This study was conducted to investigate the effects of dietary probiotic blend and liquid feed program at different nutritional densities on growth performance, nutrient digestibility, fecal score of weaning piglets.A total of 120 weaning pigs with an initial body weight of 7.05±0.93 kg per pig (21 days of age) were randomly allocated into 1 of the following 8 dietary treatments (3 replicates per treatment with 5 pigs per replicate) in a 2×2×2 factorial arrangement .During d 5 to d 15, greater average daily gain (ADG) and average daily feed intake (ADFI) (p<0.05) were observed in probiotics treatments. During d 15 to d 25, gain:feed (G:F) ratio (p<0.05) were significantly improved in probiotics, wet feed and high nutrition diet. Moreover, two interactions i) between nutrition levels and feed types, and ii) between nutrition levels and probiotics were found in G:F ratio. Furthermore, there was a significant positive interaction on G:F among those 3 factors (p<0.05). Overall, increasing ADG, ADFI, and G:F ratio were detected in probiotics treatment significantly (p<0.05). Besides, an obvious reduction on fecal score was observed in probiotics treatment from d 0 to d 5 (p<0.05). There was an interactive effect on fecal score between feed types and nutrition concentrations from d 5 to d 25 (p<0.05).These results indicated that probiotics supplementation could benefit growth performance and reduce the frequency of watery feces. Besides, wet feed program (feed:water = 1:1.25) could improve the G:F. The effect of liquid feed or probiotic could be influenced by dietary nutrition density in weaned piglets. An increased value of G:F was obtained when wet feeding a high nutrition diet  was supplemented with probiotics for 15 to 25 days. Gastrointestinal disturbances immediately post weaning can cause heavy economic loss in the pig industry. The weaning transition is a complex period during which the piglets have to cope with abrupt separation from their mothers and adapt to new environment where they are mixed with other litters. In addition, their diet will be switched from highly-digestible (liquid) milk to a less digestible and more-complex solid feed during this transition. Weaned at an early age 21 to 35 d) in intensive production systems has probably exacerbated the level of general stress in these immature animals /g, Enterococcus faecium 2×108 CFU/g). The 8 experimental diets were: TRT1, high nutrition × dry type without probiotics; TRT2, high nutrition × dry type with probiotics; TRT3, high nutrition × wet type without probiotics; TRT4, high nutrition × wet type with probiotics; TRT5, low nutrition × dry type without probiotics; TRT6, low nutrition × dry type with probiotics; TRT7, low nutrition × wet type without probiotics; TRT8, low nutrition × wet type with probiotics. This research was divided into three periods: Phase 1, d 0 to 5; Phase 2, d 5 to 15; Phase 3, d 15 to 25. All diets were formulated to contain approximately equal amounts of essential amino acids and the amino acid was on standard ileal digestibility basis. Ca, absorbable P, and Na, based on the analytical data from the feedstuffs. Different nutrition concentration diets meet or exceed the requirements suggested by the National Research Council  with an initial body weight (BW) of 7.05±0.93 kg per pig were randomly allocated into 1 of 8 dietary treatments (3 replicates per treatment with 5 pigs per replicate) in a 2×2×2 factorial arrangement with 2 levels of nutrition density apparent metabolic energy (AME) = 14.63 kJ/kg or 3,500 kcal/kg, CP = 20% vs AME = 14.23 kJ/kg or 3,400 kcal/kg, CP = 19.42%), 2 types of feed (dry vs wet), the liquid feed was prepared by mixing meal and water in a 1:2.5 ratio. The 2 levels of probiotics (0 mg/kg vs 300 mg/kg) were provided by Daehan feed mill company (2O3) was added to each diet at 0.2% as an indigestible marker to evaluate the nutrient digestibility on d 5, d 15, and d 25. Pigs were housed in an environmentally controlled nursery facility with slatted plastic flooring and a mechanical ventilation system. The temperature of the room was maintained at approximately 30°C for the first week of the experiment, after which it was reduced by 1°C per week over the next 4 weeks. Each pen (1.8 × 1.0 m) was equipped with a self-feeder and a nipple waterer to allow ad libitum access to feed and water throughout the experimental period.Chromic oxide  and G:F ratio on 0, d 5, d 15, and d 25. Fecal samples were collected from two pigs in each pen (1 gilt and 1 barrow) on the d 5, d 15, and d 25 of the experiment to determine the apparent total tract digestibility of dry matter (DM), N, and energy.Feed samples were dried at 70°C for 72 h, after which they were ground to pass through a 1-mm screen. The DM and N concentrations were determined according to the AOAC . RepreseThe fecal score was determined by the average value of five pigs of each pen by using a 5-grade score system . The staData were analyzed as a completely randomized block design, with a 2×2×2 factorial arrangement, using general linear model procedure . VariabiThe results of growth performance and nutrient digestibility of DM, energy and nitrogen are presented in The results of fecal score are appeared in B. licheniformis and B. subtilis) in the diets. Generally, probiotics or probiotics mixtures could improve ADG [Bacillus coagulans, B. licheniformis, B. subtilis, and Clostridium butyricum) levels in the diets linearly increased the ADG and ADFI for day 0 to 7 as well as ADG for day 8 to 21. However, Zhao et al [Lactobacillus reuteri and Lactobacillus plantarum) blend could not affect ADFI. Different probiotics strains and concentration of probiotics might be an important factor affecting the ADFI. Especially, since probiotics also live on nutrient, they might compete with host for nutrients in the diet or make hosts require more feed.The results of this study showed that dietary probiotics blend can increase the ADG, ADFI, and G:F ratio throughout the trial. And the results are accordance with the previous paper of our laboratory , which erove ADG –18 and irove ADG , 20 in prove ADG  documentao et al  believedThe key factor underlying the poor postweaning performance is the immediate reduction in FI due to the abrupt transition from liquid milk to less digestible feeds ,24, therEscherichia coli strains in the gut [Probiotics and nutrition density showed an interactive relationship in G:F ratio, which meant probiotics improved G:F ratio more dramatically in the high-nutrition diet. Similarly, our previous studies of Meng et al  and Yan  the gut  can mult the gut . However the gut  whose pa the gut  nutritioThese results indicated that probiotics in a supplementation diet could benefit growth performance  and reduce the frequency of watery feces. Besides, a wet feed program (feed:water = 1:1.25) could improve the G:F. Because there were two positive interactions: one between liquid program and nutrition density, the other between supplementation probiotics and nutrition density, the effect of liquid feed or probiotic could be influenced by dietary nutrition density in weaned piglets. An increasing value of G:F was obtained when wet feeding a high nutrition diet (100 kcal higher than NRC [


Salmonella enterica subsp. houtenae were sequenced using an Illumina MiSeq instrument. These isolates came from the urine and cerebrospinal fluid of a dog treated for hind-limb paresis with immunosuppressive drugs. S. enterica subsp. houtenae has also been implicated in brain infections in humans.The genomes of three clinical isolates of  Salmonella enterica subsp. houtenae were sequenced using an Illumina MiSeq instrument. These isolates came from the urine and cerebrospinal fluid of a dog treated for hind-limb paresis with immunosuppressive drugs. S. enterica subsp. houtenae has also been implicated in brain infections in humans.The genomes of three clinical isolates of  Salmonella enterica subsp. houtenae was originally isolated from a cockatiel in 1978 and has been recovered from a variety of animals, including mammals, birds, reptiles, and amphibians  mass spectrometry using the Biotyper  with flexControl v3.4 build 135.14 software. The National Veterinary Services Laboratory analyzed the organisms for serotyping and determined them to be S. enterica subsp. houtenae serotype 43:z4,z32:− (Patient specimens were spread onto a blood agar plate (BAP) with Trypticase soy agar and 5% sheep’s blood and incubated at 35°C ± 2°C in an atmosphere supplemented with 5% COz4,z32:− .Brucella broth supplemented with 10% glycerol and revived for sequencing by inoculating an aliquot of the frozen bacteria onto a BAP. They were grown in 5 ml lysogeny broth overnight at 37°C with aeration. DNA isolation from 1-ml aliquots of the overnight cultures was performed according to the manufacturer’s (Macherey-Nagel) protocol using Macherey-Nagel type B bead tubes and lysis buffer from the NucleoMag tissue DNA kit with a Qiagen TissueLyser machine. DNA quality was verified using a genomic DNA TapeStation run (Agilent).Isolates were stored at −80°C in Default parameters were used for the following steps, and manufacturer’s instructions were followed, unless otherwise stated. Illumina libraries were prepared using the Illumina Nextera DNA Flex library preparation kit and sequenced on an Illumina MiSeq v2 2 × 250-bp kit. All data were uploaded to BaseSpace v5.39, NativeApp.Core v0.9.0.2 (Illumina) for run monitoring, FASTQ file generation, demultiplexing, and adapter trimming. The sequencing output of paired-end read sets comprised 251 bp for all three isolates. Isolate 36GH had 1,616,328 reads, isolate 38CD had 1,411,316 reads, and isolate 76AB had 1,652,672 reads. This resulted in approximately 92× coverage, 80× coverage, and 94× coverage, respectively.N50 contig lengths were 220,987, 265,007, and 265,007, respectively, with an approximate GC content of 51% for all three isolates. The Benchmarking Universal Single-Copy Orthologs (BUSCO) score was used to analyze completeness, with a score of 100% for all three isolates based on the Enterobacteriales OrthoDB v9 data set  were identical in these three isolates, supporting the idea that all three isolates were clonal , 38CD (TAMU38CD), and 76AB (TAMU76AB) are available under the GenBank accession numbers JAAAGG000000000, JAAAGF000000000, and JAAAGE000000000, respectively. The raw reads are available under the SRA accession numbers SRR10876206, SRR10876207, and SRR10876208, respectively.The whole-genome shotgun sequences and raw reads for 36GH, 38CD, and 76AB can be found under the BioProject accession number


