selecting either TC but not both. The aim of our study was to comparatively investigate for the first time the A total of 35 patients with MS diagnosed according to the McDonald criteria  were incAll experimental protocols were approved by the Local Institution’s Ethical Committee of the University General Hospital of Larissa, University of Thessaly while written informed consent has been obtained from all study participants according to the declaration of Helsinki.Peripheral blood samples (20-30mL) from MS patients and HCs were collected by venipuncture in preservative-free heparin tubes (50 U/mL) and aliquots were layered onto an equal volume of Ficoll-Hypaque (10ml Lymphoprep™) density gradient solution . Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation at 300g, washed twice with RPMI-1640 , counted, and their viability, determined by trypan blue exclusion, routinely exceeded 95%. Cells were re-suspended in freezing medium containing 10% DMSO and 70% fetal calf serum (FCS), aliquoted into cryogenic vials , kept at −80°C for one day, and then stored in liquid nitrogen tanks until used.Results section). DMSO concentration in TC-supplemented cultures was less than 0.1%. The same DMSO concentration (0.1%) was also added to control cultures in the absence of TCs, to exclude a direct cytotoxic effect of DMSO. Recombinant IL-12 and IL-18 were purchased from R and D Systems Inc  and used at a final concentration of 20 ng/ml and 25 ng/ml, respectively  were purchased from Cayman Chemical Company, Michigan USA. Both antibiotics were reconstituted in DMSO, further aliquoted at small volumes and stored at a final concentration of 5 mg/ml. TCs were supplemented simultaneously with cell stimuli (see below) at a final concentration of 50μg/ml unless otherwise stated and remained in culture for 5 hours . PBMCs were seeded in 24-well plates and allowed to rest at 37°C in a CO2 incubator for at least one hour before stimulation. In this study we used the following mouse anti-human MoAbs: (FITC)-conjugated anti-CD3 (clone UCHT-1), (PE)- and (PE-Cy7)-conjugated anti-CD56 (clones C5.9 and B159), (PE)- and (PE-Cy7)-conjugated anti-CD4 (clone RPA-T4) and PE conjugated anti-CD8 (clone SK1). All MoAbs were obtained from BD Biosciences  and Merck-Millipore . Isolated PBMCs (0.5-1 x 106cells) were washed in phosphate-buffered saline (PBS) and re-suspended in staining buffer (PBS plus 1% FCS plus 0.09% sodium azide) and then incubated with labeled MoAbs specific for cell surface antigens for 30 minutes on ice and fixed with paraformaldehyde (2%). Background auto-fluorescence was monitored by equivalent 4-colour matched isotype control mouse anti-human MoAbs and formed the basis to set the cut-off for surface-positive cell discrimination. Flow cytometric analysis was performed in Guava® EasyCyte8  benchtop flow cytometer using logarithmic amplification and a forward and side scatter-based gate for total lymphocyte populations. At least 3x105 events within the lymphocyte gate were collected for accurate measurement of infrequent cell subtypes.Phenotypic assessment and enumeration of peripheral blood sub-populations was performed using standard monoclonal antibodies (MoAbs) panels and protocols as described previously , 44. BriIL-12 and IL-18 activated PBMCs supplemented with 50μg/ml minocycline or doxycycline were collected and stained with FITC-labeled Annexin V , which is used to specifically target and identify apoptotic cells , in the In order to measure intracellular IFN-γ protein production by peripheral T cells and innate NK and NKT cells, PBMCs were left untreated, or cultured in 10% RPMI supplemented with 20 ng/ml PMA plus 1μg/ml ionomycin for 5 hours in the presence of Brefeldin A . Cells were surface stained, fixed and subsequently permeabilized using commercially available Perm/Wash buffers (BD Biosciences). Intracellular IFN-γ protein was detected using APC-conjugated MoAbs (clone 4S.B3) obtained from BD Biosciences.In order to measure intracellular IL-17 protein production by peripheral Th17 cells, PBMCs were left untreated or cultured in 10% RPMI supplemented with 50 ng/ml PMA plus 1μg/ml ionomycin for 5 hours in the presence of Brefeldin A. Cells were surface stained, fixed and subsequently permeabilized using commercially available Perm/Wash buffers (BD Biosciences). Intracellular IL-17 was detected using FITC- and PE-conjugated MoAbs clones (BL-168) all obtained from BD Biosciences and Merck-Millipore.Percentages of cells expressing cell surface markers and mean fluorescence intensities (MFI) were described as median of the individuals in each group. Variation in each patient group was defined by standard deviation (SD). Differences between healthy controls and patients and between patient groups one-way analysis of variance (ANOVA) and the nonparametric Mann-Whitney test. P-values smaller than 0.05 were considered significant. All graphs and statistical calculations were performed with Graph Pad Prism 9 software.in vitro effect of either minocycline or doxycycline on IFN-γ production, antibiotics in isolation were supplemented in PBMC cell cultures at different concentrations ranging from of 0.1μg - 50μg/ml, similarly to previous reports in whole blood cultures and THP-1 human monocytes , doxycycline plasma concentrations (Cmax) of 1.5 to 7.0 μg/ml were usually reached within 3 h, and the drug had a half-life of 14 to 24 h  activation and robust IFN-γ production within 2-6hrs post stimulation in innate and adaptive cells . However, the maximal inhibitory effect was noted when IL-12 plus IL-18 and 50μg/ml minocycline or doxycycline were used (see below). In all tested concentrations there was no detrimental effect of TCs on cell viability, as assessed by microscopic evaluation and trypan blue exclusion in line with previously published observations  cells following treatment with doxycycline   decreased from 1.1 ± 0.41% to 0.53 ± 0.21% in the presence of minocycline (p = 0.004) and to 0.42 ± 0.20% in the presence of doxycycline (p = 0.001). The percentage of IFN-γ+ CD4+ T cells in RRMS patients (n=20) decreased from 0.89 ± 0.43% to 0.53 ± 0.27% in the presence of minocycline (p = 0.009) and to 0.44 ± 0.22% in the presence of doxycycline (p = 0.002). In HCs (n=10), the percentage of