Neonatal stroke is a devastating insult that can lead to life-long impairments. In response to hypoxic–ischaemic injury, there is loss of neurons and glia as well as a neuroinflammatory response mediated by resident immune cells, including microglia and astrocytes, which can exacerbate damage. Administration of the antidiabetic drug metformin has been shown to improve functional outcomes in preclinical models of brain injury and the cellular basis for metformin-mediated recovery is unknown. Given metformin’s demonstrated anti-inflammatory properties, we investigated its role in regulating the microglia activation and used a microglia ablation strategy to investigate the microglia-mediated outcomes in a mouse model of neonatal stroke.Hypoxia-ischaemia (H-I) was performed on post-natal day 8. Metformin was administered for one week, starting one day after injury. Immunohistochemistry was used to examine the spatiotemporal response of microglia and astrocytes after hypoxia-ischaemia, with or without metformin treatment. To evaluate the effects of microglia depletion after hypoxia-ischaemia, we delivered Plexxikon 5622 for 1 or 2 weeks post-injury. The regional pattern of microglia and astrocyte depletion was assessed through immunohistochemistry. Motor behaviour was assessed with the righting reflex, hindlimb suspension, grip strength and cylinder tests.Herein, we revealed a spatiotemporally regulated response of microglia and astrocytes after hypoxia-ischaemia. Metformin treatment after hypoxia-ischaemia had no effect on microglia number and proliferation, but significantly reduced microglia activation in all regions examined, concomitant with improved behavioural outcomes in injured mice. Plexxikon 5622 treatment successfully ablated microglia, resulting in a > 90% depletion in microglia in the neonatal brain. Microglia rapidly repopulated upon treatment cessation of Plexxikon. Most interesting, microglia ablation was sufficient to reduce functional deficits after hypoxia-ischaemia, mimicking the effects of 1 week of metformin treatment post-injury.These results highlight the importance of regulating the neuroinflammatory response after neonatal stroke to promote recovery.The online version contains supplementary material available at 10.1186/s12974-022-02487-x. Neonatal stroke is an important cause of brain injury that occurs in ~ 1 in 2300 infant births , 17, 45.Metformin, a commonly used drug to treat type-2 diabetes, has been shown to have pleiotropic effects in the brain. Our group, and others, have shown that metformin administration leads to expansion of the endogenous neural precursor pool in their subependymal zone (SEZ) niche, migration of neural precursor cells into stroke-injured parenchyma, contribution to neurogenesis and oligodendrogenesis and improved motor and cognitive outcomes , 48. MetMicroglia are the resident immune cells of the central nervous system and during brain maturation, microglia perform several functions, such as synaptic pruning and supporting axon myelination , 54, 60.The neuroinflammatory response after injury has also been shown to be dependent on astrocytes. Similar to microglia, astrocytes have pro-inflammatory and immunoregulatory (neuroprotective) subpopulations . AstrocyIn this study, we aimed to shed light on metformin's cellular targets following neonatal stroke. We characterized the microglia and astrocyte response, with and without metformin treatment, using a well-established model of neonatal stroke. Herein, we show that the microglia response is regionally distinct at early times post-stroke and metformin treatment reduces microglia activation, as measured by morphology and protein expression (CD68). Metformin treatment after neonatal stroke did not affect astrocyte activation. Using a novel model of microglia ablation, we show that the loss of microglia does not result in motor behaviour impairments in early post-natal mice. Most interesting, the loss of microglia protects against the functional deficits that result following neonatal stroke, mimicking the metformin-mediated outcomes after 1 week of metformin treatment post-stroke. Our work provides novel insight into metformin's anti-inflammatory effects after neonatal stroke and further highlights the role of microglia in the observed motor impairments following injury to the early post-natal brain.Transgenic GFAP::GFP mice that express green fluorescent protein under the control of the human astrocyte GFAP promoter were used for all experiments . The mice were housed on a 12-h (h) day/night light cycle with access to food and water ad libitum, and each cage contained a dome house. Mice were bred in-house, and each dam was provided with a nestlet. A total of 141 neonatal, male and female mice were used. All animal procedures were in accordance with institutional guidelines and approved by the University of Toronto Animal Care Committee, following the guidelines of the Canadian Council for Animal Care.Post-natal day-8 (P8) mice were anaesthetized with isoflurane (5% induction and 1.5% maintenance) and the hypoxia–ischaemia (H-I) injury was performed as previously described , 33, 48.Metformin  was administered daily via subcutaneous injection directly to the neonates (200 mg/kg) , 33, 48 Pups received an intraperitoneal injection of EdU   one hour before sacrifice.Plexxikon 5622    was injected daily via intraperitoneal injections from P8 to P15 at a final concentration of 50 mg/kg in sterile PBS and 20% Kolliphor40 . Control mice received vehicle injections of DMSO with sterile PBS and 20% Kolliphor40.Animals were sacrificed on P9, P12, P14, P15, P21 and P22 with an overdose of Avertin. Transcardial perfusions were performed using cold PBS followed by 4% paraformaldehyde . The brains were removed and post-fixed in 4% PFA overnight, then cryoprotected in 30% sucrose solution and stored at 4 °C until cryosectioning. Brain sections  were mounted on Superfrost Plus microscope slides  and kept at − 20 °C until processing.Slides were thawed and rehydrated in PBS for 5 min at room temperature. Slides were placed in 0.3% Triton X-100  for 20 min followed by 3 X 5-min PBS washes. For EdU, slides were incubated for 30 min with the Click-It reaction cocktail, which was composed of Tris-buffered saline  + 8.76 g NaCl  + 800 ml ddH2O; pH of 7.5), Copper Sulphate , 647 Azide  and 1X reaction buffer . Next, slides were washed with PBS and were incubated in a blocking solution made of included 500ul normal goat serum , 100 mg bovine serum albumin  and 9.5 ml of 0.3% Triton X-100  for 1 h at room temperature. Following the 1 h block, primary antibodies were prepared in the blocking solution and sections were incubated overnight at 4 °C. Primary antibodies included rabbit-anti-Iba1 (Iba1) , rat-anti-mouse CD68 (CD68) , chicken anti-GFP (GFP)  and rabbit anti-GFAP . The next day, sections were washed 3 X 5 min with 0.2% Tween 20  in PBS, followed by a 1 h secondary antibody incubation at room temperature. Secondary antibodies included goat anti-rabbit Alexa Fluor 568  for Iba1 and GFAP (used on different slides), goat-anti-rat Alexa Fluor 647  for CD68 and goat anti-chicken Alexa Fluor 488  for GFP, dissolved in PBS. Slides were washed 3 × 5 min in PBS and incubated with DAPI  dissolved in PBS for 5 min to label nuclei. A final 3 X 5-min wash in PBS was performed and slides were coverslipped with mounting medium  and stored at − 20 °C until imaging.2. The numbers of neurospheres (> 80um in diameter) were counted.The in vitro neurosphere assay was performed to assess the numbers of neural stem cells, as previously described . Briefly2) and the lateral wall of the lateral ventricles  , in the ipsilateral and contralateral hemispheres (relative to the ischaemic insult). Areas chosen extended from the crossing of the corpus callosum anteriorly to the crossing of the anterior commissure, posteriorly.Slides were imaged using an Axiovert 200 M inverted microscope  and an Axio Observer Z1 inverted motorized microscope  with a high-speed spinning disk CSU-X1 confocal scanner unit  and an Axiocam 506 high-resolution camera . Images were acquired with the AxioVision 4.8 and Zen software . Three to four sections were counted per brain. Three regions of interest were imaged in the cortex and striatum  extending from the soma. Cells in the cortex were manually counted using the Cell Counter plugin in Fiji . These counts were expressed as fold changes and percentages of cells per unit area for each of the regions.2), (2) in the striatum  and (3) and in the dorsolateral corner of the lateral ventricle  using the pixel inspection tool and measurement tool in Fiji, respectively, from > 3 sections per brain.To measure microglia intensity and soma size, images were exported as TIFF files and sections were converted to 8 bits and filtered (filter = MaxEntropy). Pixel counts and soma length measurements were generated for Iba1 + cells (1) in the cortex . All data are graphed as mean ± standard error of the mean (SEM). Statistical analysis consisted of unpaired p = 7.34 × 10–5)    and striatum (p = 0.0085) relative to Shams  of microglia when compared to H-I only-injured mice , Sham + metformin, H-I only and H-I + metformin. We observed no difference in cell density across regions over time  or between groups , a time when increased numbers of microglia were observed  and P12   and at P12  , back to control (Sham) values  and P12 , where metformin treatment after H-I resulted in a reduction in amoeboid microglia back to control (Sham injured) levels   relative to sham animals    and P12   and P12    ; however, this increase in numbers was not coincident with increased EdU + GFP + /GFP + cells , with a trend towards an increase in proliferating astrocytes    Fig. B–G. In t–9) Fig. H–M. Agaip = 0.033      Fig. M-O and S16) Fig. T–V. Hencp = 0.12)   in the cortex and striatum following 1 or 2 weeks of PLX treatment and found no change in GFP + cells      metformin treatment after H-I reduces microglia activation and (2) metformin administration for 1 week post-H-I are sufficient to reduce motor impairments in the cylinder test on P22, we predicted that metformin’s anti-inflammatory effects on microglia may be mediating the improved functional outcomes seen after 1 week of metformin treatment P8-15) and motor assessment on P22  . First, we asked whether the drug metformin, which is known to have pleiotropic effects on cells in the CNS, impacts microglia activation, with the goal of uncovering the cellular targets that underlie the beneficial outcomes observed following neonatal stroke and metformin treatment. Our findings reveal that microglia and astrocyte activation are regionally and temporally distinct within the injured brain and further that microglia activation is reduced during the time that metformin is administered post-H-I. We next developed a novel microglia ablation paradigm (PLX treatment) in neonatal mice and confirmed that the loss of microglia in the early post-natal brain is sufficient to protect against functional impairments following H-I. We found that reducing the activation of microglia—demonstrated by reduced proliferation, amoeboid morphology, CD68 expression and total numbers of microglia—through metformin treatment or microglia ablation resulted in a complete lack of motor impairments following H-I. The microglia ablation timeline mimicked the metformin administration that resulted in improved functional outcomes, implicating microglia as an important target for promoting recovery following neonatal injury.Our study investigated the effects of modifying the activation state of microglia, as measured by proliferation, morphology and protein expression, and the impact on behavioural outcomes. A number of reports have shown that metformin has anti-inflammatory properties and can improve the microenvironment after injury by modulating the release of cytokines , 65. DelMoreover, the effects of metformin pre-treatment on microglia in the neurogenic niche, when metformin has been given prior to juvenile cranial radiation, has also revealed reduced microglia activation as measured by microglia numbers and CD68 expression . ReducedWe tested the hypothesis that neonatal H-I would trigger a microglia response and that metformin would dampen this response. Our observation that metformin did not reduce the microglia population after H-I was not predicted based on reports that metformin administration in the sub-acute period following early post-natal injury  reduces Metformin significantly reduced the numbers of amoeboid microglia after H-I. Microglia from H-I and metformin-treated brains failed to display an amoeboid phenotype which could be explained by (1) metformin preventing microglia from taking on an amoeboid phenotype or (2) H-I inducing the amoeboid phenotype and metformin reverts the activated microglia to their "resting" ramified phenotype. Given the early time point after injury at which we recorded a change in morphology after H-I (i.e. 1 day post-injury), our data support the hypothesis that metformin prevents the amoeboid phenotype. Microglia shape has been shown to be dependent on the Par1b/MARK2 