In vitro, Lut inhibited the upregulation of Dex-induced phospho-STAT1, cleaved caspase9, and cleaved caspase3. In addition, Lut inhibited Dex-induced expression of Bax and cytochrome c and increased the expression of B cell lymphoma-2(Bcl-2). In vivo, Lut decreased the proportion of empty lacunae in rats with GIONFH. Taken together, these findings indicate that Lut may have therapeutic potential in the treatment of GIONFH. Further, this effect might be achieved by suppressing mitochondrial apoptosis of osteoblasts via inhibition of STAT1 activity.Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a frequently occurring type of nontraumatic osteonecrosis. A failure of the timely treatment can eventually result in the collapse of the subchondral bone structure. Luteolin (Lut), a compound extracted from Rhizoma Drynariae, is reported to possess multiple pharmacological properties including anticancer, antioxidant, antiapoptosis, and antiinflammatory properties. However, whether Lut has a protective effect on the development of GIONFH remains unclear. In this study, we evaluated the effect of Lut on Dexamethasone (Dex)-induced STAT1/caspase3 pathway  Osteonecrosis of the femoral head (ONFH) is a progressive orthopedic disease, including traumatic and nontraumatic osteonecrosis . GlucocoCaspase3 is an aspartic acid-specific cysteine protease, the activation of which could participate in apoptotic cell death . Furtherin vivo. Therefore, in the present study, we evaluated the effect of Lut on Dex-induced apoptosis and its potential mechanism in vitro and the GIONFH model in vivo.Rhizoma Drynariae, a traditional Chinese herbal medicine, is used for treating bone diseases such as osteoporosis and osteoporotic fractures . FurtherLuteolin (purity > 98%), dimethylsulfoxide (DMSO), type I collagenase, and dexamethasone were acquired from Sigma Chemical Co . Antibodies against Bax, Bcl-2, STAT1, caspase3, caspase9, C-caspase9, cytochrome c, and GADPH were obtained from Proteintech . Goat anti-rabbit IgG-HRP was obtained from Bioworld  and primary antibodies against P-STAT1, and C-caspase3 were acquired from Cell Signaling Technology . Cell-Counting Kit-8 (CCK-8) was purchased from Dojindo . The Cell Apoptosis-DNA enzyme linked immunosorbent assay (ELISA) Plus kit was obtained from Roche . Bovine serum albumin (BSA), fetal bovine serum (FBS), and alpha-minimal essential medium (α-MEM) were purchased from Healthcare life Sciences .2 pieces, rinsed with phosphate buffer saline (PBS) three times, and thereafter digested in 0.25% trypsin–EDTA solution for 30 min. Following this, the tissues were incubated with 2 mg/ml (0.1%) collagenase I for 1 h in an incubator maintained at 5% CO2 at 37°C. The tissue digestate was centrifuged at 1000 rpm for 5 min, after which the supernatant was discarded and the osteoblasts were suspended in α-MEM supplemented with 1% penicillin/streptomycin and 10% FBS. The osteoblasts were passaged when they were 80% to 90% confluent, after dissociation with a 0.25% trypsin-EDTA solution. Only passage 0 to 2 cells were used to ensure no loss of phenotype in this study.Calvariae were obtained from newborn rats  following euthanization using pentobarbital. All skull tissues were harvested from the rats under aseptic conditions, separated from other the connective tissue, chopped into 1 × 1 mmvia the CCK-8 assay, in accordance with the manufacturer’s instructions. The optical density of the solution in each well was measured at 450 nm using a spectrophotometer from ThermoFisher.The effect of Lut on cell viability was evaluated The Cell Apoptosis-DNA ELISA Plus kit  was used to analyze the degree of apoptosis of the osteoblasts.LDH release assay was used to assess Dex-induced cytotoxicity. Osteoblasts were treated with Dex (1 μM) and subsequently incubated with different concentrations of Lut (0–10 μM) for 24 h in an incubator maintained at 37°C with 5% CO2. The cell culture medium was collected and LDH activity was measured in accordance with the manufacturer’s instructions .via Western blotting. Total cellular proteins were isolated using a radio immunoprecipitation assay (RIPA) lysis buffer [with 1% phenylmethane sulfonyl fluoride (PMSF)] and the protein concentration was subsequently quantified using the bicinchoninic acid assay (BCA) protein assay kit (Beyotime). Herein, 30-ng aliquots of total protein were separated via 10%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes . After blocking with 5% nonfat dried milk for 2 h at room temperature (RT), the membranes were promptly rinsed three times with tris-buffered saline with Tween (TBST). Thereafter, the membranes were probed overnight with primary antibodies  at 4°C, followed by rinsing with TBST three times (5 min each time) and incubation with an appropriate secondary antibody  for 2 h at RT. The bands were visualized using electrochemiluminescence plus reagent  and the intensity of the blots was then quantified using the Image Lab 3.0 software .The protein levels of STAT1, P-STAT1, caspase3, caspase9, C-caspase3, C-caspase9, cytochrome c, Bax, GADPH, and Bcl-2 were detected ®594 or Fluor®488-conjugated secondary antibody (1:400) for 1 h. Following this, the cells were stained by DAPI (Invitrogen) for 1 min. Finally, the cells were imaged via fluorescence microscopy . In addition, the fluorescence intensity was evaluated by researchers blinded to the experimental groups using Image J software 2.1 .Osteoblasts were cultured on glass coverslips in a six-well plate and then incubated overnight in a conditioned medium. The cells treated with or without Lut (10 μM) for 2 h and then coincubated in the absence or presence of Dex (1 μM) for 24 h. Subsequently, the osteoblasts were washed three times with PBS and then permeabilized with the 0.5% Triton X-100  for 10 min, following which they were fixed with 4% paraformaldehyde for 15 min. The cells were then blocked with 5% goat serum for 1 h at 37°C, rinsed three times with PBS, stained with antibodies against P-STAT1 (1:200) and C- caspase3 (1:200) at 4°C overnight, and then stained with Alexa Fluor2O2, permeabilized with 0.1% Triton X-100 for 10 min, and thereafter stained using in situ cell death detection kit , followed by DAPI staining for 1 min. The apoptotic cells were imaged via fluorescence microscopy (Olympus).Apoptotic osteoblasts were detected using the TUNEL Assay Kit, in accordance with the manufacturer’s instructions. The cultured osteoblasts were collected on glass coverslips in a 6-well plate. The osteoblasts were fixed with 4% paraformaldehyde for 20 min, rinsed with PBS three times, incubated with 3% HTwelve-week-old Sprague–Dawley rats were acquired from the Animal Center of the Chinese Academy of Sciences, Shanghai, China, authorized by the Animal Care and Use Committee of Wenzhou Medical University. In all, 30 rats were divided into three groups on average: control, model, and Lut groups. The model and Lut group rats was administered intramuscular injections of 10 mg/kg Dex twice a week for 8 weeks. Meanwhile, the Lut group rats were followed by an intragastric administration of 10 mg/kg Lut twice a week for 8 weeks. The control group was injected with normal saline (NS). The femoral heads of rats were collected after 8 weeks, and their histology was studies by hematoxylin-eosin staining (HE staining), immunohistochemical analysis, and image analysis of micro-computed tomography (CT) images.The specimens were scanned on a miniature CT system . The diagnostic criteria for ONFH were as follows: flattened shape of the femoral head, cyst degeneration, fracture of trabecula, and sclerosis band . ProximaThe femoral head samples were fixed in 4% paraformaldehyde  at 4°C for 24 h. Following this, the samples were transferred 10% EDTA solution (Solarbio Science & Technology) for decalcification for 4 weeks. Finally, the samples were dehydrated and embedded in paraffin blocks. The blocks were then sliced into 5-μm sections. Femoral head samples underwent HE staining and were observed under a microscope  to identify empty lacunae, pyknotic nuclei of osteocytes, and broken bone trabeculae. The proportion of empty lacunae was assessed by established criteria as described earlier .Paraffin-embedded 5-μm tissue sections were dewaxed with xylene, hydrated with an ethanol concentration gradient, treated with 3% hydrogen peroxide, and then incubated with 0.4% pepsin at 37°C for 30 min. The slices were blocked with 10% goat serum at RT for 30 min; following this, they were incubated overnight with C-caspase3 primary antibody (1:200) at 4°C and then with HRP-conjugated secondary antibody and diaminobenzene (DAB) (Solarbio Science & Technology). The slices were stored at 4°C.The data are expressed as means ± standard deviation (SD) of at least three independent trials. Statistical Product and Service Solutions (SPSS)  were performed for all statistical analyses. The data were analyzed followed by Tukey’s test to compare control and treatment groups. For statistical comparison of more than two groups, data were evaluated using one-way ANOVA. P values < 0.05 were considered significant.The chemical structure of Lut is shown in To further evaluate the effects of Lut on STAT1/caspase3 pathway in osteoblasts, the expression levels of STAT1, P-STAT1, caspase3, C-caspase3, caspase9, and C-caspase9 were measured by Western blotting. Following stimulation with Dex for 24 h, P-STAT1, C-caspase3, and C-caspase9 levels increased considerably. However, pretreatment with Lut for 2 h, inhibited the upregulation of Dex-induced P-STAT1, C-caspase3, and C-caspase9  before stimulation with Dex (1 μM for 24 h).Immunofluorescence analysis showed that in the control group, there was almost no expression of P-STAT1 in the nucleus and C-caspase3 in the cytoplasm, but this significantly expressed induced by Dex. Further, treatment with Lut inhibited Dex-induced C-caspase3 and P-STAT1 expression  before stimulation with Dex (1 μM for 24 h). We performed Western blotting to detect the protein expression levels of Bax, cytochrome c, and Bcl-2. As shown in via ELISA, the results of TUNEL assay showed that the apoptosis rate was significantly increased in the Dex group, which confirmed that the Dex-induced death of osteoblasts occurred in the form of apoptosis. And Lut pretreatment significantly reduced the apoptosis induced by Dex  Lut directly weakened the binding of Dex to GR (inactivated hormone or competitively binded GR to Dex); (2) The activity of GR was reduced by Lut; (3) Lut inhibited the transfer of activated GR to the nucleus; (4) Lut or its active decomposition acted on the binding region of GR and genes, and inhibited the expression of related genes. In addition to apoptosis of osteoblasts, recent study showed excessive adipogenesis and fatty deposition has been considered to be another important mechanism of GIONFH , Science and Technology Program of traditional Chinese Medicine, Zhejiang Province, China (2019ZB076).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.