IFN-γ+ CD4+ T lymphocytes was reduced from 1.87 ± 0.79% to 0.80 ± 0.51% in the presence of minocycline (p = 0.0015) and to 0.71 ± 0.45% in the presence of doxycycline (p = 0.001)  Figure 1+ T cells from naive MS (n=15) or RRMS patients (n=20) (+CD8+ T lymphocytes was reduced from 3.43 ± 0.45% to 2.18 ± 0.78% in the presence of minocycline (p = 0.001) and to 2.10 ± 0.75% following doxycycline supplementation (p = 0.001) (Minocycline and doxycycline did not decrease IFN-γ producing CD8s (n=20) Figure 2+ NKT lymphocytes following IL-12 plus IL-18 stimulation in naïve MS (n=15) patients decreased from 6.49 ± 4.10% to 3.61 ± 2.39% in the presence of minocycline (p = 0.02) and to 2.52 ± 1.87% in the presence of doxycycline (p = 0.003) (+ NKT cells in RRMS patients (n=20) decreased from 7.34 ± 4.40% to 3.92 ± 2.51% in the presence of minocycline (p = 0.01) and to 2.40 ± 1.89% in the presence of doxycycline (p = 0.001). In HCs (n=10), the percentage of IFN-γ+ NKT lymphocytes was reduced from 14.73 ± 7.53% to 7.35 ± 4.27% following minocycline supplementation (p = 0.03) and to 5.41 ± 3.07% following doxycycline supplementation (p = 0.005).IFN-γ= 0.003) Figure 3+ cells in the presence of minocycline or doxycycline in naïve MS (n=15), RRMS (n=20) or HCs (n=10)  Figure 4+CD4+ T cells and CD56+CD4+ NKT cells. In patients with naïve MS the percentage of CD4+IL-17A+ T cells decreased from 1.77 ± 1.33% to 0.74 ± 0.43% in the presence of minocycline (p = 0.02) and to 0.55 ± 0.44% in the presence of doxycycline (p = 0.01) (+IL-17A+ T cells decreased from 2.14 ± 1.68% to 1.2 ± 0.9% in the presence of minocycline (p = 0.04) and to 0.76 ± 0.71% in the presence of doxycycline (p = 0.02). In HCs the percentage of CD4+IL-17A+ T cells decreased from 1.35 ± 0.51% to 0.64 ± 0.35% in the presence of minocycline (p = 0.01) and to 0.43 ± 0.29% in the presence of doxycycline (p = 0.02).We also investigated the effect of TCs on IL-17A production in PMA plus ionomycin activated PBMCs from patients with naïve MS (n=9), RRMS patients (n=9) and HCs (n=9). As shown in  = 0.01) Figure 5+IL-17A+ NKT lymphocytes in naïve MS patients (n=9) decreased from 1.49 ± 0.80% to 0.69 ± 0.33% in the presence of minocycline (p = 0.008) and to 0.48 ± 0.17% in the presence of doxycycline (p = 0.001). The percentage of CD4+IL-17A+ NKT lymphocytes in RRMS patients (n=9) decreased from 2.59 ± 0.90% to 1.09 ± 0.43% in the presence of minocycline (p = 0.009) and to 0.8 ± 0.55% in the presence of doxycycline (p = 0.006). The percentage of CD4+IL-17A+ NKT lymphocytes in HCs (n=9) decreased from 1.63 ± 0.75% to 0.87 ± 0.54% in the presence of minocycline (p = 0.04) and to 0.84 ± 0.64% in the presence of doxycycline (p = 0.04).The percentage of CD4+ T and NKT lymphocytes.The major finding of our study is that of an inhibitory effect of minocycline and doxycycline on IFN-γ and IL-17 producing cells in a cell subset-related, dose-dependent manner in patients with MS, with the most prominent effect being noted in CD4Our results are of importance for studies conducted in humans, as previous studies in MS have only been limited to EAE, with their findings emphasizing an anti-inflammatory role of antibiotics and in particular minocycline, in synergy with prednisone , IFN-β1 + T and NKT cells. It remains to be seen whether the in vitro effect of TC could be augmented following supplementation of MS-related immunomodulatory agents and whether this effect could be seen in vivo.In our cohort of patients with MS, a comparable TC-mediated reduction of cytokine production was noted between naïve and treated RRMS patients. Subsequent stratified analysis amongst patients receiving different therapies revealed no statistically significant differences regarding the levels of TC-mediated cytokine inhibition within CD4Clinical trials using minocycline, alone or in combination with conventional therapeutic agents, have produced inconclusive results, some demonstrating beneficial effect on the risk of conversion from a clinically isolated syndrome to MS , while oin vitro study. In rheumatoid arthritis, a typical autoimmune disease, both experimental and human data reveal a beneficial effect of TCs in combination with methotrexate over methotrexate alone, especially in early disease state, suggesting that timing of initiation of TCs may be of importance . If this is proved in vivo in prospectively collected biological material from MS patients under treatment with these tetracyclines, it may have potential clinical relevance. In vivo inhibition of IFN-γ (and possibly IL-17)-producing NKT cells, for example, is a favorable pre-requisite for successful remission in patients with MS (In conclusion, this is the first  with MS –59, and The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics and Scientific Committee, University General Hospital of Larissa, University of Thessaly, Faculty of Medicine, School of Health Sciences. The patients/participants provided their written informed consent to participate in this study.DF, AM, and ED contributed equally. LS, GH, and DB had the original idea. AM, ED, LS, GH and DB scripted considerable part of original draft. DF, AM, ED, CK, LS, and DB had performed extensive literature search and scripted relevant parts of the manuscript. DF, ED, VT, AA, VS, CT, and GH collected clinical and biological material and clinical data. DF, AM and CL analyzed the data. DF and DB prepared the artwork. DB, DF, and AM had edited the final version of the manuscript. DB and ED provided overall supervision. All authors performed extensive literature search and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.This research was funded in part by the Special Account for Research Grants University of Thessaly, grant number 6357, 5158 and 5847.Author ED: Allergan, Novartis, Genesis, ELPEN, Bayer, Teva, Merck- Serono, Genzyme-Sanofi, Roche, UCB - speaker or chairman horonaria, advisory or travel grants, and clinical research-educational support grants. Author DB: AbbVie, Novartis, Genesis, ELPEN, Pfizer, Aenorasis, Menarini, Kopper, ITF Hellas, Roche, MSD, GSK, Hospital Line - speaker or chairman horonaria or paid investigator or clinical research and educational support grants.The remaining 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.


