A receptor-mediated currents of NG2 glia in juvenile and adult hippocampi of mice of either sex and assessed the mode of interneuron–glial signaling changes during development. With patch-clamp and pharmacological analyses, we found a decrease in innervation of hippocampal NG2 glia between postnatal days 10 and 60. At the adult stage, enhanced activation of extrasynaptic receptors occurred, indicating a spillover of GABA. This switch from synaptic to extrasynaptic receptor activation was accompanied by downregulation of γ2 and upregulation of the α5 subunit. Molecular analyses and high-resolution expansion microscopy revealed mechanisms of glial GABAA receptor trafficking and clustering. We found that gephyrin and radixin are organized in separate clusters along glial processes. Surprisingly, the developmental loss of γ2 and postsynaptic receptors were not accompanied by altered glial expression of scaffolding proteins, auxiliary receptor subunits or postsynaptic interaction proteins. The GABAergic input to NG2 glia might contribute to the release of neurotrophic factors from these cells and influence neuronal synaptic plasticity.NG2 glia receive synaptic input from neurons, but the functional impact of this glial innervation is not well understood. In the developing cerebellum and somatosensory cortex the GABAergic input might regulate NG2 glia differentiation and myelination, and a switch from synaptic to extrasynaptic neuron–glia signaling was reported in the latter region. Myelination in the hippocampus is sparse, and most NG2 glia retain their phenotype throughout adulthood, raising the question of the properties and function of neuron-NG2 glia synapses in that brain region. Here, we compared spontaneous and evoked GABA A receptors and receive direct synaptic input from glutamatergic and GABAergic neurons [NG2 glia represent the largest population of proliferative cells in the postnatal brain outside the neurogenic niches. In white matter, the majority of these cells differentiate into oligodendrocytes and are therefore also named oligodendrocyte progenitor cells or OPCs . In cont neurons ,8,9, butA receptors are ionotropic receptors forming pentamers composed of 2α, 2β and 1γ subunits. Furthermore, 16 different GABAA receptor subunits are known , forming subunit combinations with distinct pharmacological properties. Pharmacological evidence of cell-specific subunit composition is derived from modulators acting on allosteric binding sites for benzodiazepine, barbiturate, zinc and volatile anesthetics . membrane . In rodemembrane ,15. Oligrization ,18.Previously, we have shown that at the juvenile stage, receptor responses of NG2 glia are potentiated by allosteric modulators interacting with the benzodiazepine and barbiturate binding sites. GABA-mediated currents were also potentiated by zolpidem, which binds at the α/γ2-benzodiazepine site. On a subcellular level, the γ2 subunit is located postsynaptically in NG2 glia while tonic currents are mediated by zolpidem-insensitive, extrasynaptic receptors lacking γ2 ,19. A deA receptor-mediated currents of NG2 glia in the juvenile and adult hippocampus and asked whether the mode of interneuron–glial signaling changes during postnatal development. Moreover, molecular analyses were performed to reveal mechanisms of glial GABAA receptor trafficking and clustering. We found a significant decrease in synaptic innervation of hippocampal NG2 glia between postnatal days (p) 10 and 60. At the adult stage, enhanced activation of extrasynaptic receptors was observed, indicating a spillover of GABA. This switch from synaptic to extrasynaptic receptor activation was accompanied by a downregulation of γ2 and upregulation of the α5 subunit. In the present study, we compared spontaneous and evoked GABAA receptor clustering. These γ2-containing receptors form complexes with GARLH4 and neuroligin 2 [A receptors are anchored to the cytoskeleton by radixin [In neurons, the γ2 subunit is an important interaction partner of the scaffolding protein gephyrin and crucial for postsynaptic GABAoligin 2 ,22. For oligin 2 ,24. In c radixin ,26. The We compared spontaneous synaptic currents (sPSCs) of NG2 glia in the CA1 stratum radiatum at p10 and p60 using a blocking solution containing NBQX (10 µM), D-APV (50 µM) and CGP55845 (5 µM)  were compared in the presence of the above blockers plus TTX  by near-field stimulation and assessed the efficiency of ligands for the benzodiazepine binding site changes during development . This sieceptors . Diazepaeceptors ,27. In jComparing the kinetics of ePSCs, we found that rise time and decay time slowed down with increasing age C,D. For t-test p < 0.01), reflecting the primary role of GAT-1 in regulating extrasynaptic GABA concentration due to its higher expression and more proximal localization to the synaptic cleft. These results are in line with the hypothesis that during later stages of development, NG2 glia detect GABAergic input predominantly through extrasynaptic receptors. To further pursue the hypothesis of spillover-mediated activation of NG2 glia at the adult stage, GABA transporters (GATs) were blocked. Two types of GATs are expressed in the mouse hippocampus, GAT-1 and GAT-3 ,29, withA receptors in NG2 glia. To increase ambient GABA concentration and unmask tonic currents, nipecotic acid, a broad spectrum GABA transporter inhibitor, was applied . Subsequent addition of the competitive GABAA receptor antagonist, bicuculline (20 µM), induced a positive shift in holding current, which was due to block of GABA-mediated tonic currents  while α5 remained unchanged . To further elaborate alterations of α5 and γ2 subunit expression with progressing age, we performed semiquantitative real-time RT-PCR with FAC-sorted NG2 glia. The normalized γ2 expression level was again reduced in adulthood , gephyrin (postsynaptic clusters) and radixin (extrasynaptic clusters) was performed in NG2-EYFPki mice at p60 . Z-stackA receptors in neurons [We report that GABAergic synaptic innervation of hippocampal NG2 glia decreased during postnatal maturation, indicated by reduced frequencies of spontaneous and miniature PSCs, which resembled developmental changes of the cells’ glutamatergic input . With in neurons . A simil neurons , althoug neurons ). The fo neurons . Thereaf neurons . A receptors and that extrasynaptic receptors of these cells lacked γ2 [A receptors acting as a binding partner of the scaffold protein, gephyrin [Previously, we have shown that a majority of juvenile NG2 glia in the hippocampus express postsynaptic γ2-containing GABAacked γ2 . In the acked γ2 , an essegephyrin ,37,38. Igephyrin ,40. The 2+ signaling remained unaffected [A receptors in oligodendrocyte lineage cells increased their numbers, suggesting that the synaptic input decreases numbers of NG2 glia and oligodendrocytes [A receptors [Deletion of γ2 in juvenile cortical NG2 glia entailed lower frequencies and amplitudes of sPSCs, although glial proliferation, differentiation and Caaffected . The denaffected ,43, but affected . Thus, tdrocytes . GABA redrocytes . Other sdrocytes ,45. In tdrocytes . The inneceptors ,19. Putaeceptors ,48.A receptors are located extrasynaptically [2+ elevation and by increasing the threshold for LTP induction [A receptor activation and the resulting drop of the membrane resistance may fine-tune the efficiency of the excitatory GABAergic and glutamatergic synaptic input in these cells [In CA1 pyramidal neurons, α5-containing GABAptically  to generptically ,51,52,53nduction ,55. In Nse cells ,33.A receptor clustering [A receptor subunits with NLG2, e.g., in pyramidal cells of the hippocampus [The GABAergic innervation of NG2 glia raised the question whether these synapses are similarly organized as neuronal inhibitory synapses. Typically, inhibitory synapses in neurons are formed by interactions of the γ2 subunit with gephyrin ,38,56. Austering ,24. Throustering , as wellpocampus ,22. The A receptors to extrasynaptic sites [The strong upregulation of the α5 subunit in NG2 glia with increasing age raised the question whether the cells also express radixin, which in neurons anchors α5-containing GABAic sites ,57. Inde− concentration and explains why the GABAergic input causes depolarization of the glial cells [NG2 cells in the cerebellar white matter are innervated by local interneurons, and this synaptic input regulates proliferation and maturation of the glial cells. Disruption of GABAergic activity stimulated proliferation of NG2 glia and delayed their differentiation into oligodendrocytes. This process is critically dependent on the presence of the Na/K/2Cl (NKCC1)-transporter, which delivers an elevated intracellular Clal cells . Deletioal cells . − concentration of NG2, GABA receptor activation depolarizes the cells [2+ channels and induced formation of postsynaptic gephyrin clusters and inhibitory synapses [A receptors and voltage-activated Ca2+ channels [2+ influx through these channels [Due to the high intracellular Clhe cells ,17. The synapses . Becausechannels , and recchannels ,32, it ichannels . Thus, tchannels . Possiblchannels . Experiments were performed with NG2-EYFPki mice  of both 2PO4, 87 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 25 glucose, 25 NaHCO3, and 61 sucrose (325–335 mOsm). After cutting, the slices were incubated for 15 min in preparation solution at 35 °C and afterwards transferred