Neutrophil granulocytes represent the first line of defense against invading pathogens. In addition to the production of Reactive Oxygen Species, degranulation, and phagocytosis, these specialized cells are able to extrude Neutrophil Extracellular Traps. Extensive work was done to elucidate the mechanism of this special form of cell death. However, the exact mechanisms are still not fully uncovered. Here we demonstrate that the small GTPase Cdc42 is a negative regulator of NET formation in primary human and murine neutrophils. We present a functional role for Cdc42 activity in NET formation that differs from the already described NETosis pathways. We show that Cdc42 deficiency induces NETs independent of the NADPH-oxidase but dependent on protein kinase C. Furthermore, we demonstrate that Cdc42 deficiency induces NETosis through activation of SK-channels and that mitochondria play a crucial role in this process. Our data therefore suggests a mechanistic role for Cdc42 activity in primary human neutrophils, and identify Cdc42 activity as a target to modulate the formation of Neutrophil Extracellular Traps. Since the discovery of a novel cell death mechanism in 1996 that was different from apoptosis or necrosis, considerable effort was made to further characterize this phenomenon . This noBecause of the afore-mentioned observations, the existence of two main pathways for NET formation is appreciated. The NOX-dependent NET formation has PMA as its model inducer and requires the formation of ROS. The NOX-independent pathway on the other hand is independent on NOX-generated ROS. However, the major regulators of this NETosis pathway are still largely unknown . AlthougSmall GTPases (sGTPases) of the Rho-subfamily are essential for neutrophil granulocyte migration to sites of infection by controlling cytoskeletal rearrangements , 15. SmaIn the present study, we aimed to elucidate the role of Cdc42 activity in the regulation of NET formation in neutrophil granulocytes.Primary human neutrophils were isolated from peripheral blood of healthy volunteers by Histopaque and Percoll gradient centrifugation as described elsewhere . The blofl/fl and Cdc42Δ/Δ mice. The use of conventional Cdc42 gene-targeted mice, carrying a whole-body knockout of Cdc42, is not possible, since these mice die at the embryonic day 7.5  gene -Cre mice to allow interferon (IFN)-inducible Cdc42 gene excision in hematopoietic cells , the total cellularity of bone marrow did not change but the floxed Cdc42 gene sequences and Cdc42 protein became undetectable in bone marrow cells , BM cells were mixed thoroughly and filtered through a cell strainer. This was followed by washing the cell suspension for 10 min, 863 × g at 4°C and subsequent re-suspension of the cell pellet in 3 mL 45% PercollTM. A density gradient consisting of five layers with differently concentrated PercollTM, ranging from 81 to 45% was prepared. The density gradient was prepared by gently layering the different PercollTM concentrations on top of each other, starting with 3 mL of the 81% dilution. Two milliliters of the 62%-, 55%-, and 50% dilutions were layered on top of the 81% dilution. The previously prepared whole BM cell suspension in 45% PercollTM was layered on top of the gradient, followed by centrifugation for 30 min, 733 × g, w/o brake at 10°C. After centrifugation, a typical cell distribution pattern within the density gradient could be observed. The upper ring contains remaining BM-myeloid precursor cells, while mostly granulocytes can be found in the lower separation layer. This ring was carefully collected in a falcon tube, and washed for 10 min, 863 × g, 4°C with 1 × HBSS . In order to remove most of the erythrocytes contaminating the granulocyte fraction, the cells were re-suspended in 3 mL BM-isolation medium and layered on top of 3 mL Histopaque 1119. After centrifugation for 20 min, 863 × g at RT w/o brake, granulocytes appeared at the interface of Histopaque 1119, and 1 × HBSS . The granulocytes were collected into a falcon tube, washed for 10 min, 863 × g and re-suspended in 2 mL BM-isolation medium. The cell number of BM granulocytes was determined using the Hemavet 950FS Hemocytometer .Murine neutrophils were isolated from bone marrow of Cdc42 day 7.5 . Therefo42) gene , 26. Homic cells , 25, thee to IFN . The Cree to IFN . This ise to IFN . At 5 daow cells , 26. Bonow cells . Briefly5 per well) were diluted in NET medium  and 4 mM L-glutamine  and seeded to a cellstar 96-well black-fluotrac plate . To detect the extracellular DNA of NETs, 5 μM SYTOX green was added, and neutrophils were stimulated with 20 nM PMA, 10 μM ionomycin  or the Cdc42 inhibitor casin was added (10 μM). To analyze the role of calcium in NET formation, freshly isolated human neutrophil granulocytes were re-suspended in 1 × HBSS supplemented with 10 mM HEPES, 4 mM L-glutamine (Biochrom) either with or without 1.26 mM CaCl2. The fluorescence of NET bound SYTOX green  was analyzed for a period of 4 h every 5 min at 37°C using an infinite 200 reader and Tecan i-control 1.7 Software (Tecan).Staining with the non-cell-permeable DNA intercalating dye SYTOX green  was used to study the kinetics of NET formation by PMN, incubated with or without the Cdc42 inhibitor casin (10 μM) . Freshly isolated neutrophils (2 × 106 per sample) or bone marrow derived murine (2 × 106 per sample) neutrophils were seeded into a 24-well plate in NET medium or 1 × HBSS, supplemented with 0.01% BSA, 2 mM CaCl2 and 2 mM MgCl2. NET formation by human neutrophils was induced by adding 20 nM PMA, 10 μM ionomycin or 10 μM casin. NET formation by bone marrow derived murine neutrophils was induced after priming of cells with 20 ng/ml TNFα , for 20 min, by adding 100 nM PMA, 5 μM ionomycin, or 10 μM casin. After 4 h incubation at 37°C, cells were collected and centrifuged for 10 min, 863 × g, RT. Supernatants were collected and subjected to an MPO-DNA ELISA  or an Histone H1, H2A, H2B, H3, H4-DNA-ELISA . Prior to the experiment, a Maxisorp 96-well plate was coated with the anti-MPO or anti-histone H2A, H2B, H3, H4 capture antibody, respectively, diluted in bicarbonate buffer, and incubated at 4°C, overnight. On the day of the experiment, wells were washed 3 times with 200 μl washing buffer followed by 30 min blocking with blocking buffer at RT. Thereafter, cell culture supernatants were added to the wells and incubated for 2 h at RT, while gently shaking. After incubation, wells were washed 3 times with 200 μl wash buffer, followed by incubation with a peroxidase-conjugated anti-DNA detection antibody for 2 h at RT. Thereafter, wells were washed 3 times with 200 μl wash buffer. Subsequently, 100 μl of ABTS substrate solution was applied to each well, and incubated for 20 min at RT while gently shaking (250 rpm). To stop the reaction, 100 μl of ABTS stop solution (part of the ELISA kit) was added per well, followed by measuring the optical absorbance at 405 nm (DNA detection) using a reference wavelength of 492 nm.Additionally, primary human (1.5 × 105) were centrifuged 10 min, 863 × g, and re-suspended in NET medium. Cells were seeded onto Poly-L-lysine-coated cover slips  and incubated with 20 nM PMA or 10 μM casin for 4 h at 37°C. Untreated samples were used as control.To visualize NETs, fluorescence microscopy was performed. Freshly isolated neutrophils  for 15 min at RT. Subsequently, the supernatant was removed and the air-dried coverslips were rehydrated with PBS and stained with 5 μM SYTOX green for 30 min in the dark at RT. Following washing three times with PBS, the samples were mounted with ProLong GOLD antifade reagent (Invitrogen) and analyzed using a BZ-9000-E Fluorescence Microscope .To study the influence of Cdc42 on the formation of NETs, the Cdc42 activity-specific inhibitor (casin) was used . Casin is a Pir1 analog, and confers the most active Cdc42 binding activity without binding to related RhoGTPases , 29, 30.2, w/o MgCl2 and allowed to settle for 45 min at 37°C, following priming with 20 ng/ml TNFα  for 20 min at 37°C. Afterwards, supernatants were removed and 5 μM of the mitochondrial ROS indicator MitoSOX (Thermo Fisher Scientific) was added followed by incubation for 30 min at 37°C. Supernatants were removed and cells were left untreated, or stimulated with 100 nM PMA, 5 μM ionomycin or 10 μM casin in 1 × HBSS supplemented with 0.01% BSA, 2 mM CaCl2, 2 mM MgCl2. The generation of mitochondrial ROS over time was detected using the Incucyte® Live-Cell Analysis System (Essen BioScience) for 1 h at 37°C every 4 min. The amount of mitochondrial ROS produced over time was analyzed using the IncuCyte Zoom elements 2000 software. To detect mitochondrial ROS formation in primary human neutrophils, 5 × 105 freshly isolated neutrophils per sample were pre-incubated with 5 μM MitoSOX for 30 min. at 37°C. Cells were left untreated or incubated with PMA (20 nM), ionomycin (10 μM) or the Cdc42 inhibitor casin (10 μM) for 1 h at 37°C. The fluorescence signal of MitoSOX was analyzed using flow cytometry.Sixty thousand freshly isolated bone marrow derived murine neutrophils were seeded into a 96-well glass bottom plate per well in 1 × HBSS, supplemented with 0.01% BSA, w/o CaCl5 freshly isolated primary human or bone marrow derived murine neutrophils were incubated with ionomycin , PMA , or the Cdc42 inhibitor casin (10 μM) for 1 h at 37°C. Following centrifugation for 10 min, 863 × g at RT, cells were re-suspended in 2% BSA/PBS and stained with CD11b-FITC (human)  or Ly6G-FITC (mouse)  in a 1:100 dilution for 20 min at RT. Following centrifugation for 10 min, 863 × g at 4°C, cells were re-suspended in PBS and incubated with 200 nM Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (Thermo Fisher Scientific) for 30 min at 37°C in the dark. TMRE is a positively charged, cell permeable dye that specifically binds to active, negatively charged mitochondria  were left untreated (control) or incubated with PMA (20 nM), ionomycin (10 μM) or with the Cdc42 inhibitor casin (10 μM) for 10 min at 37°C. Subsequently, cells were lysed with ice-cold 10% TCA  and incubated for 10 min on ice. Thereafter, lysates were centrifuged for 5 min, 14,000 × g at 4°C. After discarding the supernatant, the remaining pellet was washed with ice-cold acetone and centrifuged for 5 min, 14,000 × g at 4°C. The supernatant was discarded and 100 μl of boiling 1 × sample buffer , 20% glycerol , 4% SDS , 100 mM DTT , bromphenol blue ; ad 200 mL distilled water, pH 7.8, 1 × sample buffer: 100 μl PhosphoSTOP, 100 μl complete mini , 250 μl 4x sample buffer, 550 μl distilled water) was added, re-suspended and boiled for 7 min 100°C. This was followed by centrifugation for 4 min 14,000 × g at RT. The remaining supernatants of each sample were collected in fresh Eppendorf-tubes, and directly subjected to SDS-PAGE. Separated proteins were transferred from gels onto nitrocellulose membranes (15 min) using the BioRad turbo blotting device. Non-specific binding of antibodies was prevented by incubating the membranes for 1 h in blocking buffer (5% BSA in 100 mL T-TBS) at RT while shaking. To analyze citrullinated histone H3, the polyclonal rabbit anti-human histone H3 (citrulline R2+R8+R17)  was diluted in blocking buffer (5% BSA in 100 mL T-TBS) and the membrane was incubated over night at 4°C, while gently shaking. The day after, the membrane was washed three times with T-TBS for 10 min and subsequently incubated with an anti-rabbit IgG, HRP-linked secondary antibody  diluted in blocking buffer for 1 h, at RT while shaking. Afterwards, the membrane was washed three times with T-TBS for 10 min. Visualization of proteins was done by oxidation of a luminol-based substrate. Chemiluminesecence was detected using a chemiluminescence imager, and the software Fusion. Analysis of the protein bands was performed using ImageJ.Neutrophils  . We thusThe generation of ROS by the NADPH-oxidase is a hallmark of PMA induced NETosis , 33–35. 