Candida albicans. Recombinant IL-26 was bacterially expressed and studied for its microbicidal effects in culture. We show that IL-26 has strong 90% bactericidal activities against Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. Similarly, IL-26 sensitivity was also detectable in vancomycin-resistant Enterococcus species, methicillin-resistant S. aureus, and carbapenem-resistant A. baumannii clinical isolates. Additionally, a significant, albeit weak fungicidal effect against Candida albicans was observed. Activities against Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were not detectable. The proinflammatory cytokine and kinocidin IL-26 shows strong bactericidal activities against A. baumannii and, almost selectively, against Gram-positive bacteria.The cationic proinflammatory cytokine Interleukin 26 (IL-26) shows antibacterial activity and inhibits the replication of cytomegalovirus and hepatitis C virus. This study evaluates the early microbicidal activities of IL-26 against major bacterial species including multi-resistant variants and  The highly cationic proinflammatory cytokine Interleukin 26 (IL-26) is a member of the IL-10 family of cytokines. IL-26 is produced by different cell types, such as activated T cells or inflammatory fibroblasts, and stimulates the production of other proinflammatory cytokines in various cell types . IL-26 uEscherichia coli strain XL1blue, which is capable of producing IL-26 in inclusion bodies, by adding 1mM isopropyl β-D-1-thiogalactopyranoside and cultivation for 6h . Particle-bound IL-26 was eluted under increasing imidazole concentrations . Eluate fractions were tested for 19kDa bands in Coomassie-stained protein gels. Renaturation of relevant eluate fractions was achieved by dialysis for 24h twice, at 4°C .Induction of IL-26 protein synthesis was achieved in n for 6h . First, The physiological STAT3 phosphorylation activity of the newly produced IL-26 was tested with the human colon carcinoma cell line COLO-205. IL-26 (5μg/ml) was added for 45min at 37°C. Cell lysates were separated on protein gels and Western blots were probed with anti-phospho-STAT3 and anti-STAT3 antibodies  and horseradish peroxidase-conjugated anti-rabbit IgG antibodies  as secondary reagent. Signals were detected by chemiluminescence  using a C. albicans were initially grown on Columbia sheep blood agar at 37°C overnight and then for 20h at 37°C in 10ml tryptic soy broth (TSB) medium with 10mM NaCl. Then, 50μl of the culture was added to 10ml of TSB/10mM NaCl medium and incubated for 3h at 37°C for reaching the logarithmic phase of growth and the optical density at 600nm was determined. We diluted the culture with 10mM NaCl to reach target concentrations of 105CFU/ml. Next, 100μl of the microbial suspension was added to 100μl of different concentrated IL-26 solutions  to achieve final IL-26 concentrations in the wells of 50, 15, 5, 1.5, and 0.5μg/ml. We always ran negative controls with pure dialysis buffer instead of the IL-26 solution, as well as positive controls with bacteria that were previously shown to be highly sensitive to IL-26, when species with low or lacking IL-26 sensitivity were examined.Bacteria and Immediately after the start of the treatment, and after 1, 2, 3, and 4h, 20μl from each culture was sampled and diluted in a 0.85% NaCl solution and 100μl of that dilution was pipetted on two lysogeny broth (LB) agar plates each. The plates were incubated overnight at 37°C and the colonies were counted. The numbers of colony forming units per milliliter (CFU/ml) were calculated after averaging of the counting results of the two separate plates in consideration of the dilution factor. Totally, 15,000 LB agar plates were plated by hand for 66 independent experiments.p were determined by using the independent two-sample t-test. We used linear regression for calculating the minimal concentration for 90% bactericidal or fungicidal effects (MBC90/MFC90), respectively, the lethal dose or concentration for 90% killing (LD90/LC90). All results of this study are given as MBC90/MFC90, even though the use of LD90/LC90 is also common in AMP research. The use of the terms MBC/MFC may be more precise from a microbiological point of view. Results with values of p ≤0.05 were rated significant. As far as not given in numbers, significances are labeled as followed: *p≤0.05; **p≤0.01; ***p≤0.001; and ****p≤0.0001, ns=not significant.Statistical evaluation was performed with GraphPad PRISM 8 . Values of C. albicans. All experiments were performed with a standardized protocol and were highly reproducible. The anti-infective activities were analyzed quantitatively for the minimal concentration for 90% bactericidal or fungicidal effects (MBC90/MFC90).IL-26 was tested for bactericidal activities against different Gram-positive and Gram-negative strains of different antibiotic resistance phenotypes, as well as for fungicidal activities against Enterococcus faecalis ATCC 29212 cultures, an immediate and highly significant (p≤0.0001) reduction of the number of colony forming units (CFU) of approximately 98.4% was detected. After 1h, significant effects were found from 5μg/ml on. Complete eradication of all bacteria was reached at 50μg/ml IL-26, after incubation for at least 1h. The CFU numbers after 2, 3, and 4h of incubation were reduced by 97.3, 92.3, and 96.1% at 15μg ml IL-26  and 50μg/ml (99.6%). The reduction levels stayed constant over time. After 4h, the incubation with 50μg/ml IL-26 resulted in a highly significant decline of CFU numbers by 99.95% . Again, a significant immediate decline by 99.1% was achieved at 50μg/ml IL-26. After 1h, complete eradication of all bacteria was reached at 50μg/ml, whereas 99.89% were killed at 15μg/ml. These effects were stable (± 0.2% at 15μg/ml) for 2, 3, and 4h incubation time. After 3h, an additional significant but weaker effect was found at 5μg/ml, which lost its significance after 4h , which increased after 1h to a highly significant reduction by 94.6% (p<0.0001). Further increments were achieved upon ongoing incubation. CFU reductions at 50μg/ml reached 95.6, 98.0, and 93.4% after 2, 3, and 4h. After 2 and 3h, significant but weak effects were detectable at 15μg/ml  and complete killing at 50μg/ml was still