into artificial cerebrospinal fluid (aCSF) at 25 °C. aCSF consisted of the following (in mM): 1.25 NaH2PO4, 126 NaCl, 3 KCl, 2 MgSO4, 2 CaCl2, 10 glucose, and 26 NaHCO3 (305–315 mOsm). Both solutions were constantly oxygenated with carbogen (5% CO2/95% O2).Mice were anesthetized with isoflurane and decapitated. Brains were removed and cut with a vibratome (Leica VT1200S) into 300 µm thick horizontal sections in cold (4 °C) preparation solution containing the following (in mM): 1.25 NaH2, 0.5 CaCl2, 5 BAPTA, 10 HEPES, 3 Na2-ATP, and 10 TEA. Series and membrane resistance were monitored in constant intervals.For patch-clamp recording, the slices were transferred into a recording chamber and constantly perfused with oxygenated aCSF. All experiments were conducted in the stratum radiatum of the CA1 region of the hippocampus at room temperature. An upright microscope , equipped with infrared-DIC optics and epifluorescence, was used. NG2 glial cells were selected with a water-immersion 60× objective and identified by their EYFP fluorescence and their current pattern in the whole-cell configuration. Fluorescent somata enwrapping blood vessels (putative pericytes) were excluded. Cells were clamped at −80 mV. Recordings were obtained using an EPC9 amplifier , an EPC800amplifier (HEKA) or a combination of an EPC7 amplifier with an ITC16 AD/DA board (HEKA) and monitored using TIDA 5.24 software (HEKA). Patch pipettes were prepared from borosilicate capillaries  and had a resistance of 2–5 MΩ. For all experiments, except for cell harvesting (KCl-based), a CsCl-based intracellular solution was used, containing the following (in mM): 120 CsCl, 2 MgClA receptor currents. After equilibration of the blocking solution in the recording chamber, spontaneous activity was recorded for 5 min. To record miniature postsynaptic currents (mPSC), 0.5 µM TTX was added to the blocking solution. mPSCs were recorded for 5–10 min. mPSCs were identified using template-based event detection . Events detected by template search were controlled visually before being accepted for further analysis, which was performed with Igor Pro 7 software . Only recordings with at least two events were accepted for further calculations. Recordings were filtered at 1 kHz and sampled at 10 kHz.To record spontaneous postsynaptic currents (sPSC), a blocking solution containing 10 µM NBQX, 50 µM D-APV and 5 µM CGP55845 was applied via the perfusion system to isolate GABAA receptor currents. ePSCs were evoked by near-field electrical stimulation [A receptor currents, the recordings were performed in the presence of 10 µM NBQX and 50 µM D-APV, blockers of AMPA and NMDA receptors. Series and membrane resistance were monitored in equal intervals. Recordings were filtered at 3 kHz and sampled at 10 kHz.Evoked postsynaptic currents (ePSCs) were characterized to investigate pharmacological properties of phasic GABAmulation . A monopUsing Igor Pro 7 software, peak amplitude, rise time (20–80%) and decay time were quantified. The effects of diazepam and zolpidem on receptor currents were determined as the ratios of the mean peak amplitudes in the presence of the drug and the mean peak amplitudes in control condition, individually for each cell recorded. A receptors. Only cells with stable holding currents (2 min) before application of nipecotic acid were included. To investigate pharmacological properties of tonic currents, the α5 subunit-specific blocker, α5IA, was used. First, 1 mM nipecotic acid was applied (at least 3 min to determine the baseline), and then, α5IA (500 nM) was added. Two min later, 20 µM of bicuculline was added to the bath solution. The effect of α5IA was then determined by measuring the shift of the holding current induced by application of α5IA and normalizing it to the shift of the holding current induced by bicuculline. In the following, this ratio is referred to as the relative effect of α5IA. Recordings were filtered at 1 kHz and sampled at 10 kHz.To detect tonic currents, 1 mM nipecotic acid, a GABA transporter blocker, was added to the bath solution for at least 3 min to elevate the extracellular GABA concentration. Tonic currents were then unmasked by adding 20 µM bicuculline, inducing a positive shift in the holding current due to blocking of GABA2, 0.5 CaCl2, 5 BAPTA, 10 HEPES, and 3 Na2-ATP. After electrophysiological recording, the cytoplasm of individual cells was carefully aspirated into the patch pipette under microscopic control. The whole content of the patch pipette was then transferred into a reaction tube containing 3 µL DEPC-treated water, frozen in liquid nitrogen and stored at −20 °C until single-cell RT-PCR was performed.For single-cell RT-PCR, cells were harvested with patch pipettes filled with KCl-based intracellular solution consisting of the following (in mM): 130 KCl, 2 MgClA receptor subunits and for PDGFRα were used (see [2 (2.5 mM), primers , dNTPs (4 × 50 µM) and 5 U Taq polymerase  to the RT product . Thirty-five cycles were performed . An aliquot (2 µL) of the PCR product was used as template for the second PCR  using nested primers. The conditions were the same as described for the first round, but Platinum Taq polymerase  was added. Products were identified with gel electrophoresis using a molecular weight marker . As a positive control, RT-PCR for total RNA from mouse brain was run in parallel. Negative controls were performed using distilled water or bath solution for RT-PCR. The primers for the targets were located on different exons to prevent amplification of genomic DNA. To discriminate between β2 and β3 subunits, the PCR product was purified , and restriction analysis was performed . PstI cut the PCR product of β2 subunit (307 bp) into 169 and 138 bp, while BanI cut the β3 product (307 bp) into fragments of 202 and 105 bp lengths.Single-cell transcript analysis was performed as previously reported ,63. Revesed see ,19). To . To A re2+ and Mg2+) to the cell suspension, it was centrifuged at 300 g for 10 min. The pellet was suspended in 1 mL HBSS (without Ca2+ and Mg2+) and filtered through a 40 µm cell strainer. Fluorescent NG2 cells were identified using EYFP fluorescence emission at 527 nm and sorted using a FACSAriaIII flow cytometer  into tubes containing HBSS (without Ca2+ and Mg2+). After centrifugation at 2000 g for 10 min, the supernatant was discarded and the cells were suspended in 200 µL lysis/binding buffer , frozen in liquid nitrogen and stored at −80 °C.Mice  were sacrificed, brains were dissected and the whole hippocampus was isolated under microscopic control . Cell suspension was prepared by mincing the tissue, digesting in papain at 37 °C for 15 min and incubating with DNAseI for another 10 min , Miltenyi, Bergisch-Gladbach, Germany). The tissue was dissociated using Pasteur pipettes and filtered through a 70 µm cell strainer. After addition of 10 mL HBSS , RNasin  and SuperscriptIII reverse transcriptase  were added, and the reaction mix was incubated for 1 h at 50 °C . Messenger RNA was isolated from isolated cells by cell lysis in the lysis/binding buffer and by using oligo(dT)25-linked Dynabeads (Invitrogen). Beads with the adherent mRNA were suspended in DEPC-treated water (20 µL). For first strand synthesis, first strand buffer (Invitrogen), dithiothreitol , dNTPs , oligo-dTT values of the target gene with those of the reference gene, β-actin. The relative quantification of different genes was determined according to 2ΔΔCT method: T the cycle number at threshold. By quantification of target gene expression against that of β-actin, ΔCT was determined for each gene at the same fluorescence emission Rn. The reaction mixture for real-time PCR contained Takyon real-time PCR mastermix  and Taqman primer/probe mix . One µL of the RT-product was added, and the reaction volume was 12.5 µL. PCRs for the respective target genes and β-actin, as housekeeping gene, were run in parallel wells for each sample, respectively, and triplicates for each sample were performed. Water served as negative control in each run. After denaturation , 50 cycles were performed . Fluorescence intensity was read out during each annealing/extension step. The target gene/β-actin gene expression ratio was determined by comparing CMice were anesthetized by intraperitoneal injection of 70–90 µL of a xylazine (2%)/ketamine (100 mg/mL) mix in a ratio of 1:3. Afterwards, mice were transcardially perfused with 20–30 mL cold (4 °C) phosphate-buffered saline (PBS), followed by 20–30 mL 4% paraformaldehyde (PFA) in PBS. The brains were removed and stored overnight in 4% PFA at 4 °C, transferred into PBS the next day and stored for at least one day at 4 °C before slicing. Brains were cut with a vibratome (Leica VT1200S) in cold (4 °C) PBS into 70 µm thick coronal slices. Slices were stored at 4 °C in PBS containing sodium azide until staining.w/v): 0.01 4-hydroxy-TEMPO; 0.2 TEMED; 0.2 ammonium persulfate). Slices were placed in the gelling solution on a glass slide and the preparation was covered with a coverslip and incubated for 2 h at 37 °C. Coverslips and excess gel around the slice were removed, and gels incubated overnight at 25 °C in digestion buffer containing 50 mM Tris, 1 mM EDTA, 0.5% Triton-X100, 0.8 M guanidine hydrochloride, 16 U/mL of proteinase K, pH 8.0. The following day, the digestion buffer was removed, and the gels were incubated with streptavidin Alexa Fluor 647  in PBS for 2 h