pathway   on microglia in postnatal mice , 62, as , PLX3397Our data reveal that dampening or ablating the early microglia response (i.e. within the first week post-H-I) is sufficient to protect against H-I-induced functional deficits. The findings are consistent with the conclusion that metformin-mediated reduction in microglia activation following H-I results in improved functional outcomes. This study further characterizes the pleiotropic effects of metformin and identifies microglia as a target to improve outcomes after neonatal stroke. Future investigation of strategies to assess microglia activation, such as assessing changes in inflammatory factors and using complementary ablation models , will contribute to our growing understanding of the cell mechanisms that underlies microglia-mediated recovery. Modulation of microglia activation states and exploring microglia heterogeneity remain important future considerations for novel strategies for brain repair.Additional file 1: Figure S1. H-I-injured mice that receive metformin treatment do not display a forepaw preference on the cylinder task. A Experimental timeline. Mice receive H-I injury on P8. Metformin was administered from P9 to P15. The cylinder test is performed on P22. B Forepaw preference for the uninjured paw on the cylinder test. Sham mice do not show a paw preference. H-I injured mice are significantly impaired compared to Shams (-0.29±3.44% preference for the un-impaired paw in Sham mice vs. 20.77±3.21% in H-I-injured mice) (p=0.0004). H-I+MET are significantly improved compared to H-I only  and not significantly different from Shams (p=0.95). n=6–14 mice per group. Data presented as Mean ± SEM. Statistics: (B) One-way ANOVA. *p<0.050.Additional file 2: Figure S2. H-I injury leads to behavioural impairments but no change in cell density in the cortex, striatum and SEZ. A Experimental timeline. Mice received H-I on P8 and Metformin administration from P9 to P14. Immunohistochemistry (IHC) was performed on P9, P12, P14 and P21. Righting reflex was performed on P8 at one hour post H-I. B H-I results in increased latency to perform the righting reflex test relative to Sham  on P8 when performed 1 hour post-injury. C H-I, Metformin and H-I+MET treatments do not affect the total number of DAPI+ cells in the cortex. D H-I, Metformin and H-I+MET treatments do not affect the total number of DAPI+ cells in the striatum. E H-I, Metformin and H-I+MET treatments do not affect the total number of DAPI+ cells in the SEZ. n=3 mice per group for immunohistochemistry. n=11–16 mice per group for behavioural testing. Data presented as mean ± SEM. Unit area : 3x0.105 mm2. Unit area (SEZ): 3x0.045mm2. Statistics: (B) Unpaired t-test; (C-E) Two-way ANOVA. *p<0.050.Additional file 3: Figure S3. Metformin treatment reduces CD68 expression in microglia after H-I. A Experimental timeline. H-I was given onP8 and metformin administration from P9 to P14.Immunohistochemistry (IHC) was performed on P9, P12, P14 and P21. B Representativeimage of activated microglia (Iba1+ (Red) CD68+ (Cyan) double-positive cells) atP12 in the cortex in Sham mice (20x magnification). Image is representative of thefirst time point of activation. C Representative image of activated microglia (Iba1+(Red) CD68+ (Cyan) double-positive cells) at P12 in the cortex after in Met-treatedmice H-I (20x magnification). Image is representative of the first time point ofactivation. D Representative image of activated microglia (Iba1+ (Red) CD68+ (Cyan)double-positive cells) at P12 in the cortex after H-I (20x magnification). Imageis representative of the first time point of activation. E Representative imageof activated microglia (Iba1+CD68+) at P12 (first time point of activation) in thecortex after H-I+MET (20x magnification). Image is representative of the first timepoint of activation. F Quantification of Iba1+CD68+ in the cortex at P12 acrossgroups, expressed as percentage per unit area. There was a significant increaseof Iba1+CD68+ microglia after H-I relative to Sham . This increase in Iba1+CD68+cells was prevented by HI+Met . G Representative image of activatedmicroglia (Iba1+CD68+) at P9 (first time point of activation) in the striatum inSham mice (20x magnification). H Representative image of activated microglia (Iba1+CD68+)at P9 (first time point of activation) in the striatum in Met-treated mice (20xmagnification). I Representative image of activated microglia (Iba1+CD68+) at P9(first time point of activation) in the striatum after H-I (20x magnification).J Representative image of activated microglia (Iba1+CD68+) at P9 in the striatumafter H-I+MET (20x magnification). K Quantification of Iba1+CD68+ in the striatumat P9 across groups, expressed as percentage per unit area. There was a significantincrease of Iba1+CD68+ microglia after H-I relative to Sham . This increasein Iba1+CD68+ cells was prevented by HI+Met . L Quantification of activatedmicroglia (Iba1+CD68+) in the striatum at P12 across groups, expressed as percentageper unit area. There was a significant increase of Iba1+CD68+ microglia after H-Irelative to Sham . This increasein Iba1+CD68+ cells was prevented by HI+Met . M Representative image of amoeboidmicroglia (Iba1+) at P9 in the SEZ in Sham mice (40x magnification). Image is representativeof the first time point of activation in the SEZ. N Representative image of amoeboidmicroglia (Iba1+) at P9 in the SEZ in Met-treated mice (40x magnification). Imageis representative of the first time point of activation in the SEZ. O Representativeimage of amoeboid microglia (Iba1+) at P9 in the SEZ in H-I mice (40x magnification).Image is representative of the first time point of activation in the SEZ. P Representativeimage of amoeboid microglia (Iba1+) at P9 in the SEZ in H-I+MET mice (40x magnification).Image is representative of the first time point of activation in the SEZ. Q Quantificationof activated microglia (Iba1+CD68+) in the SEZ at P9 across groups, expressed aspercentage per unit area. There was a significant increase of Iba1+CD68+ microgliaafter H-I relative to Sham . This increase in Iba1+CD68+ cells was preventedby HI+Met . R Quantification of activated microglia (Iba1+CD68+) in the SEZ atP12 across groups, expressed as percentage per unit area. There was a significantincrease of Iba1+CD68+ microglia after H-I relative to Sham  85.12 ± 6.03%in H-I-injured mice, p=0.017). This increase in Iba1+CD68+ cells was prevented byHI+Met . n=3-5 mice per group. Data presented as mean±SEM. Scale Bar: 50um. Unit area : 3x0.105 mm2.Unit area (SEZ): 3x0.045mm2. Statistics:  Oneway ANOVA. *p<0.050.Additional file 4: Figure S4. Dose-dependentmicroglia ablation is seen following one week of daily injections of Plexxikon.A Experimental timeline following PLX 5622 administration  from P8 to P15. Immunohistochemistry (IHC) or the neurosphereassay (NS) was performed on P15. B No differences in pup weight were recordedacross treatment groups. C Representative image of microglia  atP15 in the cortex in Sham mice (20x magnification). D Representative image ofmicroglia  at P15 in the cortex in mice that received 10ug PLX fromP8 to 15 (20x magnification). E Representative image of microglia at P15 in the cortex in mice that received 50ug PLX from P8 to 15 (20xmagnification). F Quantification of the number microglia (Iba1+ cells/area) inthe cortex, reported as fold change. Microglia are significantly depleted with10ug/g PLX (1.00±0.11-fold change of Iba1+ cells in vehicle-treated mice vs.0.52±0.03-fold change Iba1+ cells in 10ug/g PLX-treated mice) (p=0.008) and50ug/g PLX (1.00±0.11-fold change of Iba1+ cells in vehicle-treated mice vs.0.18± 0.05-fold change of Iba1+ cells in 50ug/g PLX-treated mice (p=0.0004).PLX at 50ug/g results in a significantly greater loss of Iba1+ cells comparedto 10ug/g (0.52±0.03-fold change Iba1+ cells in 10ug/g PLX-treated mice vs.0.18±0.05-fold change of Iba1+ cells in 50ug/g PLX-treated mice) (p=0.033).Average number of Iba1+cells/unit area: Vehicle=67.17±7.21; 10ug/gPLX=35.28±2.29; 50ug/g PLX=12.42±3.2. G Representative image of microglia at P15 in the striatum in Sham mice (20x magnification). HRepresentative image of microglia  at P15 in the striatum in micethat received 10ug PLX from P8 to 15 (20x magnification). I Representativeimage of microglia  at P15 in the striatum in mice that received50ug PLX from P8 to 15 (20x magnification). J Quantification of the numbermicroglia (Iba1+ cells/area) in the striatum, reported as fold change.Microglia are not depleted with PLX at 10ug/g (1.00± 0.08-fold change of Iba1+cells in vehicle-treated mice vs. 0.77±0.08-fold change Iba1+ cells in 10ug/gPLX-treated mice) (p=0.4514) but are significantly reduced with 50ug/g PLX(1.00± 0.08-fold change of Iba1+ cells in vehicle-treated mice vs.0.25±0.04-fold change of Iba1+ cells area in 50ug/g PLX-treated mice vs.vehicle (p=0.003). PLX at 50ug/g results in a significantly greater loss ofIba1+ cells compared to 10ug/g (0.77±0.08-fold change Iba1+ cells in 10ug/gPLX-treated mice vs. 0.25±0.04-fold change of Iba1+ cells in 50ug/g PLX-treatedmice) (p=0.0034). Average number of Iba1+cells/unit area: Vehicle = 32.19±2.65;10ug/g = 28.00 ± 2.65; 50ug/g PLX = 9.83±1.36. K Representative image ofmicroglia  at P15 in the SEZ in Sham mice (20x magnification). LRepresentative image of microglia  at P15 in the SEZ in mice thatreceived 10ug PLX from P8 to 15 (20x magnification). M Representative image ofmicroglia  at P15 in the SEZ in mice that received 50ug PLX from P8to 15 (20x magnification). N Quantification of the number microglia (Iba1+cells/ area) in the SEZ, reported as fold change. Microglia are significantlydepleted with 10ug/g PLX (1.00± 0.04-fold change of Iba1+ cells invehicle-treated mice vs. 0.55±0.03-fold change Iba1+ cells in 10ug/gPLX-treated mice) (p=0.0002) and 50ug/g PLX (0.31±0.01-fold change of Iba1+cells in vehicle-treated mice vs. 0.18±0.05-fold change of Iba1+ cells in50ug/g PLX-treated mice (p=0.004). PLX at 50ug/g results in a significantlygreater loss of Iba1+ cells compared to 10ug/g (0.55±0.03-fold change Iba1+cells in 10ug/g PLX-treated mice vs. 0.31±0.01fold change of Iba1+ cells in50ug/g PLX-treated mice) (p=0.0038). Average number of Iba1+cells/unit area:Vehicle=20.03±0.87; 10ug/g PLX=11.11±0.68; 50ug/g PLX=6.19±0.10±1.36. O Nodifference in neurosphere numbers were observed across treatment groups(p=0.39). Average number of neurospheres: Vehicle = 6.33±0.96; PLX: 7.33±0.42. n=3 mice per group. Data presented as mean ± SEM. Unit area :3x0.105 mm2. Unit area (SEZ): 3x0.045mm2. Statistics: (B) Two-way ANOVA; (C-E)One-way ANOVA. (O) Unpaired t-test. *p<0.050.Additional file 5: Figure S5. Microglia depletion doesnot affect the numbers of astrocytes or SVZ-derived neural precursor cells. A Experimentaltimeline. PLX (50ug/g) was administered fromP8 to 15 or P8 to 22. Mice were sacrificedat P15 and P22 for IHC. B Representative image of astrocytes  at P15in the cortex in Sham mice (20x magnification). C Representative image of astrocytes at P15 in the cortex in PLX-treated mice (20x magnification). D Representativeimage of astrocytes  at P15 in the cortex in H-I-injured mice (20xmagnification). E Representative image of astrocytes  at P15 in thecortex in H-I+PLX-treated mice (20x magnification). F Quantification of the numberof GFP+ astrocytes in the cortex at P15 across groups represented as fold change.There was no difference in the number of GFP+ astrocytes across treatment groups. Average number of GFP+cells/unitarea: Sham = 43.9±4.11; PLX = 50.62±5.09; H-I = 67.4±10.0; H-I+PLX = 64.2± 6.34.G Representative image of astrocytes  at P15 in the striatum in Shammice (20x magnification). H Representative image of astrocytes  atP15 in the striatum in PLX-treated mice (20x magnification). I Representative imageof astrocytes  at P15 in the striatum in H-I-injured mice (20x magnification).J Representative image of astrocytes  at P15 in the striatum in H-I+PLX-treatedmice (20x magnification). K Quantification of the numbers of GFP+ astrocytes inthe striatum across groups at P15 represented as fold change. There was no differencein the numbers of GFP+ astrocytes across treatment groups . Average number of GFP+cells/unit area: Sham = 32.3±3.03; PLX = 36.0±4.46;H-I = 53.2±4.98; H-I+PLX = 56.92± 8.21. L Quantification of the number of astrocytes(GFP+) in the cortex at P22 across groups, represented as fold change. No significantdifferences were found across groups at any time point examined (p=0.69). Averagenumber of GFP+cells/unit area: P8-15 Sac 22 Vehicle = 19.83±3.06; P8-15 Sac 22 PLX= 21.42±2.69; P8-22 Sac 22 Vehicle = 25.22±3.07; P8-22 Sac 22 PLX = 30.11± 3.00.M Quantification of the number of astrocytes (GFP+) in the striatum at P22 acrossgroups, represented as fold change. No significant differences were observed acrossgroups at any time point examined. (p=0.83) Average number of GFP+cells/unit area:P8-15 Sac 22 Vehicle = 24.83±2.38; P8-15 Sac 22 PLX = 24.83±2.99; P8-22 Sac 22 Vehicle= 24.89±2.61; P8-22 Sac 22 PLX = 26.33± 4.67. n= 3 mice per group. Data presentedas mean ± SEM. Scale Bar: 50um. Unit area : 3x0.105 mm2. Statistics:(A-B) One-way ANOVA. *p<0.050.