achieved. When incubating for 3h, reductions were found at 0.5μg/ml (33.5%), 1.5μg/ml (36.5%), 5μg/ml (83.9%), 15μg/ml (99.8%), and 50μg/ml (100%), of which all were significant, besides at 1.5μg/ml. Last, after 4h, declines by 63.6% at 5μg/ml, 98.6% at 15μg/ml, and complete eradication at 50μg/ml were observed (In contrast to the antibiotic-sensitive observed .90 for immediate effects ranged from 38.5μg/ml (MRSA ATCC 33593) to 57.5μg/ml , except for the methicillin-sensitive S. aureus ATCC 6538 with an unusually high MBC90 of 136μg/ml. Two groups of similar MBC90 ranges were defined. In the first group, E. faecalis ATCC 29212, E. faecium ATCC 6057, vancomycin-resistant E. faecium ATCC 17050, and MRSA ATCC 33593 had average MBC90 values between 12.5μg/ml and 13.4μg/ml IL-26 for 1 to 3h and 19.3μg/ml after 4h. In the other group, the average MBC90 for 1 to 4h for vancomycin-resistant E. faecalis and methicillin-sensitive S. aureus ATCC 6538 ranged between 43.7μg/ml and 45.5μg/ml . Additionally, we found a 50% non-significant reduction at 15μg/ml  at IL-26 concentrations of 50μg/ml , 99.8% (3h), and 99.6%  were 374.6μg/ml for 0h, 46.91μg/ml for 1h, 48.09μg/ml for 2h, 46.8μg/ml for 3h, and 47.22μg/ml for 4h , 67.5% after 2h, 58.2% after 3h, and 78.5% after 4h . The MFC90 values ranged from 54.7 to 81.41μg/ml . Thus, the questions arise why all other Gram-negative species are non-sensitive for IL-26 and what the essential factor is for the sensitivity of A. baumannii. The lipopolysaccharides (LPS) and especially the O-antigen, which is the outer chain of the LPS, are two known factors for AMP resistance in Gram-negative bacteria  – project number 413490537 within the Kiel Clinician Scientist Programme in Evolutionary Medicine. We acknowledge financial support by the state Schleswig-Holstein within the funding programme Open Access Publikationsfonds.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.


Nanoparticles have been used for biomedical applications, including drug delivery, diagnosis, and imaging based on their unique properties derived from small size and large surface-to-volume ratio. However, concerns regarding unexpected toxicity due to the localization of nanoparticles in the cells are growing. Herein, we quantified the number of cell-internalized nanoparticles and monitored their cellular localization, which are critical factors for biomedical applications of nanoparticles.2(RITC)] in various live single cells, such as HEK293, NIH3T3, and RAW 264.7 cells, using site-specific direct stochastic optical reconstruction microscopy (dSTORM). The time-dependent subdiffraction-limit spatial resolution of the dSTORM method allowed intracellular site-specific quantification and tracking of MNPs@SiO2(RITC).This study investigates the intracellular trafficking of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC) were observed to be highly internalized in RAW 264.7 cells, compared to the HEK293 and NIH3T3 cells undergoing single-particle analysis. In addition, MNPs@SiO2(RITC) were internalized within the nuclei of RAW 264.7 and HEK293 cells but were not detected in the nuclei of NIH3T3 cells. Moreover, because of the treatment of the MNPs@SiO2(RITC), more micronuclei were detected in RAW 264.7 cells than in other cells.The MNPs@SiO2(RITC) at specific sites in three different cells using a combination of dSTORM, transcriptomics, and molecular biology were performed. These findings highlight the quantitative differences in the uptake efficiency of MNPs@SiO2(RITC) and ultra-sensitivity, varying according to the cell types as ascertained by subdiffraction-limit super-resolution microscopy.The sensitive and quantitative evaluations of MNPs@SiOThe online version contains supplementary material available at 10.1186/s12951-021-01147-1. Rhodami2, and 0.8 mg/mL collagenase type IV , pH 7.4] and extracted from the mouse body. The extracted liver was homogenized, and primary hepatocytes were isolated by centrifugation at 50×g for 5 min and layered using a 40% Percoll cushion by centrifugation at 300×g for 7 min. The isolated hepatocytes were incubated on plates and cover glasses coated with collagen type I  in growth medium [Medium 199  with 10% FBS and 1% antibiotic–antimycotic agent]. Liver non-parenchymal cells fraction, containing Kupffer cells, liver sinusoidal endothelial cells, and stellate cells, was collected from the homogenized liver by layering with 25% and 50% Percoll gradient using centrifugation at 1800×g for 30 min. The layer, containing Kupffer cells and liver sinusoidal endothelial cells, was separated into Kupffer cells and liver sinusoidal endothelial cells by selective adhesion  for Kupffer cells, platelet endothelial cell adhesion molecule  for liver sinusoidal endothelial cells , as previously described –37. ExpeA lab-made dove prism-type TIRF setup, resembling the apparatus previously used in our group, but with some modifications, was employed  software. For superlocalization, the centroid of the fluorescence spot was fitted to the two-dimensional (2D) Gaussian function  assay was performed as described in a previous study , 42, 43,2(RITC)-treated cells  were examined using a Human U133 Plus 2.0 50 K Affymetrix system , which contains 54,675 probes. Biological pathways were enriched using the web-based bioinformatics software Ingenuity Pathway Analysis . A threefold change in gene expression was used as a cut-off to generate data sets of significantly changed genes.Gene expression profiles were analyzed as described in a previous study –47. In t−ddCt method.The expressions of DNA repair-related genes were determined by using a real-time PCR kit, based on RealMOD™ SYBR Green , that employed Rotor Gene-Q  with gene-specific primer pairs  for 12 h. The cells were then stained with 10 µg/mL of Hoechst33342  in PBS for 10 min at room temperature and washed three times with PBS. Fluorescent images were acquired by Axio Vert 200 M fluorescence microscopy  at the Three-Dimensional Immune System Imaging Core Facility of Ajou University. The excitation wavelengths for Hoechst33342 and MNPs@SiO2(RITC) were 405 and 530 nm, respectively. Micronuclei were counted in five to seven randomly chosen areas on each well. More than 200 cells per experimental group were