at room temperature. For expansion, slices were then incubated in distilled water (pH 7.4 adjusted with NaOH) for 2.5 h, at room temperature, and water was exchanged repeatedly every 15–20 min. Finally, slices were mounted on poly-lysine coated µ-Slide 2 well Ibidi-chambers and sealed with a poly-lysine coated coverslip on top, adding a drop of water to prevent gel drying. Ibidi chambers and coverslips were coated by incubating with poly-L-lysine solution  for at least 45 min at room temperature by shaking, and dried then with pressured air. Imaging of regions of interest was performed on a Leica SP8 inverted confocal microscope using a 40×/1.1 NA objective and hybrid detectors. Images were deconvoluted in Leica Systems software LASAF 3.3 and processed with Fiji 1.53. Prior to imaging the region of interest for each slice, the tip of the DG was imaged with a 20×/0.75 NA objective . The expansion factor was calculated by identifying the same cells labeled with Hoechst 33342 in the tip of DG before and after expansion and averaging the expansion factor of 10 measures.The expansion microscopy (ExM) protocol for imaging proteins with conventional primary and secondary antibodies was adopted from ,65,66. Ft-test either with or without Welch correction accordingly. Differences in not normally distributed data were analyzed with Mann–Whitney-U test. Data from single-cell RT-PCR was tested for significant differences using Chi-square test. Level for significance was set as p < 0.05. Data are shown as bar plots with mean ± SD or, in case of not normally distributed data, as Tukey’s box plots with median, quartiles (25% and 75%) and whiskers (±1.5 times the interquartile range). Since no sex-specific differences were found in our analyses, data from male and female mice have been pooled.Data analysis was performed using Igor Pro 7 and pClamp 10.0 software. Statistical analysis was carried out with R 3.5.0 software. All data were tested for normal distribution using Shapiro–Wilk test. Normally distributed data were tested for homogeneity of variance using Levene’s test and differences in data were tested for significance using Student’s


Fusarium oxysporum f. sp. niveum is one of the most devastating fungal diseases affecting watermelon (Citrullus lanatus L.). The present study aimed to identify potent antagonistic bacterial strains with substantial antifungal activity against F. oxysporum f. sp. niveum and to explore their potential for biocontrol of Fusarium wilt in watermelon. Out of 77 isolates from watermelon rhizosphere, six bacterial strains—namely, DHA4, DHA6, DHA10, DHA12, DHA41, and DHA55—exhibited significant antifungal activity against F. oxysporum f. sp. niveum, as well as other phytopathogenic fungi, including Didymella bryoniae, Sclerotinia sclerotiorum, Fusarium graminearum, and Rhizoctonia solani. These Gram-positive, rod-shaped, antagonistic bacterial strains were able to produce exo-enzymes , siderophore, and indole-3-acetic acid and had the ability to solubilize phosphate. In greenhouse experiments, these antagonistic bacterial strains not only promoted plant growth but also suppressed Fusarium wilt in watermelon. Among these strains, DHA55 was the most effective, achieving the highest disease suppression of 74.9%. Strain DHA55 was identified as Bacillus amyloliquefaciens based on physiological, biochemical, and molecular characterization. B. amyloliquefaciens DHA55 produced various antifungal lipopeptides, including iturin, surfactin, and fengycin, that showed significant antifungal activities against F. oxysporum f. sp. niveum. Microscopic observations revealed that B. amyloliquefaciens DHA55 exhibited an inhibitory effect against F. oxysporum f. sp. niveum on the root surface of watermelon plants. These results demonstrate that B. amyloliquefaciens DHA55 can effectively promote plant growth and suppress the development of watermelon Fusarium wilt, providing a promising agent for the biocontrol of Fusarium wilt in watermelon.Fusarium wilt caused by  Citrullus lanatus L.) is an important tropical/subtropical horticultural crop that is cultivated worldwide due to its high nutritional and economic value. The greenhouse mono-cropping system, particularly in China, exacerbates the occurrence of destructive soil-borne diseases, including Fusarium wilt, anthracnose, and gummy stem blight [Fusarium oxysporum f. sp. niveum (Fon), has become a major constraint on the global watermelon industry [Watermelon , Sclerotinia sclerotiorum (Ss), Fusarium graminearum (Fg), and Rhizoctonia solani (Rs). These antagonistic bacterial strains were capable of promoting the growth of watermelon plants and reducing the disease severity of watermelon Fusarium wilt under greenhouse conditions. Among these antagonists, B. amyloliquefaciens DHA55 showed excellent potential for biocontrol of watermelon Fusarium wilt through its production of extracellular lipopeptides, and it can be used as an efficient biocontrol agent for the management of Fusarium wilt in watermelon.The present study aimed to identify and evaluate the antifungal efficacy of bacterial strains against 2O), vortexed vigorously, and placed in a water bath for 15 min at 100 °C. After heat treatment, the soil suspension was allowed to cool to room temperature and then serially diluted. An aliquot of 100 µL of the suspension (10−4 dilution) was spread on a Luria-Bertani (LB) agar plate and then incubated at 28 ± 2 °C for 36 h. Purified bacterial isolates were maintained on fresh LB plates.Soil samples were collected from the rhizospheres of watermelon plants grown on different agricultural lands in Hangzhou, Zhejiang province, China, and stored at 4 °C until use. To isolate bacterial strains, 20 g of rhizosphere soil was suspended in 20 mL of sterile distilled water (ddHFusarium oxysporum f. sp. niveum (Fon), Didymella bryoniae (Db), Sclerotinia sclerotiorum (Ss), Fusarium graminearum (Fg), and Rhizoctonia solani (Rs), which were obtained from the Laboratory of Crop Diseases and Insect Pests of the Ministry of Agriculture and Rural Affairs at Zhejiang University. The fungal pathogens were grown on potato dextrose agar (PDA) at 28 ± 2 °C for 5~6 d before the antagonistic experiments. The inhibition zone method was used to determine the antagonistic potential of the bacterial isolates against the indicated fungal pathogens as previously described [9 colony-forming units (CFUs)/mL or in sterile LB medium (as controls) were placed at a distance of 2.5 cm from the fungal discs. After incubation at 28 ± 2 °C for 5~6 d, the inhibition zone was recorded and the mycelial growth inhibition rate was calculated using the following formula [The tested pathogenic fungi included escribed . Briefly formula :InhibitiFon [Fon mycelial discs (5 mm) and then incubated at 28 ± 2 °C for 5 d. The growth inhibition rate was calculated by comparing the diameters of the Fon colonies grown in the cell-free supernatant-containing plates to those grown in the control plates. The experiments were conducted three times, and at least three replicates were included in each of the treatments.The cell-free supernatant of the antagonistic bacterial strains was examined for its antifungal activity against Fon . The bacFon , with mi2CO3 and 0.5% C6H3N3O7) and fixed on the inner side of the plate covers. The plates were sealed with parafilm and incubated at 28 ± 2 °C for 4 d. Control plates were maintained without bacterial culture. For the ammonia production assay, the overnight bacterial culture was inoculated into 10 mL of LB broth and incubated at 30 ± 2 °C under shaking conditions (180 rpm) for 48 h. A total of 500 µL in volume of Nessler’s reagent was added to 10 mL of LB culture to measure the production of ammonia [The phosphate (P) solubilization activity of the antagonistic bacterial strains was determined using the Pikovskaya agar assay , and pho ammonia . The act ammonia . The expC. lanatus L. cv. Zaojia) seeds were imbibed in sterile ddH2O at 50 °C for 40 min and then incubated on moist cheesecloth in the dark at 28 ± 2 °C for 3 d for germination. The germinated seeds were transferred to pots (12 cm × 10 cm) filled with a sterilized mixture of perlite:vermiculite (1:2). After planting, watermelon plants were grown in a growth chamber at 70% humidity with a photoperiod of 16 h at 28 °C in the daytime and 20 °C at night. Watermelon plants were irrigated with 0.5× Hoagland solution (pH 6.5) once a week [9 CFUs/mL. Two-week-old plants were used for growth experiments and separately supplied with 15 mL inoculum suspensions of the screened bacterial strains or with sterile LB medium as controls. At 15 d after treatment, plants were uprooted and washed gently with tap water to remove adhered soil particles, after which the growth parameters were estimated, including plant/root lengths and fresh/dry weights.Watermelon  at 28 ± 2 °C under shaking conditions at 150 rpm for 48 h. The spore suspension was adjusted to 4 × 106 spores/mL for inoculation. Watermelon plants at the two-leaf stage were treated through soil drenching with 15 mL inoculum suspensions of each of the bacterial strains, pesticide Topsin-M (as a positive control), or sterile LB medium (as a negative control). At 5 d post-treatment, the bacterial strain-, Topsin-M- or LB-treated plants were inoculated with Fon by irrigating with a similar volume (15 mL) of spore suspension (4 × 106 spores/mL) to assess