2+- free  as well as the presence of extracellular calcium play a special role in the NOX-independent formation of NETs . PMA is ntaining . NETs stntaining . In contSK-channels are the major calcium-activated potassium channels known to be present on neutrophils . NeutropIt has been described that NOX independent formation of NETs requires an increase in intracellular calcium concentration which can be induced by calcium ionophores . An incrHistone citrullination is a hallmark of NOX-independent NETosis but not NOX-dependent NETosis . IonophoAs shown in Since NETs induced by Cdc42 inhibition appear to have a specific dependency on mitochondrial ROS, we explored the role of mitochondria in more detail.Maintenance of a stable mitochondrial membrane potential (MMP) is a prerequisite of normal cell functions . The memSince MMP and mtROS production correlate with each other, we next investigated if mtROS is the reason for changes in the MMP, by using the mitochondria-targeting antioxidant mitoTEMPO. MMP changes in response to PMA, ionomycin and Cdc42 inhibition were not altered by mitoTEMPO, indicating that the changes of the MMP are not dependent on the production of mtROS . TogetheAs previously described, the MMP is generated by the respiratory chain complexes I, –III, and –IV . Complexfl/fl (considered as wild type) and Cdc42Δ/Δ (considered as Cdc42 knock out) mice. The formation of spontaneous NETs or in response to PMA and ionomycin was analyzed by measuring the amount of histone-bound DNA. Cdc42-deficient neutrophils show significantly higher spontaneous as well as PMA- and ionomycin-induced NET formation compared to cells from wild type mice  represent a major antimicrobial effector mechanism of neutrophil granulocytes due to their ability to trap and kill pathogens. It is known that the formation of NETs can be induced by many pathogens but also by sterile stimuli like cytokines, immune complexes, auto-antibodies and PMA or calcium ionophores . The prophoxin vitro, Cdc42 is unable to stimulate ROS formation by the NOX-complex . The patients/participants provided their written informed consent to participate in this study. This animal study was conducted with a protocol approved by the Animal Care Committee of Cincinnati Children's Hospital Medical Center.HT designed and performed experiments, analyzed data, and wrote the manuscript. SM assisted to perform experiments. M-DF and TL supported to design experiments, interpreted data, and revised the manuscript. All authors contributed to the article and approved the submitted version.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.


By downregulating the expression of glypican-1 in U-251 MG cells, we observed that the cellular growth and proliferation were highly reduced, in which cells were significantly shifted towards G0 as opposed to G1 phases. Cellular migration was severely affected, and glypican-1 majorly impacted the affinity towards laminin-binding of glioblastoma U-251 MG cells. This proteoglycan was highly prevalent in glioblastoma cells, being primarily localized in the cellular membrane and extracellular vesicles, occasionally with glypican-3. Glypican-1 could also be found in cell-cell junctions with syndecan-4 but was not identified in lipid rafts in this study. Glypican-1-silenced cells were much more susceptible to temozolomide than in U-251 MG itself. Therefore, we present evidence not only to support facts that glypican-1 is an elementary macromolecule in glioblastoma tumoral microenvironment but also to introduce this proteoglycan as a promising therapeutic target for this lethal tumor.Glioblastoma is one of the most common malignant brain tumors, with which patients have a mean survival of 24 months. Glypican-1 has been previously shown to be overexpressed in human glioblastoma and to be negatively correlated with patient’s survival. This study aimed to investigate how glypican-1 influences the tumoral profile of human glioblastoma using  The most frequent central nervous system (CNS) malignant tumors are the gliomas, which arise from glial precursor cells, and can be divided into oligodendrogliomas, oligoastrocytomas, ependymomas, astrocytomas, and some minor classifications . AccordiThe tumor environment possesses significant metabolic and structural differences from healthy tissue. Proteoglycans (PGs) are a class of macromolecules that show distinct patterns in cancers as opposed to physiological conditions . PGs areHeparan sulfate proteoglycans (HSPGs) are known to participate in various aspects of cell signaling. The syndecans (SDCs), with four isoforms, and glypicans (GPCs), with six isoforms, are membrane-bound and generally located in lipid rafts; they can interact with morphogens such as ligands from the Wnt, Hh, and FGF families in a manner to facilitate interaction with their receptors . These HGlypican-1 (GPC1) is, along with other GPCs, bound to the membrane through a glycosylphosphatidylinositol (GPI) anchor . It is uOur objective in this work is to elucidate further the role of GPC1 in this menacing tumor. To that end, we would deplete GBM cells of the molecule and investigate biological, biochemical, and pharmacological aspects of the modified cell lines to obtain further answers of how this HSPG could influence the tumorigenic process of GBM.GPC1 was highly expressed in the tested GBM cell lines, but there was no differential expression between them . By analyzing GPC1 core protein expression in T98G, U-251 MG, and U-373 MG cells, it was clear that all three cells have similar levels of the PG . We selected the U-251 MG cell line for the downstream experiments as these cells were much more proliferative and could reveal better discrepancies after knock-down of GPC1.Lentiviral transduction effects were analyzed by RT-qPCR, and the generated polyclonal cell lines were investigated for either GPC1 or GAPDH gene expression . The positive control (C+), GAPDH, was silenced in 98%, revealing high transduction efficiency. Only MOI 20 and 30 induced significant GPC1 silencing that differs from the negative control (C-). Nevertheless, the MOI 30 polyclonal cell line was selected for the monoclonal selection due to higher probability and number of integration events per cell.+ cells as well as a broader range of cells expressing lower levels of GPC1. Therefore, these three clones were chosen for subsequent assays.Monoclonal cell lines were generated, and GPC1 expression was assessed by RT-qPCR . The fiv-ΔCt) and then to U-251 expression levels , RT-qPCR analysis was performed to measure selected membrane-bound HSPGs’ expressions . Control cell lines were the original U-251 MG cells and the C- transduced polyclonal cell line, the negative control. Gene expression was first compared to β-actin . There is considerable variation in several HSPGs’ expression after silencing of GPC1; however, only SDC2 and -3 significantly had an inhibited expression after GPC1 knock-down, and SDC4 did reveal substantial reduction effects, but not in all clones. GPC6 was the only HSPG that was not influenced at all by the procedure, and C23’s SDC1 expression was enhanced.In an attempt to follow our group’s lead in establishing a role between GPCs and Wnt signaling, we also checked the expression of Wnt-3a, -5a and -7a ligands as well as β-catenin. Wnt-5a was the major expressed Wnt ligand , yet none of the ligands revealed any pattern associated with GPC1 expression change, although β-catenin, which is highly expressed, was significantly less present in C12 and C15.As GBM is frequently associated with extracellular matrix remodeling, we checked the expression of metalloproteinases (MMPs) 2 and 9. Although MMP2 was the major MMP expressed , a significant reduction was verified in MMP9. It is also possible to state that MMP2 did experience an expression alteration from GPC1 knock-down, although no statistically significant changes were noted between specific samples.After verifying an overall expression profile change mediated by GPC1, we proceeded to investigate how the proteoglycan would affect the tumor growth and its cells’ proliferation.By constructing a growth curve of GPC1-silenced cells and control cells for up to 96 h  and comp+ fraction (a 43.7–48.0% reduction) and Ki-67+ cell density (a 40.4–54.8% reduction), as seen in Supplementary Figure 4A and 4B. Regarding clone formation, GPC1 also affected the ability of single cells to generate clones of a minimum of 50 cells . With SDC4, there was a homogenous protein expression of the SDC despite GPC1 downregulation . SDC4 diThe confocal microscopy results indicate how overly expressed GPC1 is in GBM cells. It is located in the cell membrane, is rarely found in lipid rafts, and can be colocalized along with SDC4 and GPC3. SDC4 and GPC1 are primarily found in cell-cell junctions, and GPC3 may be found altogether with GPC1 in EVs.As previously reported, GPC1 may be essential to cellular migration . We thusNext, we investigated the adhesion aspects associated with GPC1. Three substrates that are found in the brain and are typically overexpressed in brain tumors were tested: laminin, type IV collagen, and vitronectin , 