counted using ImageJ. The statistical analysis was done by assessing the results by one-way analysis of variance (ANOVA) with Bonferroni’s multiple-comparison tests using IBM-SPSS software . In all analyses, p < 0.05 was taken to indicate statistical significance.Cells were cultured in a 24-well plate  and treated with MNPs@SiOAn inductively coupled plasma/quadrupole mass spectrometer  was used for the quantitative estimation of Co. The analysis was done with a 1600 W RF power supply, 18.0 L/min coolant gas-flow rate, 1.30 L/min auxiliary gas-flow rate, and 0.98 L/min nebulizer gas-flow rate. For quantitative estimation of Fe, an atomic absorption spectrometer  was used along with a flame atomizer system and an air-acetylene fuel with a 1.0 L/min gas flow at a 248.3 nm wavelength.6 cells were washed with ice-cold PBS and lysed with Nuclei EZ lysis buffer. The cells were harvested with a scraper and incubated for 5 min on ice. The lysed fractions were pelleted by centrifugation at 500 g for 5 min at 4 °C. The pellets were washed with Nuclei EZ lysis buffer and vortexed. Final nuclei fractions were collected by centrifugation at 500g for 5 min at 4 °C and resuspended with a Nuclei EZ storage buffer.The nucleus fraction was prepared with Nuclei EZ™ prep kit . Isolation procedures were performed according to the manufacturer’s instructions. In this step, 3 × 10@SiO2(RITC). The 50 nm MNPs@SiO2(RITC) was placed on PLL and laminin-coated cover glasses and irradiated with a 532 nm laser in the presence of a 100 mM MEA buffer. A linear dependence of first-order rate constants was observed in relation to the excitation wavelength intensities. The rate for the off state  with dSTORM imaging. Because NPs are not reagents to which specific antibodies are bound or stained for specific organelles in cells, the optimization of the concentration of NPs was determined through intuitive characterization rather than localization density [@SiO2(RITC) was considered optimum for performing a dSTORM quantitative analysis. The HEK293 cells were incubated with 0.01 µg/mL of MNPs@SiO2(RITC), and the sample was irradiated simultaneously with 532 nm and 405 nm lasers. The movie M1 shows the photo-switching  in the HEK293 cells. The photo-switching cycles were repeated over 10,000 frames, and the resultant frames were reconstructed into a single image. The cell morphology was maintained as per normal cell morphology in culture conditions until imaging and data acquisition through dSTORM analysis.HEK293, NIH3T3, and RAW 264.7 cells were incubated with three different concentrations at different incubation times  in serum-free growth media. The growth media avoided cellular damage at longer incubation times. The experiment was performed with all three types of cells under the same incubation conditions at 37 °C for energy-dependent uptake. After incubation for 1 h, all three cell samples were observed to have internalized NPs  concentration of 0.1 to 1.0 mg/mL  treatment in a dose-dependent manner. In contrast, we did not find any toxicity in cells at concentrations of 0.1 to 1.0 µg/mL of MNPs@SiO2(RITC) -treated HEK293 cells were significantly changed in 69 DNA repair-related genes in a dose-dependent manner when using IPA , RE1-silencing transcription factor (REST), DAZ associated protein 2 (DAZAP2), homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1), and ubiquilin 4 (UBQLN4) were validated using quantitative PCR -treated HEK293 and RAW 264.7 cells rather than in NIH3T3 cells, and the frequency increased in a dose-dependent manner, showing a more pronounced increase in RAW 264.7 cells  may interact with chromosomes in nuclei and induce genotoxicity in the cells.Micronuclei provide one indicator of genotoxicity and are produced when double-strand breaks occur in a nucleus and replication is accompanied by a nuclear envelope burst . MicronudSTORM as the detecting tool arises from the possibilities it offers for performing nanostructure analysis of cells. In an application of the dSTORM technique, the NPs internalized within the nuclear regions of RAW 264.7 cells were observed , three cells were incubated with NPs for different time intervals  were internalized within the nuclei of both RAW 264.7 and HEK293 cells. Even though a large number of NPs seemed to be located in the cytoplasm and accumulated around the nuclei, forming nuclear indentations, a small fraction of NPs entered the nuclei , which was the same concentration used for dSTORM imaging. ICP-QMS could not detect the NPs as a whole within the cells because of sensitivity limitations. However, the results with quantification of NPs in the nuclear region showed that NPs were internalized more in the nuclei of RAW 264.7 cells than in those of NIH3T3 cells; this conclusion matches the results obtained from dSTORM analysis. Moreover, to observe this intranuclear internalization, we performed 3D DIC combined TIRF wide-field imaging for the RAW 264.7 cells  to obtain visualizations of NP interactions with cell nuclei  were observed within the nuclei and the tracks of the NPs were traced  in single cells with imaging-fluorescence-labeled NPs. Given diffraction limitations, the task of distinguishing individual NPs in the aggregated regions of cells proves to be difficult. Therefore, the labeled dye should be of sufficient quality to support super-resolution imaging for distinguishing individual MNPs@SiO2(RITC) for performing the accurate site-specific quantitative estimation of the internalized NPs [dSTORM enabled individual MNPs@SiO2(RITC) in the aggregated region to be distinguished and provided improved resolution over the results obtained from conventional wide-field TIRF images.Intracellular aggregation presents the major concern that arises when detecting MNPsized NPs . The res@SiO2(RITC), the uptake of these NPs largely depended on endocytosis rather than phagocytosis. The buffer medium was the other factor that could influence NP uptake [On incubation, the NPs were widely sensed and internalized by macrophage cells. The macrophage cells are well known for their phagocytic uptake of particles . They caP uptake , 56. The@SiO2(RITC) internalization did not find NPs internalized in the nuclear region of cells when z-sectional images