their disease-controlling potential. The experiments were conducted three times using a completely randomized design and four replicates were included in each of the treatments. Disease scores were recorded using a five-scale rating standard . The disease severity index (DSI) was calculated as described previously [For disease assays, eviously , and bioeviously :Biocontr16S rRNA gene was amplified using a set of universal primers: 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1479R (5′-TACGGTTACCTTGTTACGACTT-3′) [B. subtilis strain SQR6 was used as a reference to design gene-specific primers (w/v) agarose gels and the target bands were purified using the AxyPrep DNA Gel Extraction Kit  and then commercially sequenced . Homologues of the obtained sequences were searched for using the BlastN and BlastX programs at NCBI. The ClustalX program was used for multiple sequence alignment [Genomic DNA was extracted using a MiniBEST Bacteria Genomic DNA Extraction Kit Ver3.0  according to the manufacturer’s instructions. DNA concentration and quality were measured using a NanoDrop 2000 . A fragment of the bacterial ACTT-3′) . For the primers . The PCRlignment , and thelignment .3OH:H2O (2:1) solution. The extract was filtered through a 0.45 μm syringe filter, dried under a rotary vacuum at 40 °C, resuspended in 2 mL of methanol, and stored at −20 °C until further experiments. The antagonistic activity of the lipopeptide extract was determined as previously described [Fon (5 mm in diameter) were placed at the center of the PDA plates and incubated at 28 ± 2 °C for 3~5 d. The colony sizes were recorded, and the inhibition rate was determined by comparing the diameters of the fungal colonies grown with the lipopeptide extract to those grown with controls [For the extraction of lipopeptides, strain DHA55 was grown in LB broth under shaking conditions at 200 rpm and 28 ± 2 °C for 3 d. The bacterial culture (100 mL) was centrifuged  at 4 °C for 20 min, and the lipopeptide-containing supernatant was collected. The lipopeptides were allowed to precipitate under acidic conditions (pH 2.0) using 2 M HCl and then incubated overnight at 4 °C. The precipitated lipopeptides were collected using centrifugation at 15,000 rpm at 4 °C for 20 min and then resuspended in CHescribed . Brieflycontrols .Fon was grown under agitating conditions in mung bean liquid medium at 28 ± 2 °C for 48 h, and spores were collected. Wells in 96-well microtiter plates were first filled with 50 µL of spore suspensions (4 × 105 spores/mL) and then with lipopeptide extract to final concentrations of 50, 75, 100, 200, and 300 µg/mL or with DMSO (0 µg/mL) for the controls, following a previous report [600 values, and the growth inhibition rate was calculated by comparing fungal growth in lipopeptide suspensions to that in controls. The experiments were conducted three times, and at least three replicates were included in each of the treatments.s report . Fungal 10H7NO3, 30% of C2H3N, and 0.1% trifluroacetic acid). Spectral data were analyzed to identify the presence of different lipopeptides in the extract.Lipopeptide extract was dissolved in 2 mL of methanol and subjected to MALD-TOF/mass spectrometry (MS) analysis to identify possible lipopeptides . BrieflyThe strain DHA55 was cultured on Tryptic Soy Blood Agar (TSBA) medium  at 28 ± 2 °C for 24 h. Lipids were extracted using the Sherlock Microbial Identification System. Lipid profiling was performed using a GC . The fatty acid methyl esters were identified based on their relative retention times compared to the standard of the methyl ester, 3-hydroxy-hexadecanoate , and the fragmentation profile obtained using MS with electronic ionization (EI-70 eV). The fatty acids were determined by comparing their retention times to the whole area of the peak. The fatty acids of the bacterial strain DHA55 were identified by searching the Sherlock bacterial fatty acid reference library RTSBA6 6.10 .Fon on cellophane membrane and on the root surface of watermelon plants was examined using an SEM . To observe the direct interaction with Fon, 5 µL in volume of Fon spore suspension (4 × 106 spores/mL) was dropped onto the cellophane membrane placed on the PDA plate and incubated at 28 ± 2 °C for 72 h. Then, 30 µL in volume of bacterial suspension (~109 CFUs/mL) was then dropped onto the Fon mycelia grown on the cellophane membrane and incubated at 28 ± 2 °C for 24 h. After incubation, samples were collected, washed with sterile ddH2O twice, and fixed with 2.5% glutaraldehyde. To examine the interactions presented by DHA55 on the watermelon root surface, plants were harvested carefully at the first true-leaf stage, washed with sterile ddH2O twice, soaked in 98% ethanol for 30 s, rinsed again with sterile ddH2O, and dried on sterile paper. The roots of the seedlings were then soaked in the suspension of the bacterial strain DHA55 (1 × 109 CFUs/mL) and incubated at 28 ± 2 °C at 150 rpm for 2 h. The DHA55 strain-treated and untreated seedlings were then grown hydroponically in a Fon spore suspension (4 × 106 spores/mL) under shaking conditions (95 rpm) at 28 ± 2 °C. Fon-inoculated and non-inoculated root samples with and without treatment with DHA55 strain were collected after 24 and 48 h, rinsed with sterile ddH2O, cut into 0.5–1.0 cm pieces, and fixed with 2.5% glutaraldehyde. All samples were prepared according to a previously described procedure for SEM analysis [The interaction between the bacterial strain DHA55 and analysis .p-value < 0.05) was performed to determine differences between the treatment means of the different datasets.Data analysis was conducted using one-way analysis of variance with the SPSS 20.0 software package. The Duncan multiple range test  was closely clustered with that of B. amyloliquefaciens InAD-160 (KY859771), showing 100% sequence identity  was observed at a concentration of 100 µg/mL of the lipopeptide extract, while the lipopeptide extracts at concentrations of 50 and 75 µg/mL also inhibited Fon growth by 39.4~68.7% , two fengycin genes (Fengycin E and D), and one surfactin gene (Surfactin) were amplified from B. amyloliquefaciens DHA55. BLASTn searching revealed that the sequences of Iturin C, Iturin D, Fengycin E, and Fengycin D amplified from B. amyloliquefaciens DHA55 showed 98.60%, 95.81%, 97.31%, and 100% nucleotide identities with their corresponding genes in other Bacillus species, with e values of 0, 0, 0, and 8 × 10−79, respectively , iturin (with peaks at 1079.895 and 1093.884 m/z), bacillomycin D , and fengycin , were detected  with different lifestyles and infection processes  have been identified in  cinerea ,68,69,70fengycin —which wans DHA55 . The seqefaciens ,72,73. Nrocesses , implyinntegrity . HoweverB. amyloliquefaciens DHA55 was the most effective strain in suppressing watermelon Fusarium wilt (Bacillus sp. strains colonize root and internal plant tissues [Fon–DHA55 strain system (B. amyloliquefaciens DHA55 adhered to and reproduced on the root surface (B. amyloliquefaciens DHA55 interacted with and disintegrated the Fon mycelia on the root surface of watermelon plants (Fon, thus making it possible to suppress Fusarium wilt in watermelon, and correlates with previous observations reporting the destructive impact of B. subtilis and B. amyloliquefaciens on F. oxysporum and Phytophthora capsici on the root surface of cucumber [B. subtilis SQR 9 has been reported to colonize the root surface, which might be triggered by rhizodeposits exuded by host plants, competing with and suppressing the proliferation of fungal pathogens on the root surface of the host plants, such as soybean and mung bean [B. amyloliquefaciens DHA55 in the roots of watermelon plants. Alternatively, some rhizobacteria have been shown to suppress disease development by modulating plant defense responses, such as activation of induced systemic resistance [B. amyloliquefaciens PMB05, B. subtilis MBI600, and B. cereus EC9 have been reported to induce systemic resistance, contributing to the biocontrol of soil-borne pathogens, such as F. oxysporum [B. velezensis F21 enhances the basal immunity of watermelon plants against Fon by increasing the expression of the defense-related genes and activities of antioxidant enzymes, such as catalase, peroxidase, and superoxide dismutase [B. amyloliquefaciens DHA55 suppresses Fusarium wilt by priming the defense response in watermelon plants.Results from disease experiments showed that ium wilt , possibl tissues ,44, whicn system . Interes surface . Importan plants G. This fcucumber ,76. Moreung bean ,77. Furtsistance ,78. For xysporum ,80,81,82Fon. The results revealed that these strains have great potential as biocontrol agents for suppressing watermelon Fusarium wilt and promoting plant growth. Among these strains, B. amyloliquefaciens DHA55 exhibited the best performance in promoting plant growth and suppressing Fusarium wilt in watermelon. B. amyloliquefaciens DHA55 produced antifungal lipopeptides that showed significant inhibitory activity against Fon in vitro and on the root surface of watermelon plants. The present study highlights the potential of B. amyloliquefaciens DHA55 as a biopesticide for controlling Fusarium wilt in watermelon, offering an eco-friendly and cost-effective alternative to chemical fertilizers and pesticides. Overall, our study provides a basis for the further development of biocontrol strategies based on B. amyloliquefaciens DHA55 to combat watermelon Fusarium wilt in the field.The present study established the antifungal activity of six antagonistic bacterial strains against five agronomically important phytopathogenic fungi; in particular,