The Syqe inhaler delivers metered doses of phytocannabinoids by inhalation and utilizes accurate quantities of ground cannabis inflorescence packaged in tamper-proof cartridges. We aimed to assess the stability of phytocannabinoids in ground cannabis before and after packaging in Syqe cartridges as well as the reproducibility of THC delivery in the aerosolized dose.Although the worldwide use of medical cannabis (MC) is on the rise, there is insufficient data regarding the long-term stability of phytocannabinoids in the plant material under different storage conditions. Specifically, there is insufficient data on the effect of storage conditions on the availability of (-)-∆Ground MC inflorescence was stored under different temperature and humidity conditions, before or after being packaged in Syqe cartridges. Concentrations of the major phytocannabinoids therein were analyzed at different time points using ultra-high performance liquid chromatography (U-HPLC). THC doses aerosolized via the Syqe inhaler were evaluated using cartridges stored for up to 2 years at 25°C. Every vapor chip contains 13.5±0.9 mg of ground MC powder.No significant changes were observed in phytocannabinoid concentrations in ground cannabis inflorescence after 3 months of bulk storage in a polypropylene container and sealed in an aluminum foil pouch at 5°C. In contrast, significant changes in phytocannabinoid concentrations were found when ground inflorescence was stored in the cartridges at 25°C for 2 years. Specifically, CBGA, THCA, and total THC concentrations decreased from 0.097±0.023, 2.7±0.3, and 2.80±0.16 mg/chip at baseline to 0.044±0.007 (55% decrease), 1.50±0.27 (44% decrease), and 2.20±0.083 (21% decrease) mg/chip following 2 years, respectively, while CBN and THC concentrations increased from 0.005±0.005 and 0.44±0.11 mg/chip at baseline to 0.14±0.006 (2700% increase) and 0.88±0.22 (100% increase) mg/chip following 2 years, respectively. Storage at 30°C revealed a steeper change in phytocannabinoid concentrations within an even shorter period. Despite the significant change of relative cannabinoid composition within the cartridge, the actual THC dose present in the aerosol remained relatively stable throughout this period and within the dosage range of 500mcg±25% required for pharmaceutical-grade inhalers.MC powder in Syqe cartridges may be stored at room temperature for at least 2 years after production without affecting the aerosolized THC dose delivered to patients by more than ±25%. Future studies should analyze additional phytocannabinoids and terpenes in the cannabis inflorescence and assess the stability of different cannabis cultivars following storage in Syqe cartridges. Cannabis sativa L. (Cannabis) started following the report of O’Shaughnessy in his 1839 book on the therapeutic effects of Indian hemp , cannabidiolic acid (CBDA), and cannabigerolic acid (CBGA). As these compounds contain a chemically unstable carboxyl group (COOH), when heat is applied they are converted by a process known as decarboxylation into (-)-∆9-trans-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG), respectively , which is formed by THC oxidation and by cannabinolic acid (CBNA) decarboxylation, has been established as a marker for Cannabis cultivars are typically classified by their THC and CBD concentrations  and it serves as an indicator for the amounts of other molecules, such as minor phytocannabinoids, terpenes, and more that vaporize into the aerosol during the vaporization of a selected THC dose. In recent years, Syqe inhaler technology was evaluated in several clinical trials  and concluded that the best conditions for preserving the original phytocannabinoid and terpene contents of inflorescences throughout long storage periods are in the form of whole (non-ground) inflorescences at 4°C . Liquid chromatography-mass spectrometry (LC-MS)-grade formic acid was purchased from BioLab Ltd. . Phytocannabinoids analytical standards (>98% purity) of THC, THCA, CBD, CBDA, CBN, and CBGA were manufactured by Restek . Data were restricted to the requirements of the Israeli Medical Cannabis Authority regulations.Pseudomonas aeruginosa, Staphylococcus aureus, and bile-tolerant gram-negative bacteria).The MC inflorescence product “Bedrocan” , which contains about 22% THC and <1.0 CBD, was used in all experiments. It was free of pesticides, heavy metals , stalks, and foreign materials. Microbiological purity was also confirmed by the manufacturer  to form a fine and homogenized powder (particle diameter <1000 μm).Before processing, the MC powder is stored at 25°C (± 2°C) and relative humidity (RH) of 60% (± 5%) in a polypropylene container, sealed in an aluminum foil pouch . All MC handling — starting from processing until a cartridge is vacuum sealed — are performed in a clean-room environment. Manufacture begins with the grinding of whole VCs are manufactured by a dedicated machine, which allots 13.5±0.9 mg of ground MC powder into each VC. Sixty VCs are subsequently assembled into each tamper-proof cartridge which is then packaged in a vacuumed aluminum foil that protects it from exposure to light and oxygen until use. The Syqe inhaler releases a single VC for each inhalation and returns it to the cartridge after use. To ensure dose accuracy, each VC is used by the inhaler only once. In this stability study, only the 500 mcg THC dose was assessed.To assess the effect of refrigeration (5°C±2°C with up to 12% RH) on its stability during the handling and hold time, samples were taken from three separate MC powder batches and assayed at baseline (BL) and at 1, 2, and 3 months after grinding. Each sample was assayed for loss on drying and for the concentration of the major phytocannabinoids  test).n=9) and after 3 (n=6), 6 (n=9), 9 (n=8), 12 (n=7), 18 (n=4), and 24 (n=4) months. At each time point, samples of the ground inflorescence within the cartridges were analyzed for loss on drying and for the relative amount (mg) of each of the major phytocannabinoids . Cartridges from the same batches were also used for aerosolization by the Syqe inhaler at BL and at 6, 9, 18, and 24 months, and the THC content of the aerosolized dose was analyzed.Once cartridges are produced, they are stored, distributed, and used at room temperature. To assess the shelf life of such cartridges under those conditions, a long-term stability study was performed. To that end, cartridges were stored at 25°C (± 2°C) and relative humidity of 60% (± 5%) in a calibrated and certified stability chamber (Memmert Constant climate chamber HPP260). Batches were tested at BL  and relative humidity of 65% (± 5%). Three different batches were analyzed at BL and at 1, 2, 4, and 6 months of storage.Cannabis inflorescences, 100 mg of ground inflorescences from the same container bulk were weighed in duplicates and combined with 25 mL methanol. The samples were extracted for 40 min in an ultrasonic bath and centrifuged at 3000 rpm for 5 min. After dilution in 70/30 ACN/water + 0.1% formic acid solution, the samples were filtered through a 0.22 μm PP filter vial. The phytocannabinoids were analyzed by an U-HPLC system with a refrigerated auto-sampler, thermostatic column oven, and ultraviolet detector . Chromatographic separation was performed using a Waters ACQUITY UPLC 1.7-μm C18 column 2.1 × 150 mm, maintained at 30°C. The phytocannabinoids were separated using gradient elution with 0.1% (v/v) formic acid in double-distilled water (phase A) and 0.1% (v/v) formic acid in acetonitrile (phase B). A constant flow rate of 0.4 mL/min was employed throughout the analysis. The gradient profile varied from 70% to 100% B in 10.5 min, held for 0.5 min in these conditions, and returned to the initial conditions. The runtime was 14 min. Quantitation of the samples was performed according to calibration curves.To analyze the six main phytocannabinoids  of 2 (decarboxylation). Figure For THC and CBD, the concentrations of the acid and its neutral counterpart were summed and reported as the total content. For example, the concentration of total THC was calculated as total THC =(THCA ∗ 0.877) + THC, with 0.877 being the molar ratio between the two compounds that correct for a change in the mass of THCA due to the loss of COTo determine the amount of volatile matter (reported as loss on drying content) in the sample 1.1g ± 0.1g of mixed MC flowers sample or ground powder was weighted and spread in a thin layer over the moisture analyzer  sample pan. The test was performed in duplicates at 90°C for 10/20 min for ground MC powder or MC flowers raw material, respectively.p<0.05 level.The R software package (V.1.1.463) with tidyverse =21.56; p<0.001) was observed  = − 7.53; p<0.001), at 1 month compared to 3 months (t(8) = − 5.93; p<0.01) and at 2 months compared to 3 months (t(8) = − 3.43; p<0.05).A significant increase in loss on drying from 5.6±0.06% at BL to 6.6±0.29% at 3 months =2.58, p=0.13), CBGA =0.06, p=0.97), CBN =0.31, p=0.81), THC =0.006, p=0.99), THCA =0.03, p=0.99), Total THC =0.29, p=0.82), and total CBD =2.59, p=0.12). As expected, due to the baseline concentrations of CBD in this cultivar (<1%), CBD concentrations were mostly undetected at all time points. These results confirm that the conditions in which the ground inflorescences are preserved maintain stable phytocannabinoid concentrations.As shown in Fig. Cannabis in Syqe cartridges, a 2-year stability study was performed.To assess the effect of storage at room temperature on phytocannabinoids content in ground =0.68, p=0.66), in CBDA content =1.90, p=0.10) and total CBD content F=0.67, p=0.67). In contrast, significant decreases were observed in CBGA =8.96, p<0.001), THCA =11.26, p<0.001), and total THC contents =9.94, p<0.001). Tukey post hoc test showed a significant decrease in CBGA content between BL and 24 months (t(40)=5.33, p<0.001) and