confocal microscopy [@SiO2(RITC) internalization in nuclei was attributed to the nuclear pore size of the cells [Earlier studies of MNPscroscopy , 45, 57 he cells , 59. Howhe cells . Further@SiO2(RITC) internalized in the cell nucleus region in both RAW 264.7 and HEK293 cells. The kinetics of internalization varied by cell type. RAW 264.7 cells consumed more than both HEK293 and NIH3T3 cells. The NPs were internalized in RAW 264.7 cells only after 5 h of incubation; meanwhile, 12 h of incubation were required by HEK293 cells. NIH3T3 cells showed high resistance towards NPs. After 12 h of incubation, there was still no sign of a fluorescence signal in the cell nuclei of NIH3T3 cells. DNA repair-related gene disturbances were analyzed in HEK293 cells and micronuclei were detected in both HEK293 and RAW 264.7 cells, indicating genotoxicity. In addition, cell viability was significantly decreased in MNPs@SiO2(RITC)-treated RAW 264.7 cells in a dose-dependent manner. Moreover, MNPs@SiO2(RITC) showed high levels of uptake in RAW 264.7 cells, as compared to HEK293 and NIH3T3 cells. Because the increase in reactive oxygen species (ROS) generation by MNPs@SiO2(RITC) in cells is linked to cell viability [Herein, we observed 50 nm MNPsiability , these r2(RITC) and SiO2 NPs in three types of primary cells  from the liver, one of the major organs for NP accumulation [2(RITC) internalization was observed in hepatocytes, followed by Kupffer cells and liver sinusoidal endothelial cells, as indicated by the intensity of MNPs@SiO2(RITC)  of MNPs@SiO2(RITC) in 10, 50, and 200-nm SiO2 NPs treated cells  treated cells. Although the number of NPs that entered the cell nuclei of RAW 264.7 and HEK293 cells was much lower than the total number of NPs in the cells, that number was sufficient for generating genotoxicity in the cells. Thus, the presence of NPs in the cell nuclei may induce dysfunctions in the nuclei and genotoxicity through the aggregation of intranuclear proteins or inhibition of RNA transcription. In addition, previous studies suggested that these biological effects of MNPs@SiO2(RITC) were caused by the silica shell rather than the cobalt ferrite core [2-coated Fe3O4 NPs induced ROS after 48 h in the cells [Changes in DNA repair-related transcriptome and formation of micronuclei, which are responses and phenomena related to DNA damage , 66, werite core –47, 67, he cells .2(RITC) using the MTS assay, and a single particle tracking approach was performed by visualizing the interaction of MNPs@SiO2(RITC) particles with the cell membrane using dSTORM. Subdiffraction-limit spatial resolution resolved individual MNPs@SiO2(RITC) and resulted in the quantification of MNPs@SiO2(RITC) in specific regions of the cells. Quantitative analysis suggests that macrophage cells are more susceptible to MNPs@SiO2(RITC) than human kidney cells and fibroblast cells at the point of NP uptake efficiency. Moreover, the MNPs@SiO2(RITC) were observed to be internalized in the nuclear regions of the cells by breaking the biological barriers. Thus, the movement was monitored, and individual NPs were tracked with ultra-high spatial resolution at the subdiffraction limit. Further studies, evaluating the mechanism of subcellular localization of MNPs@SiO2(RITC) in NP-treated cells, will help improve predictions of nanotoxicity and aid in the development of NPs as diagnostic and therapeutic agents.The major drawbacks of using engineered NPs as drug and gene carriers are the unknown influences on in vivo toxicity, pharmacokinetics, and intracellular NP internalization. In this study, cell viability was evaluated by intracellular MNPs@SiOAdditional file 1: Table S1. Ingenuity Pathway Analysis-based profiles of DNA repair-related genes in HEK293 cells treated with MNPs@SiO2(RITC). Table S2. ICP-QMS and AAS data for Co and Fe quantification in 3 × 106 cells of HEK293, NIH3T3 and RAW 264.7 at 0.01 µg/mL MNPs@SiO2(RITC). Table S3. Quantitative real time PCR primer sequences for genes encoding DNA repair related genes. Fig. S1. Characterization of mouse primary liver cells. a Morphological analysis of isolated Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells. Scale bar = 20 μm. Cell specific marker protein expression analysis for b F4/80 using flow cytometry, c albumin, and CD31 using immunoblotting. β-actin was used as the internal control. Fig. S2. (a) Physical layout and (b) schematic representation of the lab-made dSTORM setup. Following acronyms were used; L, laser; M, mirror; DCM, dichroic mirror; MS, mechanical shutter; NP, Nomarski prism; EM-CCD, electron-multiplying cooled charge-coupled device. Fig. S3. Plot of drift correction on the x- and y-axes with respect to frame illustrated with a fiducial marker. Images of raw, before drift correction and after drift correction. Fig. S4. a. The photo-switching rate constant koff was obtained by irradiation of 532 nm laser and monitoring the decrease in the fluorescence intensity over time. b. The photo-switching rate constant kon was obtained by simultaneous irradiation of the sample with 405 nm and 532 nm (200 mW/cm2) and monitoring the increase in the fluorescence intensity with time. Both koff and kon shows a linear dependence with illuminated intensities. Error bars represent the standard deviations of five measurements. c. Photoinduced restoration of RITC to fluorescence state with 405 nm activation laser. Fig. S5. Wide-field and dSTORM images of HEK293, NIH3T3, and RAW 264.7 cells incubated for 1 h with three different concentrations a. 0.1 µg/mL, b. 0.01 µg/mL, and c. 0.003 µg/mL of 50-nm MNPs@SiO2(RITC). Fig. S6. Wide-field and dSTORM images of HEK293, NIH3T3, and RAW 264.7 cells incubated for 3 h with three different concentrations a. 0.1 µg/mL, b. 0.01 µg/mL, and c. 0.003 µg/mL of 50-nm MNPs@SiO2(RITC). Fig. S7. The wide-field TIRF images of Fig. Fig. S8. The wide-field TIRF images of the RAW 264.7 cell incubated with 0.01 µg/mL of 50-nm MNPs@SiO2(RITC) for 5 h with a. serum free DMEM and b. PBS buffer. Fig. S9. Evaluation of cytotoxicity in MNPs@SiO2(RITC)-treated cells. MTS assay of MNPs@SiO2(RITC)-treated HEK293, NIH3T3, and RAW 264.7 cells. Cells were treated with 0.1 µg/mL, 0.3 µg/mL and 1.0 µg/mL MNPs@SiO2(RITC) for 12 h, followed by cell