Markedly elevated aminotransferase levels are commonly encountered among hospitalized patients. However, data regarding the trajectory of enzyme elevation and disease-specific prognosis are limited.This study included 3237 patients with at least one episode of aspartate aminotransferase or alanine aminotransferase level being higher than 400 U/L between January 2010 and December 2019 at 2 centers. Patients were classified into 5 groups comprising 13 diseases according to etiology. Factors associated with 30-day mortality were evaluated using a logistic regression analysis.The most common disease leading to markedly elevated aminotransferase level was ischemic hepatitis (33.7%), followed by pancreatobiliary disease (19.9%), DILI (12.0%), malignancy (10.8%), and viral hepatitis (7.0%). The 30-day all-cause mortality rate was 21.6%. The mortality rate for patients from the pancreatobiliary, hepatocellular, extrahepatic, malignancy, and ischemic hepatitis groups was 1.7%, 3.2%, 13.8%, 39.9%, and 44.2%, respectively. Age, etiology, and peak aminotransferase levels were independently associated with 30-day mortality.In patients with markedly elevated liver enzymes, the etiology and peak AST level are significantly associated with mortality. Elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels are a hallmark of hepatocellular injury. Both AST and ALT are highly represented in the liver. AST is also diffusely found in the heart, skeletal muscle, kidneys, brain, pancreas, lungs, leukocytes, and erythrocytes. In contrast, ALT has low concentrations in other tissues. Thus, elevated ALT is a more specific marker than AST for hepatocellular injury.Marked elevation of serum aminotransferase levels is frequently encountered in clinical practice. Recently, several studies on the etiology of the marked elevation of serum aminotransferase have been undertaken.The mortality rates are the highest in patients with ischemic hepatitis and hepatobiliary malignancy 37%–75%),7%–75%,3,Therefore, we conducted a large multicenter observational study of all patients with markedly elevated aminotransferase levels (>400 U/L) to determine their common etiologies and to investigate the factors associated with mortality.http://links.lww.com/HC9/A266): (1) age <18 years (n=74); (2) failure to classify the etiology due to lack of available data (n=746); (3) liver surgery or liver trauma (n=207); (4) loss to follow-up within 30 days (n=234); and (5) lack of consistent liver function test data (n=44). The remaining 3237 patients with at least one episode of AST or ALT level being >400 U/L were included . In cases with more than one etiology, the etiology that had the most significant effect on the elevation of aminotransferase level was selected based on the judgment of experienced hepatologists. Information on all-cause mortality for all enrolled patients according to the etiological group was recorded.Patients with markedly elevated aminotransferase levels, defined as at least one episode of AST or ALT level being >400 U/L, were classified into 5 groups comprising 13 diseases according to etiology by 2 experienced hepatologists  AST/ALT levels <1000 U/L , (2) AST/ALT levels <3000 U/L , and (3) AST/ALT levels ≥3000 U/L. Hepatic decompensation was defined as the presence of hepatic decompensated events, including hepatic encephalopathy, ascites, or documented gastroesophageal variceal bleeding. Heart failure was defined as either a history of heart failure through chart review or objective findings of impaired left ventricular and/or right ventricular function noted on echocardiogram at enrolment.2 test or Fisher exact test. For continuous variables, Student t test and 1-way ANOVA were used, as appropriate. Logistic regression analysis was performed to identify independent predictors of 30-day mortality. Variables with a significance level of p < 0.05 by univariate logistic regression analysis were subjected to multivariate analysis. The results of the logistic regression model are reported as OR with 95% CIs. Kaplan–Meier survival curves until 30 days with log-rank tests were used to compare the all-cause mortality between groups. All statistical tests were performed using 2-sided tests, with a significance level of 0.05.PASW software  was used to process the data. Categorical data were expressed as number (%), and continuous data were expressed as mean (±SD). For categorical variables, comparisons of between-group differences were performed using the χThe overall population of the study comprised 1936 (59.8%) men and 1301 (40.2%) women, with a mean±SD age of 60.2±16.9 years (Table http://links.lww.com/HC9/A267). The ischemic hepatitis group included cases with heart failure [46.3% (n=506)], pre-existing end-stage renal disease [4.9% (n=54)], and infection [28.6% (n=312)], the rates of all of which were higher than those in the rest of the groups. In the malignancy group, pre-existing liver cirrhosis (58.4%) and hepatic decompensation (51.9%) were the most frequent among all groups.The 5 etiological groups and 13 diseases according to the etiology of patients with markedly elevated aminotransferase levels are shown in Figure http://links.lww.com/HC9/A268). Progression of ALT levels occurred in 8.7% (n=234) and 5.2% (n=24) of the patients with initial ALT levels of <1000 and <3000 U/L, respectively . Overall, 45.8% (n=1477) of the patients with markedly elevated aminotransferase levels developed hyperbilirubinemia (>3 mg/dL) during the study period. Of the 3237 patients, 22.1% (n=714) were admitted to the intensive care unit and 16.7% (n=541) had hepatic decompensation at enrolment. In the entire group, the 7-day and 30-day mortality rates were 16.2% and 21.6%, respectively.At enrollment, we identified 71.9% (n=2326) of the patients to have initial AST levels <1000 U/L, 23.2% (n=751) to have initial AST levels <3000 U/L, and 4.9% (n=160) to have initial AST levels ≥3000 U/L. During the follow-up period, 62.0% (n=2008) of the patients had a peak AST level <1000 U/L, 27.9% (n=903) patients had a peak AST level <3000 U/L, and 10.1% (n=326) patients had a peak AST level ≥3000 U/L. Progression of AST levels occurred in 13.7% (n=318) and 9.6% (n=72) of the patients with initial AST levels of <1000 and <3000 U/L, respectively , pancreatobiliary, and hepatocellular groups (p < 0.001)  and ≥3000 U/L  . Patients with increasing AST and ALT levels during injury had a higher 30-day mortality rate than did those without progression of AST and ALT levels.A strong stepwise relationship was noted between peak AST levels and mortality. Patients with peak AST levels <1000 U/L (11.7%) had a lower 30-day mortality rate than did those with peak AST levels <3000 U/L  as ischemic hepatitis (33.7%), followed by pancreatobiliary disease (19.9%), DILI (12.0%), malignancy (10.8%), and viral hepatitis (7.0%). The 30-day mortality was high in the entire cohort , but most deaths occurred in patients in the malignancy and ischemic hepatitis groups.In the multivariate analysis, the peak AST level, rather than the peak ALT level, was a prognostic indicator in patients with markedly elevated aminotransferase levels. This is presumed to be because the AST/ALT ratio is higher in the malignancy and ischemic hepatitis groups (which have a poor prognosis) than that in the hepatocellular group (which has a good prognosis). Even in patients with acute viral hepatitis, fulminant hepatitis can occasionally lead to a high AST/ALT ratio (>2).Patients with markedly elevated aminotransferase levels and high mortality rates present a challenge to physicians. In a previous population-based study from Iceland,In our study of 3237 patients with markedly elevated aminotransferase levels, we used an aminotransferase cutoff of 400 U/L, whereas previous studies have used cutoff levels ranging from 400 to 3000 U/L for defining markedly elevated aminotransferase levels.This study has a few limitations. First, its retrospective design may have limited the identification of the etiology of markedly elevated aminotransferase levels. Second, there were 780 patients for whom we failed to classify the etiology of elevated aminotransferase levels due to a lack of available data. This suggests that there may have been a bias in the population studied, especially since 618 patients with cardiopulmonary arrest on arrival were also excluded; these patients had a very poor prognosis. Despite these limitations, the strength of this study is that it is the largest study on patients with markedly elevated aminotransferase levels to date and that it shows the effect of etiology and peak AST level on mortality.In conclusion, our study not only identified the various etiologies of patients with markedly elevated aminotransferase levels but also revealed that various etiologies have significant effects on mortality. By monitoring liver function, we demonstrated that the magnitude of the peak aminotransferase levels was significantly associated with mortality. Our observations suggest that it is important to establish the etiology of patients with markedly elevated aminotransferase levels, and that patients should be closely monitored for liver function.