between 3 months and 24 months (t(40) = − 4.07, p<0.01). THCA content decreased significantly between BL and 24 months (t(40)=6.72, p<0.001), 3 months and 24 months (t(40)=4.79, p<0.001), 6 months and 24 months (t(40)=3.96, p<0.01), and between 9 months and 24 months (t(40)=3.54, p<0.05). Total THC content decreased significantly between BL and 24 months (t(40)=5.83, p<0.001), 3 months and 24 months (t(40)=4.55, p<0.001), 6 months and 24 months (t(40)=3.56, p<0.05), and between 9 months and 24 months (t(40)=3.17, p<0.05).As shown in Fig. =122.18, p<0.001 and F=6.04, p<0.001, respectively). Notably, CBN amounts increased between BL and 24 months (t(40) = − 22.18; p<0.001), 3 months and 24 months (t(40) = − 18.17, p<0.001), 6 months and 24 months (t(40) = − 17.08, p<0.001), 9 months and 24 months (t(40) = − 14.82, p<0.001), 12 and 24 months (t(40) = − 11.40, p<0.001), and between 18 and 24 months (t(40) = − 3.84, p<0.01). THC content increased between BL and 24 months (t(40) = − 5.40, p<0.001) and between 3 months and 24 months (t(40) = − 3.29. p<0.05). As expected, CBD was mostly undetected at all time points, as the pre-storage amount of its precursor, CBDA, was 0.006 mg/sample or less.CBN and THC content increased significantly =1.12, p=0.19), but CBGA, THCA, and total THC content decreased significantly =33.57, p<0.001; F=22.52, p<0.001; and F=6.46, p<0.01, respectively). Tukey post hoc test showed a statistically significant decrease in CBGA content between BL and 6 months (t(12)=11.17, p<0.001), 1 and 6 months (t(12)=5.68, p<0.001), 2 and 6 months (t(12)=6.76, p<0.001), and between 4 and 6 months (t(12)=3.55, p<0.05). A statistically significant decrease in THCA content was observed between BL and 6 months (t(12)=8.39, p<0.001), 1 and 6 months (t(12)=5.00, p<0.01), and between 2 and 6 months (t(12)=3.21, p<0.05). Total THC content decreased significantly between BL and 6 months (t(12)=4.36, p<0.01). Additionally, CBN and THC content increased significantly =106.72, p<0.001 and F=44.44, p<0.001, respectively). Tukey post hoc test showed a significant increase in CBN amount between BL and 6 months (t(12) = − 19.26, p<0.001), 1 and 6 months (t(12) = − 13.30, p<0.001), 2 and 6 months (t(12) = − 10.79, p<0.001), and between 4 and 6 months (t(12) = − 5.66, p<0.001). THC content increased between BL and 6 months (t(12) = − 11.81, p<0.001), 1 and 6 months (t(12) = − 7.52, p<0.001), and between 2 and 6 months (t(12) = − 3.51, p<0.05) =9.09, p<0.001). BL, 6-, 9-, 18-, and 24-months’ values were 484.76±31.28 mcg, 486.30±50.56 mcg, 563±22.06 mcg, 597.94±16.75 mcg, and 554.67±36.00 mcg, respectively. All aerosolized THC doses were well within the 500 mcg ±25% range  that apply to pharmaceutical-grade inhalers worldwide (18). Tukey’s post hoc test did not show any significant changes between the time points analyzed.In the framework of the 2-year stability study, THC dose was analyzed following aerosolization via the Syqe inhaler. The Syqe inhaler was set to aerosolize 500 mcg THC doses from the standard amount of MC contained within the VCs. Data are shown only for time points in which three or more batches were tested . As shown in Fig. Cannabis after 3 months of bulk storage in a polypropylene container and sealed in an aluminum foil pouch at 5°C. This suggests that grinding of the bulk product may be performed at least up to 3 months prior to cartridge manufacture, without a detectable effect on phytocannabinoid composition in ground Cannabis inflorescence.Our analysis did not find significant changes in the main phytocannabinoid concentrations in ground In contrast, significant changes in phytocannabinoid contents were found when the ground inflorescence stored in the cartridges at 25°C over a 2-year period. Specifically, CBGA, THCA and total THC concentrations decreased, while CBN and THC concentrations increased. These findings are in line with those reported by Milay et al. , who repAlthough the decrease in THCA and total THC amounts in ground inflorescence stored in the vacuum-sealed cartridges were expected to be correlated with a respective decrease in aerosolized THC doses, these remained within the 500 mcg ±25% range  as required for pharmaceutical grade inhalers  and vaporization (of THC and THCA). The amount of THC in the aerosol is the sum of vaporized THC and vaporized and decarboxylated THCA. If the efficiency of THC vaporization is greater than that of the vaporization and decarboxylation of THCA then an increase in THC concentration in the plant would more than compensate for an equal loss in THCA concentration. Such overcompensation may explain the observed relative stability of aerosol THC despite a loss of total THC, as observed during storage at 25°C.Cannabis production, storage, and transport. Furthermore, patients should be encouraged to keep the cartridges at lower temperatures whenever possible.Storage of MC in the cartridges at an elevated temperature of 30°C demonstrated a more rapid degradation and decarboxylation process to an extent that would shorten the shelf life of the product. These results show that such temperatures should be avoided during pharmaceutical grade Our findings suggest that storage at 5°C suffices to maintain phytocannabinoid concentration in MC powder packaged as detailed in this study for up to 3 months. Accordingly, storage by freezing may be unnecessary for this phase of cartridge manufacture, thereby potentially simplifying the logistics of the production process, and reducing its costs. Once a cartridge is produced, it may be kept at room temperature or at a lower temperature to maintain a shelf life of at least 2 years without a significant effect on the dose delivered by aerosolization. Refrigeration may be unnecessary at this stage, potentially simplifying the distribution and storage requirements for the entire supply chain, as well as being more convenient to patients. It is nonetheless important to avoid significant exposure of cartridges to a temperature significantly higher than 25°C, in order to prevent the reduction in phytocannabinoids content.Cannabis inflorescence is smoked in a cigarette — with or without a tobacco additive — provides an important improvement to the MC field. Future studies should analyze a much wider range of known phytocannabinoids and terpenes present in the Cannabis inflorescence and assess the stability of different Cannabis cultivars following storage in Syqe cartridges.Although significant changes in phytocannabinoid amounts occurred during extended storage (up to 2 years) of cartridges containing ground Bedrocan MC inflorescence, the therapeutic aerosolized THC dose remained relatively stable, within the dosage range of 500 mcg ±25% required for pharmaceutical-grade inhalers. This precise aerosolized THC dose, which have yet to be demonstrated in scientific studies by other devices or when


Pseudomonas aeruginosa has risen in the recent decade. The development of quorum sensing inhibition (QSI) is a promising approach for controlling Pseudomonas infection. Therefore, this study mainly aimed to investigate how a plant-source material inhibits QSI to produce an antipathogenic effect for fighting microbial infections. The QSI effect of Trigonella stellata was assessed by using Chromobacterium violaceum ATCC 12472 reporter strain. Trigonella stellata exhibited high QSI activity, and an ethanolic extract of T. stellata was prepared for phytochemical isolation of the most active QSI compound. Nine pure compounds were isolated and identified as kaempferitrin (1), soyasaponin I (2), β-sitosterol-3-O-glucoside (3), dihydromelilotoside (4), astrasikokioside I (5), methyl dihydromelilotoside (6), -4, 2′, 4′-trihydroxy-7-methoxy-4′-O-β-d-glucopyranosylisoflavan (7), -4, 2′, 4′-trihydroxy-7-methoxyisoflavan , and (+)-d-pinitol (9). These compounds were screened against C. violaceum ATCC 12472, and TMF exhibited a potent QSI. The effect of TMF at sub-minimum inhibitory concentrations (MICs) was assessed against P. aeruginosa virulence factors, including biofilm, pyocyanin formation protease and hemolysin activity. TMF induced significant elimination of QS-associated virulence behavior. In addition, TMF at sub-MICs significantly reduced the relative expression of lasI, lasR, rhlI, and rhlR compared with that in untreated cells. Furthermore, molecular docking was performed to predict structural basis of the QSI activity of TMF. The study demonstrated the importance of T. stellata as a signal modulator and inhibitor of P. aeruginosa pathogenesis.The number of deaths caused by multidrug-resistant The online version contains supplementary material available at 10.1007/s11274-022-03337-x. Pseudomonas aeruginosa is an opportunistic human pathogen that can be isolated from various ecosystems, including water and soil  system  is one of the most promising approaches for reducing the pathogenicity of Trigonella stellata is a palatable and nutraceutical herb that possesses anti-inflammatory activity, antioxidant and anticancer effects  seeds inhibited QS and biofilm formation in Aeromonas hydrophila and P. aeruginosa with activity associated with caffeine production by the plant  was then inoculated with 100 µL C. violaceum ATCC 12472 culture, and plates were allowed to completely solidify before wells were cut (10-mm diameter) in the agar using a cork borer. An aliquot of the concentrated T. stellata extract (100 μL) was added to the agar wells. After 24-h incubation at 28 °C, the extent of violet color around the well was evaluated and inhibition of this was determined as a positive QSI activity. Solvent was used as a negative control . The subfractions of comparable spots were combined to yield eight fractions (TE1 to TE8). Fraction TE2 (185 mg) was chromatographed over RP-18 column and isocratically eluted with CH3OH:H2O (1:1) to provide two pure compounds: 6 