viability assessment using MTS assay. Fig. S10. Sixty-nine DNA-repair-related genes were constructed algorithmically using Ingenuity Pathway Analysis (IPA). Red and green in the genetic network indicate up- and down-regulated genes, respectively, in HEK293 cells treated with 1.0 mg/mL MNPs@SiO2(RITC), when compared with non-treated controls for 12 h. The data set of differentially expressed genes obtained from microarray data with a greater than threefold change is shown. Fig. S11. Sixty-nine DNA repair related genes showing significantly altered expression by MNPs@SiO2(RITC) on microarray analysis. DNA repair-related genes were constructed algorithmically using IPA. Red and green in the genetic network indicate up- and down-regulated genes, respectively, in HEK293 cells treated with 0.1 mg/mL MNPs@SiO2(RITC), when compared with non-treated controls for 12 h. The data set of differentially expressed genes obtained from microarray data with > threefold change is shown. Thirty-six genes were significantly changed. Fig. S12. DIC, wide-field, and dSTORM images of HEK293, NIH3T3, and RAW 264.7 cell respectively. Images were acquired after incubation of 50-nm MNPs@SiO2(RITC) for 3 h with each cell. Merged images of DIC and dSTORM for site-specific analysis of the internalized NPs. Fig. S13. DIC, wide-field, and dSTORM images of HEK293, NIH3T3, and RAW 264.7 cell respectively. Images were acquired after incubation of 50-nm MNPs@SiO2(RITC) for 5 h with each cell. Merged images of DIC and dSTORM for site-specific analysis of the internalized NPs. Fig. S14. DIC, wide-field, and dSTORM images of HEK293, NIH3T3, and RAW 264.7 cell respectively. Images were acquired after incubation of 50-nm MNPs@SiO2(RITC) for 12 h with each cell. Merged images of DIC and dSTORM for site-specific analysis of the internalized NPs. Fig. S15. DIC and super-resolution images of Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells incubated for 5 h with 2 µg/mL concentration of 50-nm MNPs@SiO2(RITC). Fig. S16. DIC and super-resolution images of Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells incubated for 24 h with 2 µg/mL concentration of 50-nm MNPs@SiO2(RITC). Fig. S17. DIC and super-resolution images of Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells incubated for 5 h with 2 µg/mL concentration of 10-nm SiO2 NPs. Fig. S18. DIC and super-resolution images of Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells incubated for 5 h with 2 µg/mL concentration of 50-nm SiO2 NPs. Fig. S19. DIC and super-resolution images of Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells incubated for 5 h with 2 µg/mL concentration of 200-nm SiO2 NPs. Fig. S20. DIC and super-resolution images of Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells incubated for 24 h with 2 µg/mL concentration of 10-nm SiO2 NPs. Fig. S21. DIC and super-resolution images of Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells incubated for 24 h with 2 µg/mL concentration of 50-nm SiO2 NPs. Fig. S22. DIC and super-resolution images of Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells incubated for 24 h with 2 µg/mL concentration of 200-nm SiO2 NPs. Fig. S23. Evaluation of cytotoxicity in SiO2 NPs-treated cells. MTS assay of a. 10-nm, b. 50-nm, and c. 200-nm SiO2 NPs-treated Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells. Cells were treated with 0.1 mg/mL, 0.3 mg/mL and 1.0 mg/mL SiO2 NPs for 12 h, followed by cell viability assessment using MTS assay.Additional file 2: Movie S1. Photo-switching of 50-nm MNPs@SiO2(RITC) internalized in the HEK293 cell after 5 h of incubation.Additional file 3: Movie S2. 2D and 3D wide-field fluorescence and DIC imaging of 50-nm MNPs@SiO2(RITC) internalized in RAW 264.7 cell.Additional file 4: Movie S3. wide-field fluorescence (left) and DIC merged wide-field fluorescence image (right) of real-time dynamics corresponds to 2000 frames of 25000 frames with 50-nm MNPs@SiO2(RITC) internalized in the cell nucleus of RAW 264.7 cell.


NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the  NPC1 or NPC2 gene. The proteins NPC1 and NPC2 accomplish the exclusion of cholesterol from late endosomes and lysosomes and are essential for maintaining cellular cholesterol homeostasis ; c.140G>T [p.C47F]. Cell line GM05659 was used as the control. Cells were cultured in fibroblast medium containing DMEM high glucose, 10% FBS, and 1% penicillin/streptomycin.2 and subsequently further cultured on Matrigel  in mTESR1 medium . The medium was changed daily and cells were passaged weekly.Human iPSCs were generated as described recently . In brie2 on poly-L-ornithine /laminin -coated dishes in proliferation medium containing 40% DMEM, 60% DMEM/F-12, 1X B27, 0.5% penicillin/streptomycin, 20 ng/mL FGF2 , and 20 ng/mL EGF . For terminal neuronal differentiation, cells were plated at a density of 45,000 cells/cm2 in differentiation medium, containing 40% DMEM, 60% DMEM/F-12, 1X B27, and 0.5% penicillin/streptomycin, which was changed every 4 days over a period of 6 weeks.Neural differentiation of iPSCs was induced by density-dependent growing of iPSCs on Matrigel  to induce generation of neural rosettes. Once neural rosettes had been formed spontaneously, neural progenitor cells (NPCs) were isolated using magnetic beads against the surface marker PSA-NCAM . NPCs were seeded at an expansion density of 100,000 cells/cm2 × 6H2O 1, CaCl2 × H2O 2, and Mg-ATP 2. pH was adjusted to 7.2. When filled, patch pipettes had a resistance of 6–8 MΩ. Cell cultures were continuously superfused with an extracellular solution consisting of (mM): NaCl 125, KCl 2.5, CaCl2 × H2O 2, MgCl2 × 6H2O 1, and NaHCO3 26, NaH2PO4 × H2O 1.25, glucose × H2O 25.Patch clamp recordings were performed as recently described . In brieCells were kept in HBSS , supplemented with 5 mg/mL magnesium and 6.6 mg/mL calcium for 6 h to simulate nutrient starvation and induce autophagy. Bafilomycin A1 from Streptomyces griseus  was used at a final concentration of 50 nM for 6 h to block the clearance of autophagosomes.