CAPRA (NCT02565992) evaluated Coxsackievirus A21 (V937) + pembrolizumab for metastatic/unresectable stage IIIB–IV melanoma.Patients received intratumoral V937 on days 1, 3, 5, and 8 (then every 3 weeks [Q3W]) and intravenous pembrolizumab 2 mg/kg Q3W from day 8. Primary endpoint was safety.ICAM1 expression, or CD3+ infiltrates. Surprisingly, the baseline cell density of CD3+CD8− T cells in the tumor microenvironment was significantly lower in responders compared with nonresponders (P = 0.0179).Median time from first dose to data cutoff was 32.0 months. No dose-limiting toxicities occurred; 14% (5/36) of patients experienced grade 3‒5 treatment-related adverse events. Objective response rate was 47% . Among 17 responders, 14 (82%) had responses ≥ 6 months. Among 8 patients previously treated with immunotherapy, 3 responded . Responses were associated with increased serum CXCL10 and CCL22, suggesting viral replication contributes to antitumor immunity. For responders versus nonresponders, there was no difference in baseline tumor PD-L1 expression, These findings suggest responses to this combination may be seen even in patients without a typical “immune-active” microenvironment.NCT02565992.The online version contains supplementary material available at 10.1007/s00262-022-03314-1. Oncolytic viruses represent a novel therapeutic modality for the treatment of cancer, and several have been or are currently being investigated as potential immunotherapies in patients with various types of cancers . OncolytIn preclinical studies, V937 demonstrated rapid oncolysis of in vitro melanoma cell cultures and in vivo melanoma xenografts in immunodeficient mice . Notably+ T cells, and programmed death ligand 1 (PD-L1) expression [Pharmacodynamic effects of oncolytic viruses in the tumor microenvironment, including increased interferon production, CD8pression  and redupression , suggestpression . An ORR pression .The anti–PD-1 monoclonal antibody pembrolizumab is a standard-of-care therapy for patients with advanced melanoma. In the phase 3 KEYNOTE-006 study, which included 834 patients with metastatic melanoma (65% were stage IVM1c), pembrolizumab demonstrated superiority over ipilimumab with respect to the dual primary endpoints of PFS and overall survival (OS), with less high-grade toxicity . The benCAPRA was a phase 1b, multicenter, open-label, single-arm study. Eligible patients were ≥ 18 years old and had histologically confirmed metastatic or unresectable stage IIIB to IV melanoma, according to the American Joint Committee on Cancer 7th edition , 15. Pat8 50% tissue culture infectious dose  in a maximum volume of 4.0 mL. The volume could be reduced below 4 mL, with a consequent reduction in dose, if too few lesions of sufficient size were available to inject. Multiple lesions were injected at each injection visit if possible. Lesions > 25 mm in diameter were injected first (2.0 mL volume), followed by lesions 15 to 25 mm in diameter (1.0 mL volume) and then lesions 5 to < 15 mm in diameter (0.5 mL volume). After the initial injection of V937, any injected lesion that decreased to < 5 mm in diameter was injected with 0.1 mL volume. Tumor diameter was measured using a ruler/calipers or calipers on ultrasound. After administration of V937, the injection site was wiped with sterile tissue, and an occlusive dressing was applied to completely cover the injection site.Patients were treated with intratumoral V937 on days 1, 3, 5, and 8 and then every 3 weeks for up to 19 sets of injections. At each injection visit, the maximum dose of V937 administered was 3 × 10Patients also received intravenous pembrolizumab (2 mg/kg solution) starting on day 8 and continuing every 3 weeks for up to 2 years or until complete response, disease progression, or intolerable toxicity, whichever occurred first. On days in which patients received both V937 and pembrolizumab, V937 was administered first.Adverse events and DLTs were assessed based on the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. AEs were assessed from the time of initiating study treatment through 30 days after cessation of study treatment. DLTs were defined as any grade ≥ 3 toxicity related or possibly related to V937 with onset on or before the day 92 visit, except for lymphopenia, which was not considered a DLT.Disease status was assessed every 6 weeks by computed tomography or magnetic resonance imaging. Response and progression were evaluated using immune-related response criteria derived from modified World Health Organization criteria, as previously described , 17.Optional melanoma tumor biopsy samples were obtained at screening by incisional, excisional, or punch biopsy; archival tissue could be used in place of a fresh biopsy sample. Tumors selected for biopsy could have been target or nontarget lesions; target lesions had to be > 20 mm in total size, and sample collection could not affect the longest diameter for purposes of tumor assessment. Biomarker analyses performed on these tumor samples included NanoString-based interrogation of gene expression with the PanCancer IO 360™ Panel  and multiplex IHC (Supplementary Table S1). In addition, Luminex cytokine analysis was performed on day 1 and day 29 paired serum samples with a 48-plex human Cytokine/Chemokine/Growth Factor Panel A  analyzed on a Luminex 2000 analyzer . Serum V937 levels, based on polymerase chain reaction, were assessed during the first 2 years of treatment . Anti-V937 antibody titers were also evaluated during the first 2 years of treatment .The primary endpoints were the occurrence of AEs, serious AEs, and DLTs. Secondary endpoints were ORR, PFS, OS, and duration of response. Biomarkers were an exploratory endpoint.α = 0.05 level of significance with 90% power to detect a difference when the true ORR was 50%. The total sample size was estimated to be 50 patients to account for those who may not complete the postbaseline lesion assessment. However, study enrollment was stopped early because of prioritization of sponsor resources.As described in detail in the Supplementary Material (see Protocol), a Simon’s 2-stage design was used for sample size justification for ORR. The historical response rate was assumed to be 28% . With 45P values were based on Welch’s t test . For ORR analysis, 95% CIs were based on the exact method for binomial data. PFS, OS, and duration of response were analyzed using the Kaplan–Meier method.Safety and efficacy analyses were performed in all patients who received treatment. Biomarker analyses, performed in all patients with data available, are exploratory; n = 20), followed by death (n = 2), physician decision (n = 2), and other . Median time from first dose to data cutoff date was 32.0  months.A total of 36 patients were enrolled between December 17, 2015 and November 4, 2019 (Supplementary Table S2). All patients received the combination of V937 and pembrolizumab. Of the 36 patients enrolled, 7 (19%) completed and 29 (81%) discontinued the study. Progressive disease was the most common reason for discontinuation  had an ECOG performance status of 0. Twenty-three patients (64%) had stage IV disease and 13 patients (36%) had stage III disease. Previous therapies included immunotherapy , adjuvant therapy , and radiotherapy . No patient had previously received chemotherapy or neoadjuvant therapy.Patients had a median age of 68.5  years and 27 (75%) were men Table . More thn = 14, 39%), fatigue , pruritus , diarrhea , dry mouth , and hypothyroidism . Serious treatment-related AEs were experienced by 3 (8%) patients and included grade 3 keratoacanthoma, grade 3 autoimmune hepatitis, grade 3 autoimmune encephalitis, and grade 5 septic shock (the latter 2 events occurring in 1 patient), all of which were considered related to pembrolizumab by the investigator. There was 1 other grade 5 AE (cardiac failure), which was not considered treatment related by the investigator. Treatment-related AEs that led to treatment discontinuation were grade 3 increased hepatic enzymes and the grade 5 septic shock, the latter of which was related to steroids that had been administered for grade 3 autoimmune encephalitis.Adverse events occurring during the study are summarized in Table n = 4, 11%); no other immune-mediated AEs occurred in more than 1 patient. Four (11%) patients experienced grade 3 immune-mediated AEs including encephalitis, hepatitis, colitis, and pneumonitis (n = 1 for each). There were no grade 4 or 5 immune-mediated AEs. No infusion reactions occurred during the study.Immune-mediated AEs, regardless of relationship to treatment, occurred in 8 22%) patients  patients and target-noninjected lesions for 10 of 16 (63%) patients  patients experienced a PFS event  and lower expression of 35 genes , toll-like receptor 7 (TLR7), and toll-like receptor 8 (TLR8).Baseline tumor samples from 12 patients were available and analyzed. NanoString analysis demonstrated significantly higher expression in pretreatment tumor samples from responders versus nonresponders of 3 genes  by multiplex immunohistochemistry (IHC) analysis  and macrophage-derived chemokine (MDC/CCL22) from day 1 to day 29 compared with nonresponders  and with pembrolizumab monotherapy in a pooled analysis (9.1%) . HypothyNotwithstanding the limitations of cross-study comparisons, based on immune-related response criteria, median PFS with the combination of V937 and pembrolizumab in CAPRA 11.9 months) was numerically greater than with V937 monotherapy in CALM 5.7 months) .7 months. The ORR.9 months+CD8− T cells in pretreatment tumor samples were actually lower in responders compared with nonresponders. Although the reason for this finding is unknown, one hypothesis is that the pretreatment tumor samples of responders included fewer regulatory cells. We observed that V937 viral load was more often detectable in responders. Robust viral replication is a major determinant of effective oncolysis [Biomarkers were explored in CAPRA to gain insight into the possible mechanism for the systemic clinical responses. Surprisingly, responses were not associated with an increased baseline expression of viral entry proteins (ICAM-1 and DAF), PD-L1, or viral signaling proteins , nor were they associated with an inflamed baseline tumor microenvironment. In fact, levels of infiltrating CD3ncolysis . Evidencncolysis . Furtherncolysis . Clinicancolysis , and CXCncolysis .Given our findings above and the supportive data in the literature, we hypothesize that V937 viral infection leads interferon-gamma–mediated secretion of CXCL10 by multiple cell types in the tumor microenvironment, especially macrophages , which pIn conclusion, the combination of intratumoral V937 plus pembrolizumab had favorable safety and efficacy in patients with advanced melanoma. The ORR was 47%, and there was a high rate of complete responses (22%) not typically seen with pembrolizumab monotherapy. Thus, the addition of V937 to pembrolizumab appeared to improve efficacy with limited impact on tolerability. Biomarker analysis showed that responses were not associated with an inflamed baseline tumor microenvironment, indicating that a pre-existing tumor immune infiltrate is not required for response to this combination. Markers of the intensity of the viral infection  were associated with response, suggesting that the downstream effects of viral replication contributed to antitumor immunity and potentiated the activity of pembrolizumab. Further evaluation of the combination of V937 plus pembrolizumab for the neoadjuvant treatment of stage III melanoma is being assessed in an ongoing phase 1/2, randomized, open-label study .Supplementary file1 (DOCX 92 kb)Supplementary file2 (PDF 1551 kb)Supplementary file3 (EPS 2405 kb)Below is the link to the electronic supplementary material.