and 7 .The ethyl acetate fraction (1.53 g) was spotted onto a 2.5 × 95 cm silica gel column and eluted with a mobile phase consisting of ethyl acetate:CHCl3OH:H2O (1:1 then 6:4) to obtain 11 fractions (TC1 to TC11). Fraction TC5 (118 mg) was applied to a standard silica gel column (2 × 95 cm) and eluted with ethyl acetate:CHCl3 (1:1) followed by ethyl acetate:CHCl3:CH3OH:H2O  to provide the purified compound 8 (10 mg). Fraction TC9 (529 mg) was chromatographed over a silica gel glass column (1.5 × 90 cm) and eluted with the gradients of ethyl acetate:CHCl3:CH3OH:H2O  to yield compound 3 (23 mg).The methylene chloride extract (1.1 g) was purified using vacuum liquid chromatography (VLC) over RP-18 silica (20 × 2 cm) and eluted with two gradients of CH2O:CH3OH , 1 L each, to yield 11 fractions (TB1 to TB11). Compound 9 (31 mg) was precipitated from the methanol-concentrated fraction TB4 (179 mg). The purification of two compounds 1 (8.1 mg) and 4 (9.8 mg) was achieved through column chromatography of fraction TB2 (108 mg) over RP-18. A white powder of purified compound 2 fraction TB6 (182 mg) was applied to an RP-18 column (2 × 30 cm) and eluted with gradients of H2O:CH3OH . Compound 5 (6.2 mg) was obtained through the RP-18 column fractionation of TB7 (110 mg) and isocratic elution with H2O:CH3OH (6:4).The butanol extract (2.5 g) was passed over a VLC packed with RP-18 silica (25 × 4 cm) and eluted with the following mobile phase compositions H2SO4:C2H5OH (10:90). All the chromatographic procedures were performed using analytical grade solvents .One- and two-dimensional (1D and 2D) NMR experiments were performed using a Varian 400 MHz, Bruker DRX-500, or Bruker Avance III 600 and 400 MHz spectrometer with deuterated solvents of pyridine, methanol, or dimethylsulfoxide (DMSO); the solvent signals served as an internal reference for adjustment. The Agilent Technologies 6200 series was used to record the positive and negative ion modes of compound masses. An AUTOPOL IV Automatic Polarimeter  was used to detect the specific rotations of the pure compounds. In addition, the Varian Cary 50 Bio UV–Vis spectrophotometer was used to measure the UV spectra. The silica gel  and RP-18  were used for the preparation of column chromatography. TLC plates were monitored with silica gel F254 sheet (Fluka) or RP-18 (Merck). The spots of the tested compounds were visualized at 254 nm (UV) and then sprayed with vanillin (1% w/v) (Sigma) prepared in concentrated HT. stellata was assessed using C. violaceum ATCC 12472. Compounds were dissolved to 5 mg/mL in DMSO. Aliquots of 50 μL of each compound were applied in each well (10 mm diameter), and then the plates were then incubated at 28 °C. After 24 h, the inhibition of induced violacein production around the wells was examined. DMSO was used as a control. Compounds possessing potential QSI with inhibition of violacein pigment production were estimated for their effect on virulence factors.QSI activity of the nine isolated compounds from 6 CFU/well of actively growing P. aeruginosa strains  of the active compound TMF was calculated using the microtiter plate assay technique. Each well received 100 µL Mueller Hinton broth, and the TMF compound was serially diluted in twofold serial concentrations  and inoculated with 1 × 10P. aeruginosa was investigated using the pour plate method after adding 1/2 MIC of TMF and comparing with that of the untreated culture. A volume of the overnight culture (1 mL) was collected and diluted 1:10. Diluted cultures were subcultured in molten LB agar, and the colonies were counted after overnight incubation at 37 °C. Pseudomonas aeruginosa growth curves (treated and nontreated) at an optical density (OD) at 600 nm were estimated at different time intervals.The viability of P. aeruginosa strains were quantified after treatment by 1/2 and 1/4 MICs of TMF in triplicates. Assessment of virulence factors was performed with and without TMF  were distributed into the plates, which were then incubated at 37 °C for 24 h. Wells were washed three times with 200 μL of physiological saline before the attached bacteria were fixed for 15 min with 150 μL of absolute methanol. After emptying and drying the plates, the bacterial cells were stained with 150 μL of 1% (w/v) crystal violet. The excess stain was washed away, and the dye was dissolved in glacial acetic acid at 33% (v/v). The absorbance of the dyed cells was determined at 490 nm , and MgCl2 0.14% (w/v))  and incubated with agitation at 200 rpm at 37 °C. The cultures were then centrifuged at 3000 rpm for 10 min at 4 °C to remove the cells. Pyocyanin was extracted with 3 mL of chloroform and vortexed until the color changed to greenish blue. The mixture was centrifuged at 3000 rpm for 10 min; 1 mL of 0.2 M HCl was added, and the mixture was shaken until the blue color turned pink. The absorbance of the pink layer was measured at 520 nm, and the concentration of pyocyanin in µg/mL was estimated using the formula: absorbance: absorbance × 17.072 , KP. aeruginosa untreated and treated (1/2 and 1/4 MICs of TMF) culture-free supernatant was determined. In brief, 500 μL of supernatant was mixed with 700 μL of washed erythrocytes and incubated for 2 h at 37 °C. The reaction mixture was centrifuged at 8000 × g for 15 min at 4 °C  was estimated using the skimmed milk assay technique. The assay was performed by adding 1 mL of 1.25% (w/v) skimmed milk to 500 μL of culture supernatants and incubating for 1 h at 37 °C. The OD was measured at 600 nm and compared with that of the blank (skimmed milk). The reduction in the OD600 of the treated cells was compared with that of the untreated cells under the same conditions , according to the manufacturer’s instructions. The SensiFAST™ cDNA Synthesis Kit  was used to synthesize complementary DNA. The Rotor-Gene Q thermocycler  was used to perform RT-PCR. The primers described in Table rpoD housekeeping gene, and the relative expression was determined using the formula 2−ΔΔCT (Livak and Schmittgen The effect of TMF on the expression of QS regulatory genes P. aeruginosa LasR ligand-binding domain (Protein Data Bank ID: 2UV0) to evaluate the binding mechanism with LasR; this structure used 3-oxo-C12-HSL as an AI  (Khan and Hossain O-β-d-glucopyranosylisoflavan (7) , -4, 2′, 4′-trihydroxy-7-methoxyisoflavan , and (+)-d-pinitol (9)   for 24 h at 37 °C. The bacterial count from untreated cultures was 112, 94, 126, 113, 98, 133, and 103 × 107 CFU/mL for Ps.A11, Ps.A12, Ps.A13, Ps.A16, PA14, PAO1, and PAO-JP2, respectively. The count from the treated cultures was 108, 95, 121, 107, 96, 129, and 100 × 108 CFU/mL for Ps.A11, Ps.A12, Ps.A13, Ps.A16, PA14, PAO1, and PAO-JP2, respectively. There was no significant difference between the bacterial count between untreated and treated cultures. Furthermore, no effect on bacterial growth was observed in cultures treated with 1/2 MIC of TMF in comparison with that in untreated cultures .The antimicrobial effect of TMF on Treatment with both the sub-MICs of TMF compound (1/2 and 1/4) significantly inhibited formation of biofilm and pyocyanin and activity of hemolysin and protease in Ps.A11, Ps.A12, Ps.A13, Ps.A16, PA14, and PAO1 as compared with that in untreated cultures. PAO-JP2 had the lowest activity among the tested strains for all virulence factors.A sub-MIC of TMF 256 μg/mL) significantly reduced biofilm formation in Ps.A11, Ps.A12, Ps.A13, Ps.A16, PA14, and PAO1 by 76.5%, 75.3%, 79.6%, 76.3%, 81%, and 81.8%, respectively. Furthermore, 1/4 MIC significantly decreased biofilm by 77.2%, 73.7%, 79.6%, 74.9%, 80%, and 77.3% in Ps.A11, Ps.A12, Ps.A13, Ps.A16, PA14, and PAO1, respectively  (p ≤ 0.001), also the 1/4 MIC of TMF significantly decreased pyocyanin by 77.7%, 68.3%, 73.4%, 65.5%, 57.7%, and 61.8%, respectively, in these strains as well  significantly inhibited pyocyanin production in all tested p ≤ 0.001) in isolates Ps.A11, Ps.A12, Ps.A13, Ps.A16, PAO1, and PA14. The 1/2 MIC decreased hemolysin production by Ps.A11, Ps.A12, Ps.A13, Ps.A16, PAO1, and PA14 by 53.8%, 53.8%, 40.8%, 52.1%, 61.8%, and 56%, respectively, whereas the 1/4 MIC (128 μg/mL) lowered hemolysin activity by 48.9%, 52.2%, 41.2%, 47.4%, 56.1%, and 55%, respectively  lowered the mRNA level of lasI and lasR by 64.5% and 34%, respectively, in the standard strain PAO1 compared with that in the untreated cultures  compared with that in the untreated PAO1 -l-homoserine lactone (AHL). In addition, other signaling molecules, such as 3-oxo-C10-HSL and 3-oxo-C12-HSL, can enhance violacein production in this bacterium , silver nanoparticles  resulted in a significant reduction in pyocyanin levels, indicating inhibition of the C4-HSL and pqsR signaling molecules of the RhlI/R and PQS signaling systems, respectively.The RhlI/R and PQS signaling systems of Pseudomonas aeruginosa produces extracellular enzymes such as protease and hemolysin that allow the bacteria to spread inside host tissues and to resist host immunity . The scoring functions and hydrogen bonds produced by the LasR active site with the surrounding amino acids could be used to determine the binding mode, affinity, and orientation of TMF . In 2017, pyridoxal lactohydrazone was discovered to reduce QS-related virulence factors in P. aeruginosa and to establish hydrogen bonds with Thr75, Trp60, Ser129, and Arg61 in the active site of LasR . Furthermore, these in vitro results were confirmed using a molecular docking study that calculated the binding affinity of TMF with LasR. Overall, our study shows that TMF can combat the pathogenesis and dissemination of Pseudomonas infection through QS interference. Further studies need to be performed to evaluate the in vivo antipathogenic effects of TMF.This study highlights the importance of the medicinal plant Supplementary file1 (PDF 2195 kb)Below is the link to the electronic supplementary material.