® X-100, 10% glycerol with cOmplete™, and Mini, EDTA-free Protease Inhibitor Cocktail . Lysates were centrifuged at 15,000× g for 25 min at 4 °C. Protein concentration of the supernatant was determined using the Pierce™ BCA Protein Assay Kit  according to the manufacturer´s instructions. Samples were boiled for 5 min at 95 °C in 5× Laemmli-buffer  and subsequently centrifuged at 22,000× g for 1 min at 4 °C. For electrophoresis Criterion™, Vertical Electrophoresis Cell with Criterion™ TGX Stain-Free™ Precast Gels (4–15%)  were used. Electrophoresis buffer contained 250 mM TRIS, 2 M glycine, and 0.1% SDS. For Western blot, the Trans-Blot® Turbo™ Transfer System with Trans-Bolt® Turbo™ Transfer Pack  was used. Afterwards, the membranes were washed in TRIS-buffered saline (TBS), containing 20 mM TRIS and 137 mM NaCl (pH 7.5) for 5 min and blocked with 5% BSA  or milk in TBS supplemented with 0.1% Tween® 20 (TBST) for 1 h. For protein detection of catalase, SOD1, SOD2, and LC3BII/I membranes were incubated with primary antibody solution (3% BSA in TBST) overnight at 4 °C and for 1 h at room temperature for detection of GAPDH and β-actin. For detection of NPC2 protein, membranes were incubated with primary antibody solution (3% milk in TBST) overnight at 4 °C. Subsequently, membranes were washed 3× with TBST and incubated for 1 h with DyLight™ secondary antibody. Precision Plus Protein Dual Xtra Standards  was used as a molecular weight marker. Finally, membranes were washed 3× with TBST and 1× with TBS and dried. The Odyssey Infrared Imaging System  was used to visualize and quantify the protein signals. Expression of β-actin was used to normalize the expression of catalase, SOD1, SOD2, and NPC2, and the expression of GAPDH was used to normalize the expression of LC3BI and II. Used antibodies are listed in Western blot was performed as recently described . In brieTo determine the subcellular cholesterol accumulation, NDCs were loaded with 60 µg/mL low density lipoprotein in differentiation medium  for 24 h. For visualization of unesterified cholesterol, cells were fixed with 4% paraformaldehyde for 15 min, washed 3 × 5 min in PBS at room temperature, and incubated with 0.1 mg/mL filipin for 45 min. After three washing steps with PBS, cells were mounted with Fluoromount-G . Filipin fluorescence intensities were quantitatively determined by taking 10 random pictures of three replicates using a Laser Scanning Microscope 780 . Analysis was performed using ImageJ  by deter® . Pictures were taken with a Biozero 8000 microscope system . Used primary and secondary antibodies are listed in Pluripotency of neural progenitor cells was proven by staining for Sox2, nestin, and Pax6. Specimens were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 1 h at room temperature in 1% BSA/PBS. Subsequently, cells were washed three times with PBS and mounted with Fluoromount-G . Differentiated cells were stained for the neuronal marker βIII-tubulin and the glia cell marker GFAP. Therefore, cells were permeabilized using 0.1% Triton X-100 for 5 min on ice. Cells were incubated with primary antibodies overnight at 4 °C in 1% BSA/PBS, followed by three washing steps with PBS. Secondary antibodies were added for 1 h at room temperature in 1% BSA/PBS. After washing with PBS, cells were stained with DAPI , washed three times, and mounted with Fluoromount-Ghttps://sourceforge.net/projects/fcsalyzer, accessed January 2021). PI-negative and DCF-positive cells were defined as ROS positive cells. An example of a FACS-analysis is shown in The DCF Cellular ROS Detection Assay Kit  was used according to the manufacturer’s manual to determine intracellular ROS level, as described recently . In brie® X-100, 10% glycerol, 0.05% SDS, supplemented with cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail ) for 30 min on ice. Subsequently, cells were frozen with liquid nitrogen and thawed 5 times at room temperature and subjected to 10 ultrasonic pulses . Afterwards, samples were centrifuged at 15,000× g for 25 min at 4 °C. The Pierce™ BCA Protein Assay Kit  was used to measure protein concentration of the supernatant. Samples were treated with or without xanthinoxidase enzyme solution, and WST-1 working solution was added. Cells were incubated for 20 min in the dark at 37 °C under agitation. Absorption was measured at 450 nm with a Spark® plate reader and SparkControl Magellan software . The SOD activity was calculated as follows: SOD activity (inhibition rate %) = [(Amax − Amin) − ]/(Amax − Amin) × 100. This value was normalized to the protein concentration of the sample.A SOD Determination Kit  was used accordingly to the manufacturer’s manual measure SOD activity, as described recently . In brieLevel of mRNAs were determined by quantitative real-time polymerase chain reaction (RT-qPCR), as recently described . In briep-values < 0.05 were considered statistically significant, with * = p < 0.05, * = p < 0.01, and *** = p < 0.001.Data are presented as mean ± SEM of at least three independent replicates. Statistical analysis was performed with GraphPad Prism 6.07 . The D’Agostino-Pearson normality test was used to test the data sets for normal distribution. Statistical significance was tested by using an unpaired t-test or ordinary one-way ANOVA test with Dunnett’s multiple comparisons test. NP-C2 patients cannot be distinguished from NP-C1 patients based on clinical presentation. Similarly, no differences were found at the cellular level with biochemical analyses. However, the extent to which this applies to nerve or liver cells is insufficiently investigated, as there are no human cell models for this. This gap is at least partially filled by the present publication. In NPC2-deficient neuronally-differentiated cells, we were able to demonstrate typical cellular pathophysiological features that have also been described for NPC1-deficient cells. However, we observed differences, in markers of oxidative stress and autophagy, between NPC2- and NPC1-deficient cells. It would be interesting to know whether there are further differences or whether the pathophysiological expression in other affected cells, such as liver cells, also differs. For this purpose, iPSC-based model systems, such as the one described here, are ideally suited and we are convinced that further work with such model systems will contribute to a better understanding not only of the pathology of NP-C2 and NP-C1, but also of other lysosomal storage diseases.