We investigated the use of titania coated ZnO tetrapods for photocatalytic degradation of two common types of microplastics, namely polyethylene (PE) microparticles and polyester (PES) microfibers. We found that the plastics morphology affects the rate of degradation, and that the use of electron scavengers is needed to maintain the reactivity of the photocatalysts over a prolonged period of time. Complete mass loss of PE and PES is achieved under UV illumination for 480 h and 624 h, respectively. In addition to pristine microplastics, the degradation of environmental microplastics sample (consisting primarily of polypropylene) was also demonstrated, though in this case longer degradation time (∼816 h) was needed to achieve complete mass loss of the samples. The pla2 [Among these processes, photocatalysis is of significant interest as it commonly shows significantly higher degradation efficiency compared to biodegradation and it is performed under milder conditions compared to chemical degradation  ,8. In ad2 .2 [2O5 [Consequently, photodegradation processes including the effect of illumination and the effects of a number of different photocatalysts on degradation/chemical changes of different plastics have been investigated in recent years , and the2 ,9,20,21 2 [2O5 ,19, and 2 [2, negligible degradation of PE was obtained under UV-A (365 nm) and visible illumination [2, namely improved crystallinity (which can lead to lower recombination losses) and higher mobility of photogenerated electrons, which can lead to improved photocatalytic activity [2 [2) providing an additional advantage of improving charge carrier separation and thus enhancing photocatalytic activity [2 photocatalysts, we have selected ZnO tetrapods, due to their exceptional optical properties (low nonradiative recombination losses) [2 shells using atomic layer deposition (ALD) to ensure conformal coating, and applied to photocatalytic degradation of microplastics. The effect of plastics composition ) and morphology  on the degradation process has been investigated. Complete mass loss has been achieved for PE MP particles, PES fibers, and environmental MPs, which predominantly consisted of polypropylene (PP).Thus, there is a well-recognized need for the development of novel photocatalysts to improve photocatalytic plastics conversion . This is2 , have be2 ,7,9. Whimination . Thus, tactivity ,46. Howeivity [2 , with chactivity ,47. For  losses) , and con losses) . The exc losses) , in agre losses)  and reac losses)  of ZnO i losses) . The ZnO22.12 precursor tetrakis (dimethylamido)titanium  was obtained Suzhou Fornano Electronic Technology Co., Ltd,. Zn powder (99.995 %) and Na2S2O8 were obtained from Sigma Aldrich. Spin trap 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was purchased from ABCam. PES was obtained from Dingwang Co., Ltd., while PE particles were extracted from the facial scrub (Neutrogena), using previously described procedure [TiOrocedure ,36. BrieEnvironmental microplastics pieces are retrieved from a water sample collected in Tsing Lung Tau  located on the Southwest coast of the New Territories in Hong Kong. The site corresponds to a remote beach in Tsing Lung Tau in a semi-enclosed bay with 2–30 m water depth. Water sample was collected at the water boundary, where the wave was about to break since macro- and micro-plastics accumulated there following the conclusion of Ref. . A total2.22O. To prepare a 10 nm black titania coating, ZnO tetrapods were placed in the powder sample holder, and the temperatures of the reactor heater and chamber heater were set to 150 °C and 200 °C, respectively. The TDMAT precursor was heated up to 75 °C to maintain the adequate vapor pressure in the bubbler. Nitrogen was used as purge and carrier gas with a flow rate of 20 sccm during the titanium/O2 precursor pulse processes. One cycle consisted of a 0.2 s pulse of TDMAT with 8 s purge time and a 0.01 s pulse of water, with 15 s purge time. The pressure before the deposition was 0.6 Torr.To prepare ZnO tetrapods, procedures in our previous work ,50 were 2.3in situ PE degradation with 2 mg PE mixed with 100 mg TiO2/ZnO tetrapod was carried out using IR-AFM (Nano IR2 from Bruker). BET surface area was determined using a Micromeritics ASAP2020 physisorption analyzer with pure nitrogen at 77 K after each sample was evacuated at 573 K for 4 h. ESR measurements were carried out on a Bruker EMXPlus. Different weights of sodium persulfate . The morphologies of the photocatalysts and microplastics samples were investigated using transmission electron microscope (TEM) equipped with EDX analyzer , a scanning electron microscope , and optical microscope (Leica). The FTIR spectrometer (VERTEX 80v from Bruker) was used for the measurements of the chemical structure of PE, PES and environmental sample before and after photocatalytic degradation, where the spectra were measured on plastics:KBr powder pellets (4 mg plastics/500 mg KBr). The investigation of the 2.42O2 for 96 h. The FTIR spectra of PE samples before and after H2O2 pre-treatment are shown in −1 [2S2O8 was added as electron scavenger [2 or PLS-LED100C, PerfectLight, 480 mW/cm2; the effect of illumination power on the degradation is small, as illustrated in 0–M)/M0, where M0 is the initial mass of the microplastics and M is the mass at the hours of irradiation.The 50 mg of catalyst was dispersed in a Petri dish containing 25 mL of de-ionized water. Before the photocatalysis, the plastics  were placed in 12 % Hwn in −1  is obsercavenger ,57, unle32/ZnO tetrapod core-shell photocatalysts, as shown in 2 interface [To construct a core-shell heterojunction photocatalyst, ZnO tetrapods were selected as the core material due to their exceptionally low defect density and high photocatalytic activity . Howevernterface .Fig. 1a)2/g for bare tetrapods, and 7.6 m2/g for TiO2/ZnO core-shell structures . The FTbrations ,36. We cl chains ,36. Mainbrations ,33. For brations . After 4expected ,64, as s2/ZnO catalysts demonstrated good performance in degrading MPs with different chemical composition and morphology , we investigated the photocatalytic degradation of environmental MPs. Obtained results are shown in 2/ZnO core-shell catalysts for photocatalytic degradation of microplastics. It should also be noted that complete mass loss does not necessarily imply complete mineralization, but it is expected that any degradation products in solution  [2 [Finally, as the TiOketones) ,41,43), ones) [2  since thones) [2 . In addiones) [2  and thusones) [2 ,50,53. Tones) [2  and/or H42/ZnO photocatalyst under 365 nm illumination. The degradation was found to be strongly dependent on the morphology of MPs, since the fibers exhibited saturation of the mass loss, with observable damage to the fibers but still maintaining the fiber integrity. The achievement of 100 % mass loss of MPs required ball milling to obtain sufficiently small fragments (∼2 μm particles) that can be efficiently degraded for MPs with large size , as well as the addition of an electron scavenger Na2S2O8 to significantly enhance the generation of ROS and thus enhance the photocatalytic degradation. Although different treatment time was needed for different types and morphologies of MPs, 100 % mass loss could be achieved for all materials investigated.In summary, we demonstrated efficient photocatalytic degradation of different MPs, namely HDPE extracted from facial scrub, PES microfibers, and environmental MPs using TiOYanling He: Investigation, Methodology, Visualization. Atta Ur Rehman: Investigation, Methodology, Visualization. Muxian Xu: Investigation. Christelle A. Not: Resources, Writing – review & editing. Alan M.C. Ng: Funding acquisition, Project administration, Resources, Supervision, Conceptualization, Writing – review & editing. Aleksandra B. Djurišić: Conceptualization, Funding acquisition, Project administration, Resources, Writing – original draft, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


LRRK2 mutation in LRRK2/PARK8 gene (genetic model). This analysis revealed that the metabolism of carbohydrates, nucleotides and nucleosides was similarly altered in the liver from the two PD mouse models. However, long-chain fatty acids, phosphatidylcholine and other related lipid metabolites were only altered in hepatocytes from G2019S-LRRK2 mice. In summary, these results reveal specific differences, mainly in lipid metabolism, between idiopathic and genetic PD models in peripheral tissues and open up new possibilities to better understand the etiology of this neurological disorder.The identification of Parkinson’s disease (PD) biomarkers has become a main goal for the diagnosis of this neurodegenerative disorder. PD has not only been intrinsically related to neurological problems, but also to a series of alterations in peripheral metabolism. The purpose of this study was to identify metabolic changes in the liver in mouse models of PD with the scope of finding new peripheral biomarkers for PD diagnosis. To achieve this goal, we used mass spectrometry technology to determine the complete metabolomic profile of liver and striatal tissue samples from WT mice, 6-hydroxydopamine-treated mice (idiopathic model) and mice affected by the G2019S- Parkinson’s disease (PD) is the second most common neurodegenerative disorder , only beSNCA (PARK1/4), Parkin (PARK2), PINK1 (PARK6), DJ-1 (PARK7), LRRK2 (PARK8) and ATP13A2 (PARK9) are responsible for the autosomal dominant or recessive mode of inheritance for PD. There are few missense variants of LRRK2/PARK8 that have been confirmed to increase the risk of PD, including variants G2019S, N1437H, R1441C/G/H/S, Y1699C, and I2020T. However, the substitution of the serine residue by a glycine residue in exon 41 of the protein kinase domain in LRRK2 (G2019S mutation) is the most common mutation across 51 countries [The bases of the etiology of this disease have not been fully deciphered, so exposure to environmental toxins, genetic factors and aging are currently accepted as the major triggers of this type of neurodegeneration . Most paountries . It is wountries . The exaountries ,19,20,21Omics analysis, especially metabolomics, is a very comprehensive tool for identifying molecular networks related to the pathogenesis of this little-known disease . There an = 5 mice/group) and liver (n = 4–5 mice/group) tissues in three different mouse models of PD and respective control mice: i, control mice ; ii, GS-PD mice (parkinsonian mice with whole body G2019S-LRRK2 mutation); iii, ai-PD mice .Mass spectrometry was used to perform a metabolomic analysis of striatum (LRRK2 and G2019S-LRRK2 transgenic mice (Tg)  were obtained from Jackson Laboratories and backcrossed in house. Genotyping was performed via PCR using the following oligonucleotide primers: 5′-ATTACCATGGTTCGAGGTGA-3′ (forward) and 5′-CAAGTGTCTGCAGGAAGGTT-3′ (reverse) for G2019S-LRRK2; 5′-CTAGGCCACAGAATTGAAAGATCT-3′ (forward) and 5′-GTAGGTGAAATTCTAGCATCATCC-3′ (reverse) for an internal-positive control. About two to three animals were housed per cage with free access to chow and liquid under a 12 h light/dark cycle. Special care was taken to minimize pain and discomfort in animals.Mouse strains and housing: All animal experiments were allowed by the “Ethics Committee for Animal Experimentation” of the Biomedical Research Institute “Alberto Sols” (CSIC-UAM) in Madrid (Spain) and performed in accordance with the European Communities Council Directive (2010/63/EEC) and national regulations (normative RD1386/2018). Adult male WT-Acute intoxication PD model: This model was induced as previously described . Using ag, 4 °C. Subsequently, the upper phase of supernatant was divided into two volumes of 300 µL, one was used for gas chromatography coupled by mass spectrometry (GC/MS) experiment in microtube, and the other was used for ultrahigh pressure liquid chromatography coupled by mass spectrometry (UHPLC/MS) experimentation.Tissue sample preparation for metabolomic analyses: About 30 mg of samples for each condition were solubilized into 1.5 mL tubes with ceramic beads with 1 mL of cold lysate buffer consisted of ISTD . They were then homogenized three times for 20 s at 5500 rpm using Precellys 24 tissue homogenizer , and centrifuged for 10 min at 15,000× Regarding GC-MS aliquots, the volume of 300 µL was transferred to glass tubes and evaporated. Subsequently, we added 50 µL of methoxyamine (20 mg/mL in pyridine) to the dry extracts and stored the samples overnight at room temperature in the dark. The next day, we added 80 µL of MSTFA and the final derivatization occurred at 40 °C for 30 min. Afterwards, samples were transferred in vials and directly injected into GC-MS.Concerning the UHPLC-MS aliquots, the volume of 300 µL was dried in microtubes at 40 °C in a pneumatically assisted concentrator . The dry extracts were dissolved with 200 µL of MilliQ water. Samples were transferred in LC vials and injected into UHPLC-MS or stored at −80 °C until injection.Targeted analysis by GC coupled to triple quadrupole (QQQ) mass spectrometry: GC-MS/MS acquisitions were performed on a 7890B gas chromatograph coupled to a triple quadrupole 7000C detector , equipped with an electronic impact source (EIS) operating in positive mode and a 30 m × 0.25 mm I.D. × 0.25 mm film thickness HP5MS capillary column (Agilent Technologies). Sample aliquots of 1 µL were inoculated into an inlet operating in splitless mode and set at 250 °C. Helium gas flow rate was fixed at 1 mL/min and the septum purge flow at 3 mL/min. The temperature was programmed as follows: 60 °C for 1 min, +10 °C/min up to 210 °C, hold for 3 min, +5 °C/min up to 325 °C and hold for 5 min. The transfer line and ion-source temperatures were 250 °C and 230 °C, respectively.Targeted analysis by UHPLC coupled to triple quadrupole (QQQ) mass spectrometry: Targeted analysis was conducted on a RRLC 1260 system coupled to a triple quadrupole 6410 detector (Agilent Technologies), armed with an electrospray source operating in positive mode. Gas temperature was set at 350 °C, gas flow at 12 L/min, and capillary voltage at 3.5 kV. Sample aliquots of 10 µL were injected on a Zorbax Eclipse XDB-C18 column , protected by an XDB-C18 guard column  and heated at 40 °C. The gradient mobile phase consisted of 2 mM of dibutyl ammonium acetate (DBAA) in water (A) and acetonitrile (B). The flow rate was set at 0.2 mL/min, and the gradient modified as follows: initial condition (90% phase A and 10% phase B) was maintained for 4 min, from 10% to 95% phase B over 3 min. The column was washed using 95% mobile phase B for 3 min and equilibrated using 10% phase B for 3 min. The autosampler was kept at 4 °C.m/z. The Q-Exactive parameters were: sheath gas flow rate 55 au, auxiliary gas flow rate 15 au, spray voltage 3.3 kV, capillary temperature 300 °C, S-Lens RF level 55 V. The mass spectrometer was calibrated with sodium acetate solution specific for low mass calibration.Pseudo-targeted analysis of intracellular metabolites by UHPLC combined to a Q-Exactive mass spectrometer. Reversed phase acetonitrile method: The profiling experiment was done with a Dionex Ultimate 3000 UHPLC system  coupled to a Q-Exactive (Thermo Scientific) equipped with an electrospray source operating in both positive and negative mode and full scan mode from 100 to 1200 Samples (10 μL) were injected on an SB-Aq column (100 mm × 2.1 mm particle size 1.8 μm) from Agilent Technologies, protected by a guard column XDB-C18 (5 mm × 2.1 mm particle size 1.8 μm) and warmed at 40 °C by a pelletier oven. The gradient mobile phase consists of 0.2% of acetic acid (A) and acetonitrile (B) in water. The flow rate was set to 0.3 mL/min. Initial condition was 98% phase A and 2% phase B. Molecules were then eluted using a gradient from 2% to 95% phase B in 22 min. The column was cleaned using 95% mobile phase B for 2 min and equilibrated using 2% mobile phase B for 4 min. The autosampler was kept at 4 °C. Peak detection and integration were carried out using the Thermo Xcalibur quantitative software (2.1.)p-values were estimated by one-way ANOVA , or Pearson’s correlation coefficients with 95% confidence intervals (Pearson’s correlation coefficient (R)). Differences were considered statistically significant when p-values were: ° (p < 0.1), * (p < 0.05), ** (p < 0.01), *** (p < 0.001) and **** (p < 0.0001).Quantification and statistical analysis: The data are reported as the means ± standard error of the mean (SEM). The number of independent data points (N) is indicated in LRRK2 mutation) or acute intoxication model  were analyzed to observe the complete metabolomic profile in striatum   D. These p value < 0.001; organic compounds; R = 0.88 and p value < 0.0001; lipids R = 0.58 and p value < 0.0001; nucleosides: R = 0.79 and p value = 0.02; carbohydrates: R = 0.87 and p value < 0.0001)  . It shou 0.0001) .p value = 0.99, p value = 0.18, We also found different modulations between both PD models when we focused on nucleotides , bile acids  and lipids  are mainly modulated in the genetic model, but not in the acute intoxication model in hepatocytes. (However, we found that nitrogen bases (GS-PD vs. ai-PD correlation: R = 0.57 and tocytes. .According to the results obtained in the liver extract, genetic models and those resulting from the acute intoxication of PD exert a similar metabolic modulation. Within the metabolic routes in which we observed a similar behavior in both models, we can highlight a decrease in maltose disaccharide carbohydrates such as the pentoses ribose, ribitol and xylitol A, as welLRRK2 mutation (As previously mentioned, genetic and acute intoxication PD models exert similar metabolic modulation in the liver, with the exception of lipid metabolism, which is highly modulated in the genetic model . Analyzimutation D.PARK gene defects and liver damage, such as the parkin defect and its relationship with alcohol-induced liver injury and steatosis in mice [The factors that contribute to the onset of PD are highly variable. Indeed, the loss of dopaminergic terminals and lack of dopamine release have been reported in the striatum from models as varied as PD patients, transgenic animal models of PD, or different toxin-induced models ,30,31,32 in mice . The preLRRK2 mutation throughout the body, and by acute 6-OHDA intoxication, are very similar in terms of metabolic changes in the striatum of diseased mice compared to healthy mice.Analyzing the metabolic profiles obtained from the striatum, we observed that most metabolic changes occur in parallel in all parkinsonian mice, regardless of the genetic origin or due to acute intoxication of the disease. In general, an increase in a wide range of amino acids, organic compounds, lipids or nucleosides was reported . It is cLRRK2 mutation [Nevertheless, when we check the metabolic profiles in the liver of GS-PD and ai-PD mice, the number of differences between both models is much greater . Analyzimutation ,40. Moremutation , and thamutation , includimutation ,40. ThusLRRK2-overexpressing liver hepatic carcinoma cells [LRRK2-KO mice, the lipid droplets accumulate more than in LRRK2-WT animals [LRRK2 mutation [Going in depth into the observed changes, we noticed a decrease in mono- and polyunsaturated LCFA and VLCFA levels exclusively in the liver of GS-PD mice, but the level of these metabolites was not modified in the acute intoxication model , as prevma cells ; convers animals ,46. Indemutation ,48; thisLRRK2 mutation has been directly linked with α-synucleinopathies [In addition, and also related to lipid metabolism, we found that PC and PE, major components of biological membranes, are exclusively decreased in hepatocytes obtained from GS-PD mice . Interesopathies  and thatopathies . α-Syn iopathies . Consideopathies , and thaopathies , it can LRRK2 KO mice showed elevated levels of ceramides in the brain [Finally, there is a relationship between ceramides and sphingolipids and PD ,55. Defehe brain . In addihe brain . For allSome limitations of this study should be addressed. To reduce the effect of estrogen modulations on metabolism, this study was performed only in male mice; however, a comparison could be made between the two genders. In addition, some metabolites of interest, such as ceramides and sphingomyelin, could not be analyzed, hence additional work may improve the information we have provided. The number of mice analyzed per group could be higher to enhance the robustness of the results; however, due to the high cost of this type of analysis in two different tissue samples and in three experimental groups, this sample size has not been allowed to increase. Overall, we intend to shed light on the general and specific metabolic changes in non-nervous tissues and thus understand if the liver may be at least partially responsible for the appearance of this neurodegenerative disease; however, it is essential to carry out experiments aimed at demonstrating any causal relationship between the liver disease, alteration, appearance and development of PD.