Arabidopsis thaliana is an important and long-established model species for plant molecular biology, genetics, epigenetics, and genomics. However, the latest version of reference genome still contains a significant number of missing segments. Here, we reported a high-quality and almost complete Col-0 genome assembly with two gaps (named Col-XJTU) by combining the Oxford Nanopore Technologies ultra-long reads, Pacific Biosciences high-fidelity long reads, and Hi-C data. The total genome assembly size is 133,725,193 bp, introducing 14.6 Mb of novel sequences compared to the TAIR10.1 reference genome. All five chromosomes of the Col-XJTU assembly are highly accurate with consensus quality (QV) scores > 60 (ranging from 62 to 68), which are higher than those of the TAIR10.1 reference (ranging from 45 to 52). We completely resolved chromosome (Chr) 3 and Chr5 in a telomere-to-telomere manner. Chr4 was completely resolved except the nucleolar organizing regions, which comprise long repetitive DNA fragments. The Chr1 centromere (CEN1), reportedly around 9 Mb in length, is particularly challenging to assemble due to the presence of tens of thousands of CEN180 satellite repeats. Using the cutting-edge sequencing data and novel computational approaches, we assembled a 3.8-Mb-long CEN1 and a 3.5-Mb-long CEN2. We also investigated the structure and epigenetics of centromeres. Four clusters of CEN180 monomers were detected, and the centromere-specific histone H3-like protein (CENH3) exhibited a strong preference for CEN180 Cluster 3. Moreover, we observed hypomethylation patterns in CENH3-enriched regions. We believe that this high-quality genome assembly, Col-XJTU, would serve as a valuable reference to better understand the global pattern of centromeric polymorphisms, as well as the genetic and epigenetic features in plants. Arabidopsis thaliana Col-0 genome sequence was published in 2000 A. thaliana. However, centromeres, telomeres, and nucleolar organizing regions (NORs) have been either misassembled or not even been sequenced yet due to the enrichment of highly repetitive elements in these regions e.g., centromere or telomere regions, however, remain mostly unassembled due to the limitations in read length and the error rate associated with sequencing of long reads. Although ONT sequencing has overcome read length limitation and can generate ultra-long reads (longest > 4 Mb) (https://nanoporetech.com/products/promethion), the associated 5%–15% per base error rate A. thaliana Col-0 genome, but the consensus quality (QV) scores of all five chromosomes, ranging from 41 to 43, were lower than those of the reference TAIR10.1 (ranging from 45 to 52) The A. thaliana centromeres contain megabase-sized islands of 178-bp tandem satellite DNA repeats (CEN180) A. thaliana reference genome assemblies Centromeres mainly consist of satellite DNAs and long terminal repeat (LTR) retrotransposons A. thaliana genome assembly, we introduced a bacterial artificial chromosome (BAC)-anchor replacement strategy to our assembly pipeline and generated the Col-XJTU genome assembly of A. thaliana. We completely resolved the centromeres of Chr3, Chr4, and Chr5, and partially resolved the centromeres of Chr1 and Chr2. The Col-XJTU assembly of A. thaliana genome was found to be highly accurate with QV scores greater than 60, which were obviously higher than those of TAIR10.1 and another recently deposited assembly To obtain T2T Arabidopsis-type telomeric repeat unit (CCCTAAA/TTTAGGG) on one end, while two contigs had the 45S rDNA units on one end , which was estimated using GenomeScope v. 1.0 k-mer 17 histogram computed by Jellyfish v. 2.3.0 We assembled ONT long reads using NextDenovo v. 2.0, and initially generated 14 contigs (contig N50 = 15.39 Mb) A. Of the one end A. Contig one end A. We pol one end A. To fur one end B and C.  one end B. To gua one end C. The fi one end  and a reelements . The hetA. thaliana centromeres were surrounded by transposon-enriched sequences rather than protein-coding gene-enriched sequences (The assembly sizes of Col-XJTU centromere 1 (CEN1), CEN2, CEN3, CEN4, and CEN5 were 3.8 Mb, 3.5 Mb, 4.0 Mb, 5.5 Mb, and 4.9 Mb, respectively . The sizequences C.A. thaliana genome compared to TAIR10.1 (contig N50 = 11.19 Mb) (The Col-XJTU assembly (contig N50 = 22.25 Mb) improved the contiguity of the 1.19 Mb) , and we 1.19 Mb) . Benchma1.19 Mb) . The syn1.19 Mb)  but with1.19 Mb) . The QV 1.19 Mb) , which a1.19 Mb) . Moreove1.19 Mb) , largely1.19 Mb) .n = 33) and telomeres (n = 2)  were lifted-over from TAIR10.1  using Liftoff . We then (n = 2) A. Only 1 (n = 2) . Interes (n = 2) , especia (n = 2) . The hig (n = 2) , indicatA. thaliana was estimated using physical mapping and cytogenetic assays; however, such estimation resulted in the generation of incorrectly annotated and unknown regions, such as 5S rDNA and CEN180 repeat regions in situ hybridization and physical mapping Previously, the centromere composition of We observed that CEN1, CEN2, CEN3, and CEN4 contained highly similar CEN180 arrays , and theA functional region of centromere is defined by the binding of epigenetic modifications with CENH3 Traditionally, long-read sequencing technologies commonly suffer from high error rates A. thaliana centromeres is 2–5 folds larger than that of the rice centromeres (0.6–1.8 Mb) A. thaliana centromeres. We combined the dual long-read platforms of ONT ultra-long and PacBio HiFi to produce the high-quality A. thaliana Col-XJTU genome with only two gaps in CEN1 and CEN2. We assembled a 3.8-Mb-long CEN1, which is smaller than the 9-Mb region estimated by physical mapping A. thaliana genome was deposited with a ∼ 5-Mb-long CEN1 sequence, which is still smaller than the physical map size k-mers (SUNKs) assembly method The size of A. thaliana[36]. Our high-quality centromere assembly of A. thaliana reveals that the CEN180 arrays enriched with CENH3 occupancy are hypomethylated compared to the pericentromeric regions. Although the primary function of centromeres is conserved between animal and plant kingdoms, the centromeric repeat monomers are highly variable in terms of sequence composition and length, and little sequence conservation is observed between species Diverse methylation patterns have been observed in the centromere sequences of two human chromosomes upon completion of the human genome A. thaliana. This genome will serve as the foundation for further understanding molecular biology, genetics, epigenetics, and genome architecture in plants.In conclusion, our novel assembly strategy involving the combination of ONT long reads and HiFi reads leads to the assembly of a high-quality genome of the model plant A. thaliana accession Col-0 was obtained from the Shandong Agricultural University, China as a gift. The A. thaliana seeds were placed in a potting soil and then maintained in a growth chamber at 22 °C with a 16 h light/8 h dark photoperiod and a light intensity of 100–120 µmol·m−2·s−1. Young true leaves taken from 4-week-old healthy seedlings were used for sequencing.The https://samtools.github.io/bcftools), and tabix v. 1.9 The Illumina short read data of our Col-0, public wild-type Col-0 (JGI Project ID: 1119135), and another accession AT1741 (SRA: ERR2173372) were mapped to the reference genome TAIR10.1 (RefSeq: GCF_000001735.4). We used a series of software packages, including bwa v. 0.7.17-r1188 DNA was extracted using the Qiagen Genomic DNA Kit  following the manufacturer’s guidelines. Quality and quantity of total DNA were evaluated using a NanoDrop One UV–Vis spectrophotometer  and Qubit 3.0 Fluorometer , respectively. The Blue Pippin system  was used to retrieve large DNA fragments by gel cutting.For the ultra-long Nanopore library, approximately 8–10 µg of genomic DNA was selected (> 50 kb) with the SageHLS HMW library system (Sage Science), and then processed using the Ligation sequencing 1D Kit  according the manufacturer’s instructions. DNA libraries (approximately 800 ng) were constructed and sequenced on the PromethION (Oxford Nanopore Technologies) at the Genome Center of Grandomics . A total of 56.54 Gb of ONT long reads with ∼ 388× coverage were generated including ∼ 177× coverage of ultra-long (> 50 kb) reads. The N50 of ONT long reads was 46,452 bp, and the longest reads were 495,032 bp.https://github.com/Nextomics/NextDenovo) was used to assemble the ONT long reads with parameters: ‘read_cutoff = 5k’ and ‘seed_cutoff = 108,967’. Nextpolish v. 1.3.0 The long-read assembler NextDenovo v. 2.0 (https://github.com/lh3/gfatools) was used to convert sequence graphs in the GFA to FASTA format.SMRTbell libraries were constructed according to the standard protocol of PacBio using 15 kb preparation solutions . The main steps for library preparation include: 1) genomic DNA shearing; 2) DNA damage repair, end repair, and A-tailing; 3) ligation with hairpin adapters from the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences); 4) nuclease treatment of SMRTbell library with SMRTbell Enzyme Cleanup Kit; and 5) size selection and binding to polymerase. In brief, the 15 µg genomic DNA sample was sheared by gTUBEs. Single-strand overhangs were then removed, and DNA fragments were damage repaired, end repaired, and A-tailed. Then, the fragments were ligated with the hairpin adapters for PacBio sequencing. The library was treated with the nuclease provided in the SMRTbell Enzyme Cleanup Kit and purified by AMPure PB Beads. Target fragments were screened by BluePippin (Sage Science). The SMRTbell library was then purified by AMPure PB beads, and Agilent 2100 Bioanalyzer  was used to detect the size of the library fragments. Sequencing was performed on a PacBio Sequel II instrument with Sequencing Primer V2 and Sequel II Binding Kit 2.0 at the Genome Center of Grandomics. A total of 22.90 Gb of HiFi reads with ∼ 157× coverage were generated, and N50 of the reads was 15,424 bp. HiFi reads were assembled using hifiasm v. 0.14-r312 Hi-C library was prepared from cross-linked chromatins of plant cells using a standard Hi-C protocol; the library was then sequenced using Illumina NovaSeq 6000. A total of 21.14 Gb of Hi-C reads with ∼ 158× coverage were generated. The Hi-C sequencing data were used to anchor all contigs using Juicer v. 1.5 We introduced a BAC-anchor strategy to fill the remaining gaps in ONT-HiC assemblies. Briefly, for each gap, we first identified two BAC sequences flanking the gap locus that were aligned concordantly (identity > 99.9%) to both the ONT-HiC assembly and a HiFi contig, and then replaced the gap-containing contigs with corresponding HiFi contigs. We used the same method to polish ONT-HiC assemblies with HiFi contigs. The BAC sequences we used as anchors are list in (QV/−10)) × 100 https://github.com/mrvollger/aln_plot) command: bash cmds.sh CEN CEN.fa 5000.Two genome assemblies were aligned against each other using nucmer v. 4.0.0 (-c 100 -b 500 -l 50) https://github.com/skoren/bacValidation) with default parameters, which recognizes a BAC as ‘resolve’ within the assembly with 99.5% of the BAC length to be aligned to a single contig. BAC libraries were downloaded from European Nucleotide Archive (ENA), and the BACs used to validate five chromosomes are listed in We validated the assemblies using bacValidation (We applied TandemTools http://www.repeatmasker.org) , and 5S rDNA was detected by TideHunter v. 1.4.3 (https://github.com/yangao07/TideHunter) and predicted by rRNAmmer v.1.2 The software Liftoff v. 1.6.1 (-mm2_options = ‘-a --end-bonus 5 --eqx -N50 -p 0.5’) We first used QUAST v. 5.0.2 We chose seven tissues for gene expression analysis http://www.bx.psu.edu/~rsharris/lastz/) LASTZ  were mapped to the Col-XJTU assembly using the ‘mem’ algorithm of BWA Nanopolish v. 0.13.2 with the parameters ‘call-methylation --methylation cpg’ was used to measure the frequency of CpG methylation in raw ONT reads. The ONT reads were aligned to whole-genome assemblies via Winnowmap v. 2.0 https://ngdc.cncb.ac.cn/biocode/tools/BT007246).SNP calling and ChIP-seq analysis pipelines are available for public use at BioCode (https://ngdc.cncb.ac.cn/gwh. The genome annotation has been deposited in https://dx.doi.org/10.6084/m9.figshare.14913045. The raw sequencing data for the PacBio HiFi reads, ONT long-reads, Illumina short reads, and Hi-C Illumina reads have been deposited in the Genome Sequence Archive https://ngdc.cncb.ac.cn/gsa.The whole-genome sequence data reported in this study have been deposited in the Genome Warehouse Bo Wang: Methodology, Software, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Xiaofei Yang: Methodology, Supervision, Writing - review & editing. Yanyan Jia: Resources, Methodology. Yu Xu: Software, Formal analysis. Peng Jia: Software, Formal analysis. Ningxin Dang: Methodology, Formal analysis. Songbo Wang: Methodology, Formal analysis. Tun Xu: Formal analysis. Xixi Zhao: Formal analysis. Shenghan Gao: Methodology. Quanbin Dong: Resources. Kai Ye: Conceptualization, Methodology, Supervision, Funding acquisition, Writing - review & editing. All authors have read and approved the final manuscript.The authors have declared no competing interests.