26. GBMAfter verifying that GPC1 influences several biological aspects of GBM, we proceeded to investigate whether this PG would have any consequence on the role of the primary antineoplastic agent used with GBM patients – temozolomide (TMZ).Control cells were not susceptible to TMZ . While UEven though HSPGs have been shown to play central roles in cancer, few studies have linked their effects to GBM . Our hypThe transcriptional profiles of selected HSPGs, β-catenin and MMP9 were affected due to GPC1 in GBM cells. The heparanome is a sophisticated machine in which a plethora of enzymes and their many isoforms not only synthesize HS but modify these GAG chains; this, in turn, creates an intricate puzzle containing negative and positive feedback loops that may be affected by altering the expression of a single component . This haCellular proliferation and, consequently, tumor growth are critical characteristics of GBM . Thus, oSubsequently, we investigated not only GPC1 localization in GBM cells, its regionalization in lipid rafts, and its association with SDC4 and GPC3; we also investigated whether the downregulation of GPC1 would influence its ability to interact with those molecules. SDC4 and GPC1 are believed to play together a fundamental role in myoblast proliferation, differentiation and migration during myogenesis by mediating the signaling of fibroblast growth factor 2 . GPC1 anWe then proceeded with our investigation on the GPC1 influence on GBM cellular migration and adhesion. Our data suggested a fundamental role of this PG in tumoral cells’ migration. When GPC1 was silenced, migration activity was highly reduced or almost abolished, as seen in the C23 clones. These results are of great interest as migration is one of the primary factors associated with the proliferation of GBM . Althougin vitro model. Additionally, adhesion kinetics was substantially affected, particularly in the C23 clone, impinging alterations on collagen IV binding intensity. Laminins are a predominant family of proteins in the ECM that is tightly involved in angiogenesis, invasiveness, stemness, and metastasis in cancer [4 chains of collagen is crucial for Schwann cell adhesion, yet no effects were obtained with collagen IV [GPC1 did also reveal effects on cellular adhesion. Our data indicate that GPC1 may mediate interaction with laminin in this n cancer . Collagen cancer . In glion cancer –66. Moren cancer –70. Nevelagen IV . Indeed,lagen IV . These olagen IV , 74. The6-methylguanine-DNA-methyltransferase (MGMT) or demethylation of this enzyme’s promoter [Lastly, we were compelled to correlate GPC1 with TMZ susceptibility in GBM cells. GBM cells revealed the need for high concentrations of TMZ to promote cell death when in elevated confluence. U-251 MG cells had an IC50 of (7.27 ± 0.50) mM, and C-’s IC50 could not be calculated as a result of limited response to TMZ. GPC1-silenced cells had IC50 from two to six times lower than the tested control cell lines. TMZ is a prodrug that spontaneously converts itself to MTIC, a methylating agent, that can transfer methyl groups, in particular, to the DNA, resulting in errors in the DNA mismatch repair (MMR) system. The accumulation of DNA nicks inhibits proliferation by blocking the G2/M cell cycle boundary , 76. Ourpromoter , 79. Thepromoter . Additiopromoter –82; and promoter . We cannThis study aimed to investigate whether GPC1 could modulate GBM tumoral behavior. Our findings concluded that this GPC is a pivotal macromolecule in this environment, inciting neoplastic growth, proliferation, migration, adhesion, and chemotherapy resistance. By downregulating the expression of GPC1, the tumoral profile was severely hindered, indicating that this GPC is a vital element in GBM tumorigenesis.® CRL-1690™) cells were donated for this work. U373-MG cells were cultivated in MEM supplemented with 10% of FBS and 100 µg/mL of penicillin and streptomycin (Gibco®). U-251 MG and T98G cell lines were kept in DMEM with the same supplementation mentioned above. The cells were grown at 37°C in a humidified atmosphere with 5% CO2.U373-MG  cells were purchased from Sigma-Aldrich  while U-251 MG (Sigma 09063001) and T98G .4 cells were added for each 500 µL of the lentivector solution. Lentivectors without an annealing sequence (negative control), cells untreated with lentivectors (double negative control), and shRNA for GAPDH (positive control) were also employed. After 72 h, the medium was changed to complete DMEM supplemented with 2 µg/mL of puromycin for the selection of infected cells. Polyclonal, puromycin resistant cell lines were obtained and characterized by RT-qPCR.U-251 MG cells had the GPC1 gene silenced using the SMARTvector Human Lentiviral GPC1 shRNA . All shRNA sequences used are shown in Supplementary Table 4. In brief, lentivectors for different MOI evaluations  were diluted in DMEM containing polybrene to a final concentration of 2 µg/mL . Cells were harvested, and 5 × 10g for 5 min, after which the supernatant was collected. The plated cells were detached and diluted to 5 cells/mL in the conditioned medium. The cells were plated in a 96-well plate with 100 µL in each well (0.5 cell per well). After one week, wells containing single colonies were further examined. After 50% confluence was observed, colonies were expanded, generating their own monoclonal cell lines. This experiment produced 20 viable monoclonal GPC1-silenced cell lines.For the generation of monoclonal cell lines, the most suitable polyclonal cell line was selected and grown in 100-mm dishes. After 80% confluence was present, the conditioned medium was collected and centrifuged at 1000×2, 500 µM of dNTP mix, 80 U of ImProm II Reverse Transcriptase , 20 U of RiboLock RNase Inhibitor and 500 nM of oligo (dT)15 primer (Promega) in ImProm II Reverse Transcriptase 1× buffer (Promega). The reaction was conducted at 25°C for 5 min for primer annealing, 42°C for 60 min for transcriptase activity and at 70°C for 10 min for inactivation.All tested cells, including controls and generated clones, were plated on 60-mm dishes. After reaching 80% confluence, the medium was removed, and 1 mL of Trizol (Invitrogen™) was added and incubated for 5 min. RNA was extracted using the Direct-zol RNA purification kit , and cDNA was obtained from 300 ng of RNA template, in a reverse transcription reaction of 20 µL, containing 3 mM of MgCl® Green Master Mix (Thermo Fisher Scientific) and 500 nM of specific forward/reverse primer pairs, as listed in Supplementary Table 5. qPCR reaction was conducted in 7500 Real-Time PCR System .Real-time PCR was performed in 10 µL reactions, in which 100 ng of cDNA were mixed with 5 µL of PowerUp™ SYBR4 cells were plated in 24-well plates coated with 13 mm borosilicate coverslips. After 48 h, the medium was drained, and wells were washed with PBS. Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with PBS and a 0.1 M glycine solution, cells were blocked for 1 h with 3% BSA in PBS and incubated with primary antibodies at 1:200 in 1% BSA overnight at 4 °C. Antibodies used in this experiment were anti-GPC1 , anti-GPC3 , anti-GFAP , anti-SDC4 , and anti-flotillin-1 . The wells were then washed with PBS and incubated with the secondary antibodies at 1:250 in 1% BSA for 1 h at room temperature, in which anti-goat 488 (A11055), anti-mouse 647 (A31571) and anti-rabbit 647  were used for labeling. Cells were stained with DAPI at 2.5 µg/mL in 0.05% saponin for 15 min and further washed with PBS. Subsequentially, coverslips were rinsed in water and mounted in Fluoromount™  in clean slides. Slides were imaged in Leica Confocal Microscope TCS SP8 CARS.U-251 MG, C-, C12, C15, and C23 cells were harvested, and 2 × 10570). Blank wells were subtracted, and data were normalized from wells in which MTT was absent.U-251 MG, C-, C12, C15, and C23 cells were harvested, and 3,000 cells were plated in 96 well plates. After 24, 48, 72 and 96 h, MTT at 5 mg/mL was added and incubated for 4 h. The medium was then removed, and the formazan precipitate was solubilized with DMSO. The plates were agitated, and wells had their absorbance read at 570 nm  at 1:200 in blocking buffer overnight at 4°C. The wells were then washed with PBST (0.1% TWEEN® 20) and incubated with the anti-mouse 647 at 1:250 in PBST for 1 h at room temperature. Cells were stained with DAPI at 2.5 µg/mL in PBST for 15 min and further washed with PBS. Subsequently, coverslips were rinsed in water and mounted in Fluoromount™ on clean slides. Slides were imaged using a Leica TCS SP8 CARS Confocal Microscope .For the cell-proliferation assay, U-251 MG, C-, C12, C15, and C23 cells were harvested, and 1 × 10ications . The med3 in PBS) and collected in 5-mL tubes. Cells were resuspended in 500 µL of staining buffer, and to each tube, 10 µL of a solution containing 50 µg/mL of propidium iodide  and 100 µg/mL of RNase A (Sigma-Aldrich) was added. The tubes were left on ice for at least 30 min before assessment in a BD Biosciences FACSCalibur cytometer .For cell cycle analysis, cell lines were plated in 100-mm dishes, and after 24 h were serum-deprived for 6 h. After reaching 80% confluence, cells were detached with 500 µM of EDTA and collected by centrifugation . Cells were then fixed in cold 70% ethanol while in agitation and left at –20°C for at least 2 h. Cells were washed twice in staining buffer  in 25% (v/v) methanol for 10 min in the dark, then destained with type I water. After drying overnight, colonies were assessed for formations with more than 50 cells present .4 cells were plated in 24 well plates for 48 h. A linear scratch was made with a pipette tip of 200 µL, and the wells were washed with complete medium once. The cells were then incubated in medium supplemented with 10 µg/mL of mitomycin, and after 0, 6, 12, and 24 h the gap was imaged, and its area was quantified using the ImageJ software.U-251 MG, C-, C12, C15, and C23 cells were harvested, and 4.5 × 104 cells of U-251 MG, C-, C12, C15 or C23 cells were added to the wells and incubated for 2 or 4 h. The cells were then washed twice with PBS, fixed with methanol, stained with crystal violet, and destained with type I water. After drying, the wells were imaged, and the dye was solubilized with 10% acetic acid for absorbance quantification at 570 nm.Laminin and collagen IV, both at 20 µg/mL, and vitronectin at 5 µg/mL (Sigma Aldrich) were added to 96 well plates and left for 1 h at room temperature. The content was then drained, and wells were blocked with 1% BSA for 30 min at 37°C. The plates were washed with sterile PBS, and 2 × 104 cells were plated in each well of a 96-well plate. After 24 h, the medium was replaced by variable concentrations (eight points ranging from 0–3 mM) temozolomide (Sigma Aldrich). After an additional 24 h, the MTT metabolic activity assay was performed as previously described, in which OD570 values were normalized to untreated cells’ absorbance.U-251 MG, C-, C12, C15, and C23 cells were harvested, and 2 × 10p < 0.05.Data were analyzed using GraphPad Prism 8.0, Microsoft Office Excel 2016, and Minitab 18. Two- and one-way analysis of variance (ANOVA) was used, combined with Dunnett’s test, for comparison to control samples; Bonferroni’s test was used for particular contextual analysis. For model assessment, experimental data was either linear or non-linear fitted according to the least-squares fitting method. Statistical significance was considered when


Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonus rhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree. Dendroctonus-bark beetles (Curculionidae: Scolytinae) play a fundamental role in the structure of coniferous forests, because they colonize and kill sick, damaged, and weakened trees, contributing to community succession, nutrient cycling, and canopy thinning [Larix, Picea, Pinus, and Pseudotsuga from North and Central America [thinning . However America ,3,4.Dendroctonus-bark beetles are phytophagous insects that spend most of their life cycle under the bark of host plants, where they reproduce, feed on phloem, and interact with microbial symbionts and invertebrates. These symbionts have been isolated from the exoskeleton, galleries, and gut of these bark beetles [Dendroctonus species is known [ beetles ,7,8,9,10is known . Some ofis known , cellulois known , recycliis known ,15,16.The phloem is a conduction system that transports different molecules and ions such as mRNAs, hormones, sugars, and nutrients such as trace elements and amino acids towards different plant body parts. As constitutive tissue is formed by different cell types , which represent a substrate rich in organic acids and non-structural carbohydrates , as well as in structural carbohydrates . In particular, starch is the main carbohydrate reserve of the plants, synthesized from sugars produced during photosynthesis both in autotrophic and heterotrophic tissues  .I. typographus the presence of two α-amylase genes one of them producing two isoforms as a result of alternative splicing.Starch is stored in plants as insoluble particles or granules and is composed of amylose and amylopectin . AmyloseDendroctonus rhizophagus Thomas & Bright, an endemic species to the Sierra Madre Occidental in Mexico, which colonizes and kills seedlings and saplings (<3 m height and ~10 cm diameter) of several pine species [D. rhizophagus is annual, univoltine, and atypical among Dendroctonus species. This species does not perform massive attacks on trees. Just one pair of insects colonizes and kills individual trees. In particular, we molecularly characterized the enzyme AmyDr, determined its number of isoforms, analyzed the relative expression of α-amylase AmyDr gene through different developmental stages, provided functional evidence that both native and recombinant α-amylase AmyDr of this bark beetle are capable of hydrolyzing starch, and determined the effect of metal and non-metal ions on recombinant α-amylase activity.In this study, we analyzed the α-amylases of  species ,27. The D. rhizophagus identified a single transcript that was annotated as an α-amylase. Therefore, isoforms of this protein were not present in D. rhizophagus. The α-amylase gene (the protein hereafter named AmyDr) had an open reading frame of 1452 bp that encodes 483 amino acid residues with a predicted N-terminal signal peptide of 16 amino acid residues. AmyDr has seven potential O-glycosylation sites and one single potential N-glycosylation site, a predicted molecular mass of around 53.15 kDa, theoretical pI of 4.54, and extracellular localization.A single α-amylase gene was identified, cloned, and sequenced. All the sequenced clones showed a pairwise nucleotide identity ~98%, nucleotide substitutions were mainly synonymous and a few no synonymous. The mapping of the cDNA sequences of this gene against the transcriptome of Dendroctonus species formed a consistent  and monophyletic group, different from other scolytines, as is the case of Ips and Hypothenemus genera. AmyDr had a mean amino acid identity >90% with α-amylases of other Dendroctonus species and an identity of around 80% with α-amylases of scolytines. Three chrysomelid sequences of species Callosobruchus chinensis and Zabrotes subfasciatus were clustered into the α-amylases group from Curculionidae, which share in the Cl-binding site the substitution of lysine by arginine with scolytines.Phylogenetic inference analysis by maximum likelihood  showed tTenebrio molitor (TMA). The structural model of AmyDr presented 97.64% of the amino acids with a score ≥0.2 in the 3D/1D profile and an average overall quality factor of 81.9172. The analysis of torsional angles Phi and Psi showed 91.9% (372) of residues in the most favoured regions, 7.4% (30) residues in additional allowed regions, 0.5% (two) residues in generously allowed regions, and 0.2% (one) residues in the disallowed regions . Also, this domain has binding sites to calcium ion  and binding sites to chlorine ion . The overall alignment of AmyDr and α-amylases of other coleopterans showed that the residues involved in the catalytic process and binding site to calcium are highly conserved. However, in the binding site to chlorine ion, AmyDr has one residue mutated (ARG to LYS) in position 319 , and the Tukey test indicated that differences were found among adult gut, pre-imago-larvae gut, and pupae gut (p < 0.05) . These differences were focused only between the adult, larvae, and pupae vs. pre-imago (p < 0.05).The l stages . Signifi < 0.05) . On the Sf9 cells culture, compared to non-infected cells, showed the overexpression of a polypeptide with a molecular weight of ~53 kDa (3)2 and KNO3 at 1 and 5 mM, maintained also with CaCl2 and NH4Cl at both concentrations, and slightly with NH4Cl at 5 mM , similar to the α-amylase from other scolytines, such as D. ponderosae , Ips typographus , and Hypothenemus hampei , curculionids, such as Antohonomus grandis  and Sitophilus oryzae , other coleopterans, such as Tenebrio molitor  and Tribolium castaneum , and other insects, such as Spodoptera frugiperda , Drosophila melanogaster , and Apis mellifera . The theoretical pI of AmyDr (4.54) is similar to the pI of coleopteran α-amylases (pI < 7) [Our findings showed that AmyDr is a functional enzyme encoded by an ORF of 1452 nucleotides and is present both in the gut and head-pronotum of D. rhizophagus, which agrees with the one found in other Dendroctonus species such as D. valens (MN782510), D. frontalis (GAFI01015829.1), and D. ponderosae (BT126692), and that reported in H. hampei and other curculionids such as Odoiporus longicollis (AHN92452.2) and Cosmopolites sordidus (AKN63428.1). However, this result is different from those found in A. grandis, I. typographus, and Rhynchophorus ferrugineus in which two Amy genes have been documented [Dendroctonus species studied so far, the genus Dendroctonus presented the lowest number of α-amylases genes.Only one AmyDr isoform is present in the gut and head-pronotum of lens MN7810, D. frus AKN634.1. Howevcumented , as wellD. rhizophagus, as well as from other Dendroctonus species and scolytines such as D. ponderosae (GAFW00000000), D. frontalis (GAFI00000000), D. valens [Tomicus yunnanensis (GFJU00000000), referenced to D. ponderosae genome, did not show evidence of isoforms. The absence of isoforms in Dendroctonus-bark beetles suggests that the selection has favoured the occurrence of amylases with major functional plasticity, stability, and efficiency, but not the occurrence gene duplications. In contrast, in the bark beetle I. typographus two functional α-amylases have been reported, one of them includes two isoforms [Ips confusus transcripts (GIWS00000000), after our analysis developed on this project.The sequence analysis of AmyDr clones showed mainly synonymous substitutions along the sequences, suggesting the presence of allelic variants of the protein. In addition, transcriptome analyses of . valens , and Tomisoforms , an evenisoforms  by gene isoforms  or horizisoforms . In factDendroctonus-bark beetles feed only on phloem. Stable and functionally generalist enzymes may be more efficient in an environment heterogeneous such as the subcortical region of the pine tree, which is degraded and changed as colonization proceeds and the tree dies.In other insects, it has been postulated that the existence of gene copies of α-amylase could be associated with an increase in their adaptive capacities to different food sources or to the occurrence of plant inhibitors ,34. AlthThe phylogenetic analysis of AmyDr revealed the existence of a close relationship with the α-amylases of Curculionidae, and from these to those of Chrysomelidae, suggesting a common evolutionary history between both families of α-amylases. In addition, the presence of a short “flexible loop” with the absence of GHGA/GHG motif near the catalytic cleft in AmyDr and α-amylases from weevils and leaf beetles, suggests that this condition is conserved. Thus, like in other insect orders, two conditions exist in Coleoptera, namely one α-amylase group without motif and with a shorter “flexible loop” and the other group having a “flexible loop” with the presence of the GHGA/GHG motif . While bDendroctonus, Ips, Hypotenemus), weevils , chrysomelids , and the two leaf beetle species analyzed  is slightly stronger (binding energy −17.2 kJ mol−1 and hydrogen bonds with distances of ≤2.2 Å) than that of Cl− (−16.74 kJ mol−1 and ≤2.1 Å), as previously demonstrated in marine bacterium A. haloplanktis [On the other hand, in contrast to both chlorine-dependent (arginine-bearing α-amylases) and chlorine-independent (glutamine-bearing α-amylases) α-amylases, AmyDr has a lysine  in the third residue of the binding site to chlorine ion. This mutation is shared with other scolytines species  were collected at Mil Diez locality (23°48′28.12″ N 105°24′11.63″ W), Pueblo Nuevo municipality, Durango state, Mexico, from 2018 to 2019, and were transported immediately to the laboratory to get fifth-instar larvae (L5), pupa (PU), pre-imago (PI), and pre-emergent adult (AD) of D. rhizophagus. The gut was