Mucor circinelloides and M. hiemalis were tested for their ability to produce xylanase from tangerine peel by submerged fermentation. Experiments on five variables were designed with Box–Behnken design and response surface methodology. Analysis of variance was exercised, the xylanase output was demonstrated with a mathematical equation as a function of the five factors, and the quixotic states for xylanase biosynthesis was secured. In addition, xylanase was partially purified, characterized, and immobilized on calcium alginate beads. The optimum parameters for xylanase production by M. circinelloides and M. hiemalis were consisted of incubation temperature (30 and 20°C), pH value (9 and 7) incubation period (9 and 9 days), inoculum size (3 and 3 mL), and substrate concentration (3 and 3 g/100 mL), respectively. M. circinelloides and M. hiemalis demonstrated the highest xylanase activities after RSM optimization, with 42.23 and 35.88 U/mL, respectively. The influence of single, interchange, and quadratic factors on xylanase output was investigated using nonlinear regression equations with significant R2 and p values. The partial purification of M. circinelloides and M. hiemalis xylanase yielded 1.69- and 1.97-fold purification, and 30.74 and 31.34% recovery with 292.08 and 240.15 U/mg specific activity, respectively. Partially purified xylanase from M. circinelloides and M. hiemalis demonstrated the highest activity at neutral pH and 60 and 50°C, respectively. The immobilized M. circinelloides and M. hiemalis xylanase retained 84.02 and 79.43% activity, respectively. The production of xylanase from M. circinelloides and M. hiemalis utilizing RSM is deemed profitable for the decomposition of the agro-industrial wastes.Xylan is the primary hemicellulosic polymer found in lignocellulosic agricultural wastes and can be degraded by xylanase. In the current research,  Biotechnological processes on filamentous fungi have enabled the industrial utilization of their ability to produce valuable enzymes due to their ease of propagation and increased production of extracellular enzymes with specific characteristics such as stability over a wide range of pH and temperature –3.Mucor, are mainly saprobic fungi that can grow well on various agro-industrial wastes with the potential to produce extracellular hydrolytic enzymes that can be utilized in a variety of industrial applications [The class Zygomycetes, order Mucorales, including the genus ications .β-1,4-linked xylopyranose monomers and comprises a significant renovatable biocluster containing up to 20-35% of the dry mass of agricultural residues [β-1,4-xylanases and exo-β-1,4-xylanases) are a candidate to degrade xylan into xylose and xylooligomers [The elevated dilation of agricultural junks apostasy has resulted in the cumulation of significant amounts of lignocellulosic wastes across the globe . Lignoceresidues . Two mailigomers . Xylanasligomers , and juiligomers .The amount of fungal enzyme manufactured is determined by the conditions of the fermentation process and the need to optimize these conditions for low enzyme output cost . The funMucor spp. using agro-industrial by-products as a cheap substrate in submerged fermentation conditions. The optimization of xylanase production, as well as characterization and immobilization of partially purified xylanase, was investigated.This study aimed to evaluate the production of high-value xylanase by Mucor circinelloides AUMC 6696.A (Accession no. MT509983) and M. hiemalis AUMC 6031 (Accession no. MT365791) [T365791)  were uti4, 1; NaNO3, 2; MgSO4.7H2O, 0.5 and CaCl2.2H2O, 0.5) was supplemented with 10 g/L of xylan powder as a carbon source for xylanase production. The pH was set to 7, and the impregnated plates were preserved at 28 ± 2°C for five days and then screened for enzymes production [Czapekʼs agar medium (g/L: KH2POoduction . After i7 spores/mL) were utilized to inoculate 250-mL flasks holding 100 mL submerged broth. The impregnated flasks were preserved at 28 ± 2°C for seven days on both shaking and static conditions. Subsequently, broth media were centrifuged, and supernatants were maintained for further enzymatic analysis.Pomegranate peel, tangerine peel, and wheat straw were desiccated at 65°C for 24 h, then squelched to grist, and used as substrates (10 g/L) in Czapekʼs mineral salts broth. The broth containing flasks was adjusted to pH 7 and autoclave sterilized. The strains' spore suspensions  was determined corresponding to the subsidiary equation:k is the digit of factors and C0 is the digit of central points equal to 6.Response surface methodology (RSM) tactic using Box–Behnken design (BBD) was exercised to determine the optimum factors for boosted xylanase production, including A, temperature; B, pH; C, incubation period; D, inoculum size; and E, substrate concentration . Forty-sY) was evaluated by utilizing a second-order polynomial equation to determine the quixotic states of the xylanase biosynthesis. Y (response variable); β0 (intercept); β1, β2, β3, β4, and β5 (linear coefficients); β11, β22, β33, β44, and β55 (square coefficients); β12, β13, β14, β15, β23, β24, β25, β34, β35, and β45 (interaction coefficients); and A, B, C, D, E, A2, B2, C2, D2, E2, AB, AC, AD, AE, BC, BD, BE, CD, CE, and DE (levels of independent variables). The corresponding coefficients of variables, interaction variables, and contour graphs were obtained by Minitab 19® software. By analyzing the regression equation and constructing the response plots, the ideal values of the tested variables were secured. The coefficient of limitation R2 was used to express the fineness of profit of the polynomial equation, and the (F) test was used to determine its statistical significance level.The impact of variables on the simulation  was subjoined to the enzyme solution until the volume ratio between enzyme solution and acetone reached 1 : 1; 1 : 2; 1 : 3; 1 : 4, and 1 : 5 (v/v). The solution was left at -20°C overnight to allow protein precipitation. The precipitates were gathered by centrifugation at 10000 rpm for 15 min and resuspended in a small volume of . These samples were used for determining the activity of xylanase, purification factor, and enzyme recovery yield . Protein+, Mg2+, Ba2+, and Ni2+) for 1 h at optimum temperature and pH. Xylanase activity was assessed after processing with diverse detergents comprising tween 80 and 20 at concentrations of 1 and 5% v/v, urea (1 and 5% w/v), and Na2CO3 (50 and 75 mM) compared to control (100% activity) [The optimum temperature for partially purified xylanase activity was determined to be in the range of (30-90°C), and the thermal stability was determined after premaintaining the enzyme at each temperature degree for one hour before screening. Ideal pH estimation was carried out at optimum temperature utilizing various buffers with values (3-11), and the pH stability was assessed after preserving these pH values for 1 h before the screening. In addition, xylanase activity was estimated after maintaining the enzyme with different metal ions (10 mM of Kctivity) , 26.2 at 4°C. Calcium alginate beads were rinsed by double-distilled H2O to remove unstriated enzyme units. Then, the beads were desiccated and stored in phosphate buffer. The entrapped beads were activated using glutaraldehyde for covalent binding of xylanase onto the beads [Ai is the immobilized enzyme activity and Af is the free enzyme activity.The immobilization of partially purified xylanase was performed by combining enzyme solution with an equal volume of 3.0% sodium alginate solution. Calcium alginate beads were formalized by adding the mixture drop-wise in 0.2 M CaClhe beads , and thehe beads . (4)ImmoA scanning electron microscope  was utilized to examine the outer surface forms of calcium alginate beads before and after xylanase immobilization.All tests and measurements were repeated three times. Using the SPSS, software program (version No. 16), and one-way ANOVA, the values were expressed as the mean ± SD at the 0.05 significance level.M. circinelloides and M. hiemalis had a high potential to produce xylanase qualitatively on a solid assay medium. They were then quantitatively assayed under SmF and produced 21.77 and 15.28 U/mL, respectively, on the tangerine peel as a substrate under static condition  and M. hiemalis (35.88 U/mL) was obtained from runs No. 27 to 46, respectively. The run No. 27 for M. circinelloides consisted of incubation temperature (30°C), pH value (9), the incubation period (9 days), inoculum size (3 mL), and substrate concentration (3 g/100 mL). In comparison, the run No. 46 for M. hiemalis consisted of incubation temperature (20°C), pH value (7), the incubation period (9 days), inoculum size (3 mL), and substrate concentration (3 g/100 mL).The highest xylanase activity of both M. circinelloides and M. hiemalis is shown in Tables p value <0.05 were deemed significant. The model F value of 58.10 and 17.59 for M. circinelloides and M. hiemalis, respectively, indicated that the model is significant. Values of “Prob > F” <0.05 pointed model terms are significant. In this case, A, B, C, E, A2, B2, D2, E2, AB, AC, AE, BC, BE, CD, and CE are significant model terms for M. circinelloides, while for M. hiemalis, A, B, C, D, E, A2, B2, AC, AD, AE, BE, CD, CE, and DE are significant model terms. The “Lack of Fit F-value” of 0.83and 0.58for M. circinelloides and M. hiemalis, respectively, indicated that the lack of fit is insignificant concerning the pure error. Nonsignificant lack of fit is proper for the model to be convenient. The resulted multiple correlation coefficient  that nigh to 1 betokened preferable interconnection between experimental and predicted values and elucidated the model accuracy with upgrade response.Analysis of variance (ANOVA) for the xylanase quadric model of M. circinelloides  was obtained via crude filtrate precipitation with acetone at ratio 1 : 4  and decreased gradually at 50-30°C and 70-90°C, while xylanase activity of M. hiemalis was highly active at 50°C  and decreased gradually at 40-30°C and 60-90°C. The relative xylanase stability from both M. circinelloides and M. hiemalis was high at 30°C and decreased in the range of 40-90°C  (After incubation with 10 mM Kion (5%) .M. circinelloides enzyme had an immobilization yield of 84.02%, while M. hiemalis enzyme had a yield of 79.43%  by utilizing rice straw waste, while orange peel exhibited low xylanase production (0.17 U/mL). Cui and Zhao [Penicillium sp. WX-Z1 demonstrated a gradual rise in wheat bran concentration. Statistical analysis of xylanase production from Penicilliumoxalicum ZH-30 by RSM showed that the linear, quadric terms and initial pH and temperature interaction significantly impacted the optimal conditions for raised xylanase biosynthesis at pH 7.38 and temperature 31.1°C. Under optimal conditions, the portended and experimented xylanase activities were 14.33 and 14.50 U/mL, respectively [In the current research, the low onfirmed . For xylectively . Atalla ectively  reportedand Zhao  highlighectively .Aspergillus nigerAN-13 using wheat bran under submerged fermentation. In submerged fermentation with corncob, Aspergillus niger KIBGE-IB36 was found to be a high producer of xylanase [Aspergillus niger strain BG on 84% humidified wheat bran with a pH of 2.5 at 37°C and incubation for 66 h. Statistical analysis of xylanase output by Aspergillus foetidus in submerged fermentation with soybean scraps revealed that variables pH and the interaction of pH and temperature influenced xylanase biosynthesis, with good xylanase activity (13.98 U/mL) at pH 7.0, 28 °C, and 120 rpm for 168 hours [Fusarium sp. BVKT R2 demonstrated that the optimal conditions were sorbitol 1.5%, yeast extract 1.5%, pH of 5.0, temperature of 32.5 °C, and shaking of 175 rpm.Box–Behnken design statistical analysis by Cao et al.  demonstrxylanase  and diffxylanase . RSM metxylanase  reported68 hours . Respons68 hours  for maxiAspergillus niger 3-fold and 1.41-fold purification was attained with about 6.2% yield, and the highest activity of the purified xylanase was observed at pH 6 and 50°C. The produced xylanase exhibited high thermal and pH stability, with more than 90% residual activity between 30 and 40°C and pH 3-9 after incubation of 24 h, with half-lives of 30 min at 50 and 60°C [P. oxalicum ZH-30. Supplementation of Tween 80 as additional surfactant improved (72.4 ± 1.42 U/g) the titer of xylanase on sugarcane bagasse by T. viride-IR05 [2+ with carboxylate groups of sodium alginate helm to formulate mechanically firm grids of alginate gel that fulfill the demands for efficient hydrogel systems acting as carriers of bioactive molecules in several industrial applications [Talaromyces thermophilus xylanase covalently bound by glutaraldehyde to chitosan, chitin, amberlite, duolite, florisil, and gelatin gave immobilization yield of 89.0, 87.8, 89.3, 81.1, 96.2, and 98.8%, respectively [High temperature increases the solubility of reactants and products by decreasing viscosities, resulting in faster hydrolysis , and proand 60°C . Liet aland 60°C  reportedide-IR05 . Regardiications . Immobilectively .M. circinelloides and M. hiemalis. The experimental results are consistent with predicted responses. The produced enzyme was partially purified, characterized, and immobilized. The optimum parameters for xylanase production by M. circinelloides and M. hiemalis consisted of incubation temperature (30 and 20 °C), pH value (9 and 7) incubation period (9 and 9 days), inoculum size (3 and 3 mL), and substrate concentration (3 and 3 g/100 mL), respectively. The partial purification of M. circinelloides and M. hiemalis xylanase yielded 1.69- and 1.97-fold purification, and the immobilized xylanase retained 84.02 and 79.43% activity, respectively. Response surface methodology was effective and satisfactory and investigated many factors simultaneously. More research is needed to scale up enzyme production for a wide range of applications.The present study utilized the response surface methodology via the Box–Behnken design to improve xylanase production by