dorsally extracted and separated from the fat body and the Malpighian tubules; the head-pronotum and gut sections were separately placed into Eppendorf vials with 200 µL of Trizol® , and macerated completely using a sterile pestle. Three biological replicates of each stage were processed. As negative control, we used eggs collected directly from the galleries with fine-tip forceps. Three egg groups (n = 150) were integrated and placed in Eppendorf vials in the field, and several consecutive washes were done with distilled water and PBS buffer, and finally, they were stored in 200 µL of Trizol®. In the laboratory, they were completely macerated using a sterile pestle. For all the samples, vials were topped with 1 mL of Trizol® and stored at −80 °C for posterior RNA extraction and subsequent RT-qPCR.Naturally infested Arizona pines (D. rhizophagus’ putative α-amylase found in the transcriptome of this species [D. ponderosae (XM_019912291.1) through Blastn, and to an α-amylase  from the bark beetle Ips typographus reported by [Total RNA was isolated from the whole adult body using the RiboPure™ RNA Purification kit  following the manufacturer’s instructions. The cDNA was synthesized from 2 µg of total RNA using the High-Capacity cDNA Reverse Transcription kit  as per manufacturer’s protocol, and stored at −20 °C. Forward primer F1 5′ TGGCGATCAGTTCTAGACCAA 3′ and reverse primer R1 5′ ATTGCAACGTGCTCTCATAA 3′ were designed according to UTRs of a  species . This seorted by .The PCR reaction was performed using Dream Taq Polymerase  and the following conditions: 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, 57.5 °C annealing for 30 s, and extension at 72 °C for 2 min, with a final extension step at 72 °C for 20 min. PCR products were cloned into pGEM T Easy Vector  following the manufacturer’s instructions. A total of 30 clones with insert were sequenced in both senses at Macrogen, Inc. .D. rhizophagus [AmyDr) was deposited in the GenBank (accession number MN782509). The deduced amino acid sequence was obtained with the ExPasy Translate Tool (http://www.expasy.org/tools/dna) and the Protein isoelectric point calculator (http://isoelectric.org) was used to predict the physicochemical characteristics of the enzyme, including molecular mass (kDa) and isoelectric point (pI). Moreover, subcellular localization and signal peptide predictions were realized with the ProtComp-AN software, v.9.0 and SignalP 5.0 server . The NetNGlyc v.1.0 (http://www.cbs.dtu.dk/services/NetNGlyc) and NetOGlyc v.4.0 (http://www.cbs.dtu.dk/services/NetOGlyc) servers were used to predict potential N and O glycosylation sites.The sequence obtained by PCR was confirmed by BlastX. To search different isoforms, the putative α-amylase sequence was mapped against all transcripts of adults and pupae transcriptomes from zophagus  using GMzophagus  with a khttp://www.atgc-montpellier.fr/phyml/). The more appropriate model of protein evolution was determined using the Akaike information criteria  and Bayesian information criteria  [Spodoptera frugiperda (AAO13754.1) was used as outgroup.Multiple protein alignment with reference coleopteran sequences was performed with Muscle software, v.3.8.31  and a ph2834.65) . Both cr2834.65) , and theTenebrio molitor , which is the only structural model of α-amylases in insects elucidated by X-Ray [http://servicesn.mbi.ucla.edu/SAVES) using Verify3D [http://mordred.bioc.cam.ac.uk/~rapper/rampage.php). To identify active and binding sites with calcium and chlorine ions, a multiple alignment without signal peptides of AmyDr sequence from D. rhizophagus and several α-amylases from coleopterans and lepidopterans was performed in ESPript software, v.3.0 [The 3D structure of the α-amylase was obtained by homology modelling with Modeller software, v.9.16 , using aby X-Ray . The besVerify3D , Errat [Verify3D , ProchecVerify3D , and WhaVerify3D . The RamVerify3D  in the Re, v.3.0 . The 3D e, v.3.0 .http://www.cambridgesoft.com) and were geometrically optimized with the Gaussian software, v.5.0.9 [7 energy evaluations. The interactions between the ligands and the α-amylase were visualized using ADT software, v.4.2, and the figures were created using the PyMol™ software, v.2.2.0.Predicted interactions between amylopectin and amylose with ions were evaluated using docking analyses. Ligands were drawn in ChemDraw software, v.15.0 ( v.5.0.9  using AM v.5.0.9 . To perf v.5.0.9 . All docD. rhizophagus (1:1 sex ratio) were placed in holes drilled (0.5 cm) into the fresh stumps of Arizona pines (40 cm high and ~15 cm diameter) and secured with steel mesh and staples. The beetles were fed for 12 h at room temperature in dark conditions. Then, the head-pronotum was removed directly from the insects, and the guts were dorsally extracted after the elytra and wings. The head-pronotum and gut from insects were separately homogenized in 100 µL 1 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 10,000× g for 15 min at 4 °C. The pellets of each sample were measured, normalized, and loaded by duplicate with the sample buffer  and incubated at 37 °C for 10 min. The samples were loaded twice into an 8% polyacrylamide gel copolymerized with 1% (w/v) of starch  using a Mini PROTEAN II system with a running buffer free of SDS at pH 8.3. Native-PAGE was conducted at 4 °C for 4 h at 60 V. After electrophoresis, the gel was rinsed with distilled water for 15 min, and the zymogram assay was performed in 50 mM citrate buffer, pH 5.0, for 1 h at 40 °C.Ten adult beetles of 2O at a flow rate of 5 μL min-1 during 10 min. Then, peptides were loaded and separated on a 3C18-CL-120 column ; 75 μm X 150 mm, 120 Å pore size, 3 μm particle size, in an HPLC Ekspert nanoLC 425  using as mobile phase A, 0.1% TFA in H2O, and mobile phase B 0.1% TFA in acetonitrile (ACN) under the following linear gradient: 0-3 min 10% B, 60 min 60% B, 61–64 min 90% B, 65 to 90 min 10% B at a flow rate of 250 µL min−1. Eluted fractions were automatically mixed with a solution of 2 mg mL−1 of α-cyano-4-hydroxycinnamic acid in 0.1% TFA and 50% ACN as a matrix, spotted in an Opti-TOF plate of 384 spots using a MALDI Ekspot  with a spotting velocity of 20 s per spot at a matrix flow rate of 1.6 μL min−1. The spots generated were analyzed by a MALDI-TOF/TOF 4800 Plus mass spectrometer . Each MS Spectrum was acquired by an accumulation of 1000 shots in a mass range of 850–4000 Th with a laser intensity of 3800. The 100 more intense ions with a minimum signal-noise of 20 were programmed to fragmenting. The MS/MS spectrums were obtained by the fragmentation of selected precursor ions using collision-induced dissociation and acquired by 3000 shots with a laser intensity of 4300. Generated MS/MS spectrums were compared against the Swiss-Prot database by the Protein Pilot software, v.2.0.1, using the Paragon algorithm. Search parameters were: Carbamidomethylated cysteine, trypsin as a cut enzyme, all the biological modifications and amino acid substitution set by the algorithm; as well as phosphorylation emphasis, and Gel-based ID as special factors. The detection threshold was considered in 1.3 to acquire 95% of confidence. Also, the identified proteins observed a local false discovery rate of ≤ 5%. In a bid to minimize redundancy, identified proteins were grouped by the ProGroup algorithm in Protein Pilot software, v.2.0.1. Analyses were carried out by the “Unidad de Genómica, Proteómica y Metabolómica” at LaNSE, CINVESTAV, CDMX, México.The gel was cut into two longitudinal sections. The first section was stained with Lugol’s solution for 5 min and incubated in distilled water with shaking to visualize the amylolytic activity. The second section was not stained but was matched with the first section once activity was visualized. To determinate the presence of α-amylase peptides from the head-pronotum and gut samples using MALDI-TOF/TOF, the lane was divided into four parts (S1–S4). Due to amylolytic activity displayed in the section S3 in both samples, it was divided into two subsections  for the gut sample and  for the head-pronotum sample. These sections were cut and digested with trypsin to identify peptides according to Shevchenko et al. . The resD. rhizophagus life cycle  and the different tissues (head-pronotum and gut). Total RNA from the samples was isolated using the RiboPure™ RNA Purification kit . The RNA concentration and purity (A260/A280 ratio) were determined using an ND-2000 spectrophotometer  and its integrity was checked in 1% agarose gel electrophoresis. The cDNA synthesis was done using the High-Capacity cDNA Reverse Transcription Kit  from 2 μg total RNA in a final reaction volume of 20 μL, as per manufacturer’s protocol. TaqMan® probes and primers were designed by Applied Biosystems (® hydrolysis probes were labeled at the 5′ end with the reporter dye 6-carboxyfluorescein (FAM) and joined at the 3’ end to a non-fluorescent quencher molecule group MGB (Minor Groove Binder). The reactions were carried out with a Step One® TM Real-Time PCR System  in a final volume of 20 μL: 20X primer and probes with a final concentration of 900 nM of each primer, 250 nM of the TaqMan probe, TaqMan® Universal Master Mix II  and 5 μL of 100-fold dilution of cDNA. All samples were placed in Fast Optical 48-well reaction plates MicroAmp®™ . Standard manufacturer’s amplification conditions were used: 50 °C for 2 min, 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 60 s. PCR contamination was undetected in the no template control NTCs (unintended amplification products) and three technical replicates were performed for each biological replicate from each development state. A set of ten guts or ten head-pronotum were used in each of the replicas.The relative expression of the α-amylase was evaluated in the different developmental stages of the osystems . The TaqGAPDH; MN782508) was used as a reference gene since it showed a stable expression according to pupa and adult transcriptomes of D. rhizophagus [GAPDH and Amy genes were estimated with the relation: efficiency = (10−1/slope−1)100, where the slope values were −3.58 and −3.52 respectively, and R2 value of 0.99. To evaluate the specificity, all amplicons were visualized in 1% agarose gel electrophoresis. All experimental procedures were performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines  and different developmental stages . Statistical tests were performed with Past software, v.3.26 [p < 0.05 were considered significant.The relative expression values of the α-amylase gene were determined using the 2t method , where t, v.3.26 . Values AmyDr was recovered from DH5α cells with the construction pGEM_AmyDr, using the GenElute TM Plasmid Midiprep kit  following manufacturer’s protocol. The AmyDr open reading frame (ORF) was PCR-amplified with primers F1_DR1_SPIF (5′GTCGACTGGATCCGGATGCAGCTAGGAATTCTG3′) and R1_DR1_SPIF (5′ATCTCGAGTGCGGCCTTATAATTTTGCATTTACATG3′) using a Q5 High-Fidelity DNA Polymerase  following manufacturer’s protocol. The amplification product was cloned into pENTR4 using In-Fusion HD EcoDry  and transformed into Stellar™ Competent Cells (E. coli HST08)  following the manufacter’s instructions. The recombinant plasmid was confirmed by sequencing prior recombination into the linearized BaculoDirect™ C-term vector  by LR recombination with LR Clonase™ II . Sf9 cells were infected with the recombinant Bac_AmyDr vector to obtain a high-titer P3 viral stock by successive amplifications of P1 and P2 stocks following manufacturer’s protocol. Plasmid with recombinant AmyDr P3 viral stock (500 μL), previously produced, was used to infect 1 × 106Sf9 cells mL−1 in 30 mL of Sf900 II serum-free media  suspension cultures for 72 h at 27 °C. Cells expressing recombinant AmyDr were harvested with Cell Lysis Buffer  glycerol, and 1.1 mM EDTA) supplemented with 75 μL of phenylmethylsulfonyl fluoride (0.1 M PMSF), 15 μL of protease inhibitor cocktail (PIC) , and 30 μL of dithiothreitol (0.1 M DTT), and then centrifuged at 3000× g at 4 °C for 10 min. The supernatant was isolated and tested for functional AmyDr. As negative control of amylases expression, 30 mL of 1 × 106Sf9 cells mL−1 in Sf-900 II SFM suspension culture without viral stock and processed as mentioned before was used.Recombinant Sf9 cells without viral stock were precipitated overnight at 4 °C and 80% (NH4)2SO4 saturation. The pellet was recovered by centrifugation at 8800× g at 4 °C for 20 min, suspended in 5 mL of 50 mM, pH 5.0 citrate buffer, and dialyzed at 4 °C for 12 h in a cellulose membrane  using the same buffer. Proteins obtained were isolated in 10% SDS-PAGE.The total protein from AmyDr crude extract and the negative control from Protein analyses were performed in 10% SDS-PAGE using a Mini PROTEAN II system. Proteins in the gel were visualized using Coomassie Brilliant Blue R-250 and recorded with a MiniBIS Pro documentation system . Protein molecular weight (MW) was estimated using as reference broad-range molecular weight protein standards .−1 starch, 23 g L−1 agar dissolved in deionized water), where 50 μL of amylase crude extract were added in a 5 mm hole, previously made with a Pasteur pipette on starch agar plates. These were incubated at 37 °C for 12h and stained with Lugol’s solution to document the enzyme activity around the hole.The amylolytic activity of AmyDr was determined on 1% starch agar plates 2, and KNO3 at two final concentrations of 1 and 5 mM each. Likewise, as described before, the reaction mixture was added in a 5-mm hole on the starch agar plates, incubated at 37 °C for 12 h, and stained with Lugol’s solution to document the enzyme activity around the hole. Amylolytic activity was compared to a control plate with only metal and non-metal ions, and a control plate with Sf9 cells including metal and non-metal ions, which were the negative control of this assay.To evaluate the effect of metal ions, such as Ca


Lactobacillus plantarum NIBR97, including novel bacteriocins, has potential as a natural alternative to chemical disinfectants. It exhibits significant antibacterial activities against a broad range of pathogens, and was observed by scanning electron microscopy (SEM) to cause cellular lysis through pore formation in bacterial membranes, implying that its antibacterial activity may be mediated by peptides or proteins and supported by proteinase K treatment. It also showed significant antiviral activities against HIV-based lentivirus and influenza A/H3N2, causing lentiviral lysis through envelope collapse. Furthermore, whole-genome sequencing revealed that NIBR97 has diverse antimicrobial peptides, and among them are five novel bacteriocins, designated as plantaricin 1 to 5. Plantaricin 3 and 5 in particular showed both antibacterial and antiviral activities. SEM revealed that plantaricin 3 causes direct damage to both bacterial membranes and viral envelopes, while plantaricin 5 damaged only bacterial membranes, implying different antiviral mechanisms. Our data suggest that the cell-free supernatant from L. plantarum NIBR97, including novel bacteriocins, is potentially useful as a natural alternative to chemical disinfectants.The recent pandemic of coronavirus disease 2019 (COVID-19) has increased demand for chemical disinfectants, which can be potentially hazardous to users. Here, we suggest that the cell-free supernatant from  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the global pandemic of coronavirus disease 2019 (COVID-19), is the foremost concern among recent global health issues ,4,5. AltEscherichia coli, Salmonella enterica, and Listeria monocytogenes, and thus many studies have highlighted their application as natural alternatives to artificial preservatives and antibiotics . Th. ThLactoormation . Collectct on it , which iE. coli Top10 strain , harboring each Pln gene cloned, showed significant antibacterial activities against both Gram-negative and Gram-positive bacteria, whereas very little antibacterial activity was detected in the negative control, that is, treatment with the cell-free supernatant from the strain without the Pln genes  and Dr. Tae Sung Jung . The Lactobacillus plantarum strains KCTC33131, KCTC21004, and KCTC13093, as well as the susceptible bacteria Escherichia coli ATCC 10536 and Staphylococcus aureus ATCC 6538, were purchased from KCTC . The human influenza A/H3N2 was provided by the Korea Centers for Disease Control and Prevention . The plasmids for lentiviral packaging  and for a positive control of transduction (pSIH1-H1-siLUC-copGFP) were purchased from Cellbiolab  and System Biosciences , respectively. The five synthetic peptides—plantaricin 1 to 5—were purchased from Cosmogenetech Inc. .As susceptible bacteria to AMPs, L. plantarum NIBR97 was incubated at 37 °C for 24 h in an MRS liquid medium -glucose, 2 g/L dipotassium hydrogen phosphate, 5 g/L sodium acetate trihydrate, 2 g/L triammonium citrate, 0.2 g/L magnesium sulfate heptahydrate, and 0.05 g/L manganous sulfate tetrahydrate). The cultural broth was centrifuged for 20 min at 2000× g, and the centrifugal supernatant was collected and then sterilized by a 0.22 μm filtration. The sterilized fluid was either applied directly for the examination of antimicrobial activity or fractionated and stored at −80 °C until use. The assessment of antimicrobial activity on a microtiter plate was performed by some modification of the dilution assay of Wiegand et al. [d et al. . The MICd et al. .Escherichia coli ATCC 10536 and Staphylococcus aureus ATCC 6538 were adjusted into 1.5 to 5.0 × 108 CFU/mL after pre-culture, and 10% sucrose was used as an interfering agent, 0.25 M KH2PO4 (pH 7.2) was used as a neutralizing agent, and 20 mg/mL proteinase K was used to degrade the AMPs. For the bactericidal activity assay, we mixed 100 μL of prepared susceptible bacterial solution, 100 μL 10% sucrose, and 800 μL cell-free supernatant  from L. plantarum NIBR97 and reacted the mixture at 20 °C for 5 min. An aliquot (100 μL) of the reaction solution was mixed with 800 μL 0.25 M KH2PO4 (pH 7.2), 5 μL proteinase K, and 100 μL distilled water, and then reacted at 20 °C for 5 min. The surviving cells were counted by serial dilution of the treated solution and incubation on an Luria-Bertani (LB) plate.The susceptible bacterial strains S. Enteritidis was treated with the cell-free supernatant  from L. plantarum culture or the synthetic peptides, Pln 3 (1 μg/μL) or Pln 5 (1 μg/μL), for 0, 1, and 8 h, and the lentivirus was assessed with the cell-free supernatant  for 5 min and with the synthetic peptides Pln 3 (5 μg/μL) or Pln 5 (5 μg/μL) for 24 h. The treated bacteria and viruses were observed by a scanning electron microscope according to previously described procedures [The ocedures .5 viruses/μL) with 0.9 mL of the cell-free supernatant  for 10 min, 30 min, and 18 h at 25 °C. After the co-incubation, the cell-free supernatant-A/H3N2 mixture was 10-fold serially diluted to infect Madin–Darby canine kidney (MDCK) cells (3 × 104 cells per well) and, thereafter, the cell culture infectious dose (CCID50) and the viral reduction were determined by cytopathic effect (CPE) and plaque assays, as previously described [For the antiviral test, we co-incubated 0.1 mL of the A/H3N2 soup (2–4 × 10escribed . 6 cells per well) using lipofectamine 2000 . The lentiviral supernatants were harvested 72 h after transfection, filtered through Millex-GP 0.45 µm filters , and concentrated using Retro-Concentin Retroviral Concentration Reagent . The titer of lentiviruses was measured with a QuickTiter Lentivirus Titer Kit  and stored at −80 °C.For the production of GFP-labeled lentivirus, we transfected 5 μg of pRSV-Rev, 5 μg of pCMV-VSV-G, 5 μg of pCgpV, and 15 μg of pSIH1-H1-siLUC-copGFP plasmids into HEK-293T cells (6 × 106 lentiviruses/μL) with 2 μL of test sample for 5 min and 24 h at 25 °C. After the co-incubation, 2 μL from the total 4 μL of the test sample–lentivirus mixture was infected in HEK-293T cells (1 × 104 cells per well). Expression of the copGFP protein was observed at day 3 after infection with a Zeiss 510 fluorescence microscope .For the anti-viral test, we co-incubated 2 μL of lentivirus soup . Gene ontology annotations of the ORFs were assigned by Blast2GO software [Genomic analysis of ocedures . De novoocedures  and the ocedures , identifocedures , and thesoftware . In addisoftware ,35. Finap < 0.05) between sample types. All the analyses were performed by the SAS statistical software package , for which differences were considered significant at p < 0.05.The mean values were separated by the probability difference option according to significant differences. The results are exhibited as least square means with standard deviations. Duncan’s multiple range tests (MRT) were applied for verification of significant differences (L. plantarum NIBR97, producing novel bacteriocins, has superior antibacterial and antiviral activities during both short and long exposures, which suggests that it is potentially useful as a natural material to completely or partially replace chemical disinfectants.Together, our data showed that the cell-free supernatant from