Microbulbifer sp. ALW1 was revealed to be able to effectively degrade Laminaria japonica thallus fragments into fine particles. Polysaccharide substrate specificity analysis indicated that ALW1 could produce extracellular alginate lyase, laminarinase, fucoidanase and cellulase. Based on alignment of the 16 S rRNA sequence with other reference relatives, ALW1 showed the closest relationship with Microbulbifer aggregans CCB-MM1T. The cell morphology and some basic physiological and biochemical parameters of ALW1 cells were characterised. ALW1 is a Gram-negative, rod- or oval-shaped, non-spore-forming and non-motile bacterium. The DNA–DNA relatedness values of ALW1 with type strains of M. gwangyangensis , M. aggregans , M. maritimus , M. okinawensis  and M. rhizosphaerae  were 28.9%, 43.3%, 41.2%, 35.4% and 45.6%, respectively. The major cell wall sugars of ALW1 were determined to be ribose and galactose, which differed from other closely related species. These characteristics indicated that ALW1 could be assigned to a separate species of the genus Microbulbifer. The complete genome of ALW1 contained one circular chromosome with 4,682,287 bp and a GC content of 56.86%. The putative encoded proteins were categorised based on their functional annotations. Phenotypic, physiological, biochemical and genomic characterisation will provide insights into the many potential industrial applications of Microbulbifer sp. ALW1.Marine bacterium Key points. Microbulbifer sp. ALW1 has Laminaria japonica degradation capability.Genomic information of strain ALW1 is useful for analysing the polysaccharides degradation process.Microbulbifer sp. ALW1 could be potentially applied in producing functional materials. Marine macroalgae play a crucial part in the ecosystem. Seaweeds synthesise a wide variety of polysaccharides including alginate, agar, carrageenan, laminarin, fucoidan and cellulose, for use as cell wall components or storing nutrients  and the Japan Collection of Microorganisms (JCM) with accession numbers CICC 23,821 and JCM 33,586, respectively. Kelp (L. japonica) was purchased from a market in Xiamen, China. Sodium alginate was purchased from Sinopharm Chemical Reagent Co., Ltd. . Laminarin and fucoidan were purchased from Shanghai Yuanye Biotechnology Co., Ltd. . Carboxymethyl cellulose–sodium salt (CMC-Na) was purchased from Sigma-Aldrich .4]2SO4, 2 g/L K2HPO4, 1 g/L MgSO4·7H2O, 0.1 g/L FeSO4·7H2O, 5 g/L L. japonica thallus flakes) at 30 °C with stirring at 80 rpm for 72 h. The integrity of the thallus flakes was captured using a Canon 50D digital camera . The morphology of the thallus flakes was examined under a BA200 microscope of Motic China Group Co., Ltd. .The refreshed cells of strain ALW1 from our laboratory stock were sub-cultured in 400 mL of growth medium  in place of the thallus flakes at 30 °C for 72 h with shaking at 180 rpm. The cell solution was centrifuged at 12,000×g for 15 min at 4 °C, and the supernatant was collected to determine the activities of extracellular enzymes from strain ALW1 against different polysaccharides. The polysaccharides tested included alginate, laminarin, fucoidan and cellulose. Enzyme activity was determined following the procedures of the 3,5-dinitrosalicylic acid (DNS) method . The substrates tested included sodium alginate, laminarin, fucoidan and CMC-Na. After incubation at 30 °C for 1 h, the reaction was stopped by adding 1 mL of DNS reagent. The reducing sugar was determined at 540 nm using the spectrophotometer of Varian Cary 50 . The unit of enzyme activity was defined as the amount of enzyme that released 1.0 µg of the reducing sugar  per hour under the measured conditions.The ALW1 cells were cultured in the medium described above with 5 g/L fine particles of milled After the cells were cultivated at 30 °C for 48 h on 2216E   and other validated species of genus Microbulbifer sp. ALW1 was conducted using the single-molecule real-time (SMRT) sequencing platform of PacBio Co., Ltd.   according to the manufacturer’s instructions. The DNA–DNA hybridisation between strain ALW1 and the type strains from reference were carried out following the method described by Ley et al.  using a Microbulbifer sp. ALW1 has been deposited in GenBank , obtaining accession number CP047569.The complete genome sequence of L. japonica thallus fragments and the ALW1 cells were co-incubated for 72 h, the thallus fragments were broken down into the finest particles , M. aggregans CCB-MM1T (97.7%), M. okinawensis ABABA23T (97.7%), M. maritimus TF-17T (97.5%) and M. gwangyangensis GY2T (97.3%). The DNA–DNA hybridisation relatedness values between strain ALW1 and the type strains were 45.6% , 43.3% , 35.4% , 41.2%  and 28.9% , implying that strain ALW1 is a member of a genomic species different from the reference species of genus Microbulbifer.The phylogenetic relationship of 16 S rRNA sequence indicated that strain ALW1 was more closely related to fer Fig. . The topN-acetylglucosaminase, β-fucosidase and α-mannosidase. In the API 20NE test, ALW1 could utilise glucose, arabinose, N-acetylglucosamine and D-maltose. It could hydrolyse aescin and gelatin. The main quinone was ubiquinone-8 (100%). The main polar lipids were phosphatidyl glycerol, phosphatidyl ethanolamine, unidentified aminolipids (UAL 1–2), unidentified phospholipid, unidentified lipid, glycolipids (GL 1–2) and unidentified phosphatidyl glycolipid. The major fatty acids of strain ALW1 were C18:1ω7c/C18:1ω6c, iso-C15:0, C16:0 10-methyl/iso-C17:1ω9c and C16:1ω7c/C16:1ω6c. The comparison of the physiological and biochemical characteristics between ALW1 and the type strains of closely related species of genus Microbulbifer were summarised in Table Microbulbifer.After colonies of ALW1 cells were incubated at 30 °C for 48 h on 2216E agar plate, the cells were circular to slightly irregular, opaque and yellow, and had a wet surface , Microbulbifer agarilyticus S89 (GenBank: AFPJ00000000.1) and Microbulbifer variabilis ATCC 700,307 (GenBank: AQYJ00000000.1), which can degrade algal polysaccharides (Lee et al. Microbulbifer sp. ALW1 was widely collinear with those of Microbulbifer sp. HZ11 and ATCC700307 (Fig. Microbulbifer sp. ALW1 and ATCC700307 were highly similar, and more species-unique genes were detected in the CDSs of Microbulbifer sp. HZ11 (Fig. Microbulbifer sp. ALW1 will facilitate a better understanding of the molecular mechanism of brown algae metabolism by this strain and provide insight into the potential biotechnological applications of strain ALW1 in various fields, such as food, cosmetic, pharmaceutical, biofuel and fertiliser manufacturing industries.The phylogenetic relationship, DNA–DNA hybridisation and strain characteristics analyses indicated that strain ALW1 could be assigned to a separate species of genus 307 Fig. . The fou307 Fig. . The CDSZ11 Fig. . The genMicrobulbifer sp. ALW1 have been characterised in our previous work, including a novel laminarinase MaLamNA (Hu et al. Microbulbifer sp. ALW1. These alginate lyases could be used for many applications, such as the production of active oxygen species, preparation of algal protoplasts, analysis of alginate structure, and medical treatment of cystic fibrosis. A new gene of exo-oligoalginate lyase AlgL17 belonging to PL17 has been cloned from strain ALW1 and characterised in our previous study (Jiang et al. GHs are involved in the hydrolysis and/or transglycosylation of glycosidic bonds present in glycosides, glycans and glycoconjugates (Berlemont and Martiny Microbulbifer sp. ALW1, common carbohydrates in nature, such as cellulose of the seaweed cell wall components, would be destroyed through cleavages (Li et al. Based on the abundance of CAZymes, the process of seaweed degradation through the action of cohort enzymes in strain ALW1 was proposed Fig. . ThroughMicrobulbifer sp. ALW1 characterised in this study was capable of degrading brown algal cell wall polysaccharides, including alginate, laminarin, fucoidan and cellulose. The physiological and biochemical characteristics of strain ALW1 suggested that it might be a novel species of genus Microbulbifer, distinct from previously annotated reference species. The genetic information of strain ALW1 and the predicted carbohydrate active enzymes responsible for the polysaccharides’ metabolism enriched our understanding of the bioactivity of strain ALW1 and provided a strong support for its industrial utilisation for algal waste cleanup and bioresource recovery.To summarise, the marine bacterial strain


The resulting crystals formed in the monoclinic space group P21/c, and adopting the common E configuration.The title compound was prepared  18H16O3, was prepared by a solventless base-promoted Claisen–Schmidt condensation and, upon recrystallization from ethanol, obtained in 56% yield. The dihedral angle between the indanone ring system and the benzene ring is 2.54 (4) ° and the C atoms of the methoxy groups deviate from the benzene ring by 0.087 (1) and 0.114 (1) Å. In the crystal, π-stacking is the predominant intermolecular force, with the molecules stacking into columns running parallel to the b axis of the unit cell.The title chalcone, C They are important pharmacophore scaffolds and can possess anti-inflammatory, anti-fungal, anti-cancer, and anti-malarial biological activities  exist [POWZUX  isolated via vacuum filtration. Recrystallization from 95% ethanol solution via slow evaporation afforded the target chalcone, (E)-2--1-indanone (1b) as colorless needles, . Melting range: 174–175°C. IR, 1H and 13C NMR spectroscopy and single-crystal X-ray analysis (see supporting information) confirmed the product identity.A 25 mL beaker equipped with a stir bar was charged with 3,5-dimethoxybenzaldehyde  and warmed to 60°C. To the liquified aldehyde was added 1-indanone  and solid NaOH . The reaction mixture was stirred for 30 minutes at 60°C. The resulting reaction mixture was neutralized with 4 mL of 1 Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2414314620007592/bt4094sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314620007592/bt4094Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620007592/bt4094Isup3.smiSupporting information file. DOI: Click here for additional data file.10.1107/S2414314620007592/bt4094Isup4.cmlSupporting information file. DOI: 10.1107/S2414314620007592/bt4094sup5.pdf1H NMR data. DOI: 10.1107/S2414314620007592/bt4094sup6.pdf13